Research Report 2007 2008

Transcription

Leibniz-Institut für Molekulare Pharmakologie
im Forschungsverbund Berlin e.V.
Leibniz-Institut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V.
Research Report 2OO7 2OO8
Scientific Board of the FMP
Prof. Dr. Annette G. Beck-Sickinger
Universität Leipzig
Institut für Biochemie
Brüderstr. 34
04103 Leipzig
Prof. Dr. Bernd Bukau
Universität Heidelberg
ZMBH
Im Neuenheimer Feld 282
69120 Heidelberg
Prof. Dr. Michael Freissmuth
Universität Wien
Institut für Pharmakologie
Währinger Str. 13a
1090 Wien
Prof. Dr. Christian Griesinger
(Chairperson since 22.01.2007)
Max-Planck-Institut für Biophysikalische Chemie
Karl-Friedrich-Bonhoeffer-Institut
Am Fassberg 11
37070 Göttingen
Prof. Dr. Hans-Georg Joost
Deutsches Institut für Ernährungsforschung
Arthur-Scheunert-Allee 114
14558 Nuthetal
Prof. Dr. Gerd Klebe
Institut für Pharmazeutische Chemie
Marbacher Weg 6
30325 Marburg
Prof. Dr. Frauke Melchior
(Deputy Chairperson since 22.01.2007)
ZMBH
Universität Heidelberg
Im Neuenheimer Feld 282
69120 Heidelberg
Prof. Dr. Eckhard Ottow
Bayer Schering Pharma AG
Bayer Health Care
13342 Berlin
Prof. Dr. Herbert Waldmann
Max-Planck-Institut für Molekulare Physiologie
Otto-Hahn-Str. 11
44227 Dortmund
4 Members of the scientific board
Contents
Interview with Acting Director Hartmut Oschkinat. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6
Research Highlights
Introduction Thirteen ways of looking at a protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11
A virtual puzzle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
15
Doorways to the brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
25
A battery for the ear . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
35
Hubs, networks and partners that moonlight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
43
A molecular construction kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
53
Science groups
Structural Biology
Protein Structure H. Oschkinat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
62
Solution NMR P. Schmieder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
64
Structural Bioinformatics G. Krause . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
66
Drug Design R. Kühne. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
68
Solid-State NMR B. Reif . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
70
Protein Engineering C. Freund . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
72
In-Cell NMR P. Selenko . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
74
Signal Transduction/Molecular Genetics
Protein Trafficking R. Schülein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
76
Anchored Signalling E. Klußmann . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
78
Cellular Imaging B. Wiesner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
80
Molecular Cell Physiology I. Blasig . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
82
Biochemical Neurobiology W. E. Siems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
84
Physiology and Pathology of Ion transport T. J. Jentsch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
86
Cytokine Signalling K.-P. Knobeloch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
88
Molecular Myelopoiesis D. Carstanjen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
90
Chemical Biology
Peptide Synthesis M. Beyermann . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
92
Peptide Lipid Interaction/Peptide Transport M. Dathe/J. Oehlke . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
94
Mass Spectrometry E. Krause . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
96
Synthetic Organic Biochemistry V. Hagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
98
Medicinal Chemistry J. Rademann . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Screening Unit J. P. von Kries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Biophysics of Membrane Proteins S. Keller . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Protein Chemistry D. Schwarzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Microdialysis Service R. Richter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Administrative and Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Structure of the FMP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Map of the Campus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
Imprint . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Contents 5
Interview
with Hartmut
Oschkinat
You have just taken over as head of the
FMP after this position was held for many
years by Walter Rosenthal. Tell me a little
bit about how the FMP developed under
his guidance.
Walter Rosenthal has done an excellent job
in shaping the institute as a place that writes
molecular pharmacology with a capital
“Molecular.” He strongly supported the structural component of our research, aimed at
understanding the molecular basis of biological
processes relevant to pharmacological problems such as the identification of potential new
drug targets. A particular focus has been the
investigation of proteins and their interactions.
Recently, the rising movement of chemical biology has been something we needed to take
part in; it is essentially a pharmacological field.
We built the screening facility and have
launched a number of projects – within the
6 Interview
institute or as collaborations – that support the
process of developing small molecules that
modulate biological functions. In this respect,
the FMP has acquired considerable visibility in
the discovery and development of non-commercial compounds.
This is particularly supported by groups that
are focused on medicinal chemistry, screening,
and peptide modifications, which have been
installed over the past few years. These people
and their areas are becoming important in
collaborative projects on campus and, in the
long run, with developments such as the creation of a new systems biology institute, currently housed right next door. We’re well situated to take part in a new “systems approach”
to the investigation of pharmacologically
relevant pathways, and we anticipate that
combining our efforts will help to define
new targets.
Finally, we’re not a small institute anymore. The
FMP has developed into a major institute that
is now attracting a surprising number of talented young researchers, which is essential to our
future success.
The FMP has departments like structural
biology which play an interesting role in
examining the smallest fundamental units
of life, far removed from the level of the
whole organism... But drugs act on the
entire animal.
Structural biologists are farthest away from the
organism, but closest to the site of “action.”
Most people now realize that to achieve the
level of very fine control through a drug – or in
a small bioactive compound that you wish to
use as a tool – the structural view is very helpful, and often essential. And an increasing
number of companies are working on drug
design from a structural basis.
What gaps do you still see at the FMP that
you’d like to fill?
We’d like to build on the existing concept and
extend it toward something that we’re calling
the “rational modulation and monitoring of
biological processes.” Monitoring is the crucial
word here. Most biological investigations
require ways to visualize processes in cells or
organisms, and that demands the development
of specialized, small molecules. For example, if
you want to watch protein interactions, you
can cross-link them and use mass spectrometry
to analyze the components of multimolecular
machines. Or you can attach fluorescent labels
to proteins, and here there has been a need to
expand on the repertoire of fluorescent molecules such as GFP and increase their brightness.
The kind of optimization that you carry out on
these molecules is essentially the same process
used in drug design, and it may lead to the
Interview 7
Interview 7
development of tools for research and even
diagnosis.
The new campus MRI facility, where we can
examine both animal models and humans, is
one place where these themes come together.
We have already attracted Leif Schröder from
Berkeley, who is setting up a research group
funded by the European Research Council. Leif
builds molecular “cages” that can trap xenon
gas, which gives off a specific signal for imaging. The idea is to attach these cages to molecules that dock onto particular proteins. The
xenon diffuses into the cages and becomes visible, which could potentially helpful, for example to identify specific cell types, even tumors.
If you can find a protein that distinguishes one
cell from other types – some cancer cells produce particular metalloproteases, for example –
and attach such a cage to it, you can make the
cell visible and distinguish it from its healthy
counterparts. That’s just one example of the
many ways these technologies can be used.
Alongside enhancing our chemistry and screening groups, I think we need one or two more
high-profile groups on the biological side,
bringing in themes of pharmacological relevance. An example of this kind of area is protein synthesis and degradation – the delicate
balance that cells achieve between making
new molecules and taking them apart. This
system is disturbed in a number of diseases –
Alzheimer’s is a good example.
However, it should not be forgotten that we
do have highly recognized groups with a “biological” orientation, and otherwise we work on
themes within a network of collaborations that
begins here on the campus, extends to many
Berlin and German institutes, and stretches
across the world. This situation is useful and
very productive and will continue. The FMP’s
role in such projects is often the chemical biology focus. The in-house projects in this area
8 Interview
are usually linked to outside groups that have
additional biological expertise. Obviously we
work closely with the MDC, but there is also
quite a bit of interaction with companies here
on campus and elsewhere.
Do you foresee any significant changes of
orientation in the near future?
No, but we do have some important decisions
to make. For example, we need to decide what
the focus of our chemistry will be. Whether we
put emphasis on the optimization of compounds, discovery through screening, the
development of new tools... All these activities
are important, and allow us to build bridges to
some very interesting projects. It’s an area in
which we have the chance to have a special
impact. It is very important to continue integrating the FMP further into Berlin’s scientific
scene and this has to be done as well as
possible.
It is not really our task to create fully developed
pharmaceutical products. We would like to do
things that are explorative, on the “risky” side
– projects that the pharmaceutical industry
would not take on. An example is our work on
protein-protein interactions. Those are of high
interest to drug developers, but a strategy to
develop inhibitors for these interactions is lacking, so industry is not likely to step in. We
hope that our work will reveal such strategies,
as part of our mission to work on projects that
aim at a “pre-proof of concept.”
An important aim of the FMP is to generate an
equivalent to the drug development pipeline in
a way appropriate for academic institutes. This
means we shouldn’t run things on an industrial
level, but within the context of an interfacing
facility that will push the development of compounds – using intermediate technologies – to
a stage where industrial companies would be
interested. The screening unit is obviously one
important facility. Add to that our ability to
optimize compounds through medicinal chemistry and structural research. And if plans for an
in vivo Pathophysiology Laboratory here on
campus come to fruition, we will be optimally
set up to test compounds in animal models.
The second project is to develop the connection between structural and systems biology,
and our NMR groups have an important role in
this. We’ll get a 1.1-GigaHerz NMR machine,
which will use specially designed cryo-probes
and give us the highest field solid state NMR
machine currently available.
How has it been to become director?
A bit like having a cold shower! On the one
hand, it’s an interesting change, after so many
years of working with a research group. On the
other hand, it’s challenging, because there is a
lot to do. Since these new responsibilities are a
temporary solution for the FMP, I’m trying to
maintain the group. This comes at a time when
there are lots of papers to write. In both the
group and the FMP administration, I have
excellent colleagues who have put in a great
deal of extra effort to help, and that makes a
big difference.
One thing I’ve had to learn is how the different
science organizations in Berlin work – the Free
University, the Leibniz Association, the
Forschungsverbund. They all function very differently, and to work with them you have to
understand their mode of doing things and
how their communications networks operate.
That’s vital to the integration I was talking
about earlier.
Is this a particularly difficult time to step
in, given the overall economic situation of
Germany and the world?
In Germany we’re in a bit of a privileged situation in this respect. There is a strong realization
among decision-makers that science is an area
of opportunity, that both basic and more
applied research are sources of innovation. If
somebody has a good idea and can express it,
there are good opportunities to find funding.
There are numerous funding bodies and
schemes, and the percentage of successful
applications is high. We are taking advantage
of these opportunities. But money doesn’t necessarily translate into innovation. The challenge
is to develop our own culture here in Buch,
and to do original things. “To get orchids, you
have to cultivate seedlings.”
The major tasks at hand are related to two
ESFRI projects that we are on our way to taking part in. One is called EU OPENSCREEN and
the other INSTRUCT. We have an important
role to play in these projects, and it’s vital that
we get the start-up phase right. These projects
fit in well with our evolution into a truly
European center for the development of small
molecules for use in manipulating biological
functions. As a part of these activities, we will
be mounting a much larger screening operation, for which we foresee the construction of
a new building.
Interview 9
Research Highlights
Introduction:
Thirteen ways of looking at a protein
D
uring the 20th century, scientists
learned to look at life in new ways. A
major change was to see the cell as a
self-organizing system – a collection
of tens or hundreds of thousands of different
types of molecules whose activity is dictated by
their physical and chemical properties. These
characteristics determine which molecules dock
onto each other in a bustling, dynamic way to
produce highly complex structures: DNA molecules that survive longer than a person’s lifetime,
membranes, cells, organisms, and ecospheres.
The greatest challenge for biology has been to
try to piece together a story that begins with the
laws of chemistry and physics and works its way
upward mechanistically to explain higher levels
of biological structure. Molecules are at the root
of all aspects of the lives of humans and other
organisms – how they normally function, and
what goes wrong during diseases. The fields of
structural biology and biochemistry, which are
the main areas of research at the FMP, start at
the bottom end of the spectrum and push their
way up the ladder of organization and scale.
There is no upper limit; sometimes these projects
provide sudden and sharp insights into the
processes by which bodies arise from a single
cell, why people become sick and die, or how
species have evolved over vast stretches of time.
How we perceive life scientifically depends on
the methods that are available to investigate it,
and the amazing revolution in biotechnology of
recent years is providing an ever-deeper, evermore-intricate view of organisms. Fifty years
ago, a unique blend of physics and chemistry led
to the discovery of the double-helix structure of
DNA. This gave scientists their first glimpse of
the interconnectedness of the whole system:
regions of DNA called genes contain the recipes
for RNA molecules, which are used as templates
to build proteins. For many years, technical limitations suggested that this was a fairly straightforward, linear process. In the meantime, new
methods have revealed that it is a branching,
labyrinthine path with many layers of regulation,
feedback loops, and dead ends. Mapping the
route between gene and protein has become a
central activity of biomedical research: if a
process goes wrong, restoring it usually requires
understanding where it has gone astray.
Another change has been a revolution in understanding how proteins – often called the “worker molecules of the cell” – go about their work.
A few decades ago, researchers became proficient at isolating single proteins and other types
of molecules and unraveling their contributions
to processes such as cell differentiation, viral
infections, and disease. In the drama of life,
some individual proteins were seen as lead
actors, and others as a less essential supporting
cast. Today this view has changed with the realization that most molecules carry out their functions as parts of large molecular machines, often
containing dozens of molecules, which constantly and dynamically rebuild themselves to carry
out their tasks. Whether a machine gets built
and how it behaves depend on which components are present, and whether they are available or tied up in other machines.
This perspective, too, is the product of new
technologies, which still have limitations. It has
become clear that many things which happen in
the cell are due to quantities – not only whether
the cell makes a particular molecule, but how
much it produces. Until very recently, it has been
almost impossible to take an accurate cellular
census and count the population of a given molecule. That problem has been an intense area of
research that is finally yielding to clever new biochemical methods described in these reports.
Another great area of change has been the ways
computers are used to analyze complex interactions and weave them into complex, dynamic
models. Many more examples of the marriage
between technology and illuminating breakthroughs are found in these pages.
The stories in this book are products of the current state of the art in structural biology, chemistry, and many other fields; told just a few years
ago, they would have been much different. The
questions scientists ask and the kinds of stories
they tell about life are deeply influenced by the
approaches they can bring to each problem. This
becomes obvious from a glance backward at the
FMP’s research reports since the institute was
founded in 1992. Year by year, these books
record a shift in the way technologies have
opened up new ways of asking and answering
questions and produced new concepts.
Artists have known for a long time that understanding the world depends on our senses, the
culture that surrounds us, and the mental models we construct to comprehend the world. A
constant theme of 20th-century art, from painting to sculpture, fiction to poetry, was to illuminate objects, characters, and situations from
shifting perspectives. An example is Thirteen
Ways of Looking at a Blackbird, published in
1917 by the American poet Wallace Stevens.
This work, which is reproduced below, serves as
the inspiration for the titles of the feature stories
in this report: each chapter looks at proteins
from a slightly different structural or functional
point of view. In the context of biological
12 Introduction
research, the number 13 shouldn’t be taken literally: there are many more ways to regard biological processes, and each provides important
insights into life. But this approach provides a
“red thread” to show how all of these topics are
linked. Today’s biology is a merger between disciplines that began as different scientific cultures
– studies of cells, medicine, evolution, embryology, chemistry, physics, and others – which have
been steadily coming together in the investigation of living systems.
It is surely unusual to begin a scientific report
with a poem, but this reflects the evolving relationship between research and other parts of
society. As the pace of technologies and discovery increases, there is a growing potential of
biology to infuence human health and other
fields. The length of time between a fundamental discovery about cells and applications is
decreasing, placing a greater burden on scientists and their institutes to explain their work to
nonspecialists in clear, understandable terms.
The FMP regards this as a critical component of
its mission and is promoting a stronger relationship to the public through education and new
communication activities, including projects such
as this book.
Thirteen Ways of Looking at a Blackbird
(1917)
Wallace Stevens
I
V
VIII
Among twenty snowy mountains,
I do not know which to prefer,
I know noble accents
The only moving thing
The beauty of inflections
And lucid, inescapable rhythms;
Was the eye of the blackbird.
II
Or the beauty of innuendoes,
But I know, too,
The blackbird whistling
That the blackbird is involved
Or just after.
In what I know.
I was of three minds,
Like a tree
In which there are three blackbirds.
IX
VI
When the blackbird flew out of sight,
Icicles filled the long window
It marked the edge
III
With barbaric glass.
Of one of many circles.
The blackbird whirled in the autumn
The shadow of the blackbird
winds.
Crossed it, to and fro.
X
It was a small part of the pantomime.
The mood
At the sight of blackbirds
Traced in the shadow
Flying in a green light,
IV
An indecipherable cause.
A man and a woman
Even the bawds of euphony
Would cry out sharply.
Are one.
VII
A man and a woman and
O thin men of Haddam,
XI
a blackbird
Why do you imagine golden birds?
He rode over Connecticut
Are one.
Do you not see how the blackbird
In a glass coach.
Walks around the feet
Once, a fear pierced him,
Of the women about you?
In that he mistook
The shadow of his equipage
For blackbirds.
XII
The river is moving.
The blackbird must be flying.
XIII
It was evening all afternoon.
It was snowing
And it was going to snow.
The blackbird sat
In the cedar-limbs.
Introduction 13
A virtual puzzle
Jens Peter von Kries,
Simone Gräber,
Andreas Oder
T
he well was six or seven meters deep.
From above it looked like an inauspicious
hole, but in reality it was a treasure
trove. It belonged to an archeological site on the
Atlantic coast of France that had been unoccupied for about 1800 years. When the Romans
abandoned the town, they threw everything into
the wells: garbage, pots, the bodies of animals –
one even held human remains. The archeologist
supervising the dig lowered us down on a rope
and harness. You stood knee-deep in water,
reached down to your feet and grabbed whatever your fingers encountered, and loaded it into a
bucket, which was hauled up so that the contents could be run through a sieve. Back in the
daylight, we inspected our harvest: a toga pin, a
broken knife, other miscellaneous objects, and
small shards of pots and bones.
We had neither the time nor the tools to sort the
thousands of fragments and try to reconstruct
individual pots or the skeletons of single animals.
Today that process would be much easier because
it has been automated by computer programs
that are capable of coping with millions of “puzzle pieces.” Bits of pottery or other objects are
photographed or scanned, then turned into virtual, three-dimensional representations. The computer places two of them side by side and rotates
them in an attempt to dock them onto each
other. If no fit is found, it moves on to the next
piece.
A similar but more sophisticated method has
become a central tool in the search for new
drugs at the FMP, supported by experts in drug
design, medicinal chemistry and high-throughput
screening. Here the puzzle pieces are not potshards but proteins and other molecules. Their
three-dimensional topography and the chemistry
of their surfaces – the edges of the puzzle pieces
– determine whether two molecules fit and bind
to each other. Interactions between proteins and
other molecules are central to all biological
processes and explain the activity of drugs. If
scientists can identify a target – such as a protein
that plays a key role in the development of a disease – the next step is to try to find a substance
that can dock onto it and influence its activity.
This may lead to the development of a new drug
or a tool to investigate biological processes.
In the past, such findings were mostly the result
of trial and error; success came through experiments in test tubes and lines of cells grown in the
laboratory. “Many large pharmaceutical companies have libraries of hundreds of thousands of
substances that can be tested for a potential
impact on proteins,” says Jens von Kries, who
runs the screening unit established and shared by
the FMP and the MDC. “What companies do –
and what we do in the facility here – is to bring
those substances into contact with a target, either
in the test tube or in living cells that use the protein. But such screens are expensive and time
consuming.”
The result may be a marketable drug or a useful
research tool. “But even substances that have
powerful effects on proteins first have to pass
substantial hurdles,” says Jörg Rademann, head
of the FMP’s Medicinal Chemistry lab. His group
and Jens’ share the second floor of the Medical
Genomics Building, a sleek black structure on the
east side of campus. “Once a ‘hit’ has been
found during a screen, an interdisciplinary team
of biologists, chemists and modelers steps in to
improve it: to make it more effective, ensure that
it influences cells and organisms in a very specific
way, and that it isn’t toxic.”
Major pharmaceutical companies go through the
same steps, but in the current economic climate
they rarely go on fishing expeditions unless there
A virtual puzzle 15
Stefanie Grosskopf,
Jörg Rademann
is a strong likelihood of success. Institutes like the
FMP, where profit is not the main motivation, are
able to cast a wider net.
ease. The FMP has been steadily consolidating the
elements of this pipeline, often in collaboration
with the MDC.
Identifying a molecule that contributes to a disease process, finding a substance that can influence its activity, and turning it into something
useful are essential parts of drug discovery. In
many cases, it is necessary to invent new tools
just to expose the mechanisms underlying a dis-
Yet academic institutes have limited resources –
both in terms of money and personnel – and usually can only afford a library of compounds that is
a fraction of the size of a company’s. One result
has been the establishment of partnerships with
industry that give the FMP access to much more
extensive chemical libraries. Another effect has
been an intensive search for shortcuts in the
process of matching proteins to substances –
such as computer programs that treat the problem as a massive puzzle.
Recently these themes have come together in a
project involving the screening platform, the
medicinal chemistry group, and scientists at the
MDC. If things go as the partners hope, the result
may one day be a new tool to control a protein
involved in a wide range of cellular processes –
including defects that lead to cancer.
The biological theme of the project stems from
years of work by Walter Birchmeier’s group at the
16 A virtual puzzle
Identifying a molecule that contributes to a disease process, finding a substance
that can influence its activity, and turning it into something useful are essential
parts of drug discovery. In many cases, it is necessary to invent new tools just to
expose the mechanisms underlying a disease.
MDC. Walter is the institute’s former director; he
has now stepped down to devote more time to
research. Most of his work has focused on signaling pathways – routes of molecules by which
information is sent through cells. Such pathways
usually begin with receptor proteins on the cell
surface, which bind to molecules on the surfaces
of neighboring cells or are secreted by them.
Once activated by one of these partners, called a
ligand, the receptor sends information to the cell
nucleus via a series of other proteins. There the
signal changes the cell’s pattern of active and
silent genes, causing it to make new molecules
that alter its structure and behavior. Signals tell
the cell how to specialize, when to divide, and
when and where to migrate – so they are crucial
in the healthy operation of the body and are frequently culprits in diseases such as cancer.
Walter’s lab and many others have spent years
identifying the ligands, receptors, and signaling
molecules responsible for specific events in the
lives of cells.
One focus of this research has been a pathway
that begins with a receptor called Met. “This protein binds to a partner called HGF/SF – which
stands for hepatyte growth factor/scatter factor,
reflecting a couple of its important functions,”
Walter says. “Met can transmit a variety of signals. These pathways activate several genetic programs in cells that are important during embryonic development and disease.”
Such programs can become confused if something goes wrong – for example, if Met or another molecule undergoes a mutation. As a result,
the pathway may become permanently active at
the wrong time, or inactive and unable to transmit information. Cells may not specialize properly,
or they may receive a signal to divide or migrate
all the time. Overactivation can lead to cancer,
and mutations in Met or other proteins along the
pathway are very often found in patients' tumors.
“Frequently during embryonic development, cells
need to detach themselves from a tissue and
migrate somewhere else to help form body structures,” Walter says. “This is also an important factor in cancer; metastases happen when cells leave
a tumor and migrate to another part of the body
to build new tumors. We’ve learned that signals
which trigger both of these processes often pass
through Met. It’s a good explanation for why
defects in the pathway are linked both to cancer
and a developmental disorder called Noonan’s
syndrome.”
In 2000, Walter’s lab discovered that Met signals
pass via a protein called Shp2, which seems particularly susceptible to changes that cause cancer.
Over 30 percent of people who suffer from certain forms of childhood leukemia have mutations
in the molecule. It is also the culprit in Noonan’s
syndrome, which results in problems in the formation of the heart, short stature, changes in the
structure of the face, and other physiological
problems.
This suggests that Shp2 might make a good target for a drug, but first the molecule’s many functions in the healthy body need to be thoroughly
A virtual puzzle 17
Phosphatases are known as difficult targets. No company has come up with a
potent inhibitor so far, despite major investments over the last decade.
understood. Shp2 is a tyrosine phosphatase – it
trims groups of phosphate atoms from other proteins. Since these molecules are involved in signals related to cancer, one way to manipulate
them might be to control Shp2. In a collaboration
with the University of Oxford and Novartis (NDDI,
Boston) the Screening Unit had set up a panel of
biological test systems and other phosphatases to
search for compounds that could affect the
behavior of Shp2.
“Phosphatases such as Shp2 are known as difficult targets,” he says. “No company has come up
with a potent inhibitor so far, despite major
investments over the last decade. There are two
main obstacles: an inhibitor has to target one
specific phosphatase, rather than gumming up a
whole range of similar molecules that are busy
doing important things in the cell. And you have
to find a way to get the inhibitor into the cell –
which means slipping it through the membrane.
The inhibitors that have been found so far often
fail one of these tests, and sometimes both. At
the FMP we have developed methods to attack
both problems.”
The search for an Shp2 inhibitor was taken on by
Klaus Hellmuth, a postdoc in Walter’s group. Jens
worked with him to enlist the computer in narrowing down a list of substances that might bind
to the protein. Virtual screening can save an
enormous amount of time, Jens says, by discarding substances that are unlikely to bind and helping focus on the best candidates. But before this
could be done, the researchers needed information about the shape of the main piece of the
puzzle: Shp2 itself.
Alexandra Klaus,
Walter Birchmeier
18 A virtual puzzle
“Most pharmaceutical research in oncology is
currently aimed at kinases – proteins which add
phosphate groups to other molecules,” Jörg says.
“In other words, the companies concentrate on a
class of proteins that behave exactly the opposite
of phosphatases. If we could find an agent that
works actively and specifically against a phosphatase, we might have new opportunities for
cancer treatment.” It’s a bit of a circular situation:
the researchers won’t know whether phosphatases will make good targets or not until they
find an inhibitor. That would be a risky change of
focus for a company, he says – in a way, making
the problem more interesting for the FMP.
Today’s scientists have access to a wide range of
information about the structures of proteins and
other molecules. This data has usually been
obtained by structural biologists through crystallography experiments, a major technique used on
campus, in which proteins have been transformed into crystals and then illuminated with
X-rays. Other structures have been obtained by
looking at specific regions of proteins using
nuclear magnetic resonance, or NMR, one of the
major techniques used at the FMP. Either method
can produce three-dimensional maps of the
atoms within proteins, revealing the chemical and
physical features that allow them to bind.
Unfortunately, scientists have never been able to
get a look at Shp2 in the necessary arrangement.
In 1998, Steven Shoelson’s lab at Harvard Medical
School had obtained crystals of Shp2 in a lockeddown form, where one module of the protein
moves into a position that blocks its interactions
with some other molecules – unfortunately, it
obscures just the surface Klaus hoped to see. The
situation was like holding a puzzle piece already
snapped onto another, unwanted piece, one that
blocked attempts to snap on others. To get
around the problem, Klaus turned to a similar
phosphatase, about which more was known.
panies have developed inhibitors of the protein, used
in treating the disease. Along the way, researchers
have obtained a structure of this domain bound to an
inhibitor – showing the way their surfaces interact.
The domains in PTP1B and Shp2 weren’t identical, but
they were close enough that one could shed light on
the other. Klaus and his colleagues studied how subtle
differences probably affected the protein’s structure,
and what that would mean in the search for an
inhibitor. He developed a new homology model of the
protein domain, supported by Gerd Krause – a protein
Sometimes two pieces of a single puzzle have
similar shapes. And different puzzles may have
pieces with the same forms because they were
cut by the same machine. In an analogous way,
the “gene factory” of evolution has given
humans and other species similarly shaped molecules.
This is the case with Shp2. The module that carries out its phosphatase activity is called the PTP
domain, and it is found in a wide variety of other
proteins. Particularly similar to the module in
Shp2 is a domain in another molecule called
PTP1B.
There the scientists had a bit of luck. PTP1B has
been heavily studied due to a role that it plays in
diabetes. Its normal job is to “reset” a receptor
protein that the hormone insulin docks onto. In
diabetes type 2 that is undesirable, so drug com-
A model of the structure of the PTP domain of Shp2, the region where an inhibitor
would probably need to bind. The codes show the positions of amino acids that play
a key role in allowing other molecules to bind.
A virtual puzzle 19
modeler at the FMP – and used it to look for substances that might fit.
“This was where the computer puzzle-solving
method really came into play,” says Jens. “We
had a huge library of chemical recipes for substances. Those were stored in the computer as
two-dimensional diagrams. That’s fine for giving
their chemical recipe, but in reality they are threedimensional, and that’s what Klaus needed. So the
first thing he had to was convert these flat representations into objects with volume. Once that
was done, the program could start trying to snap
them into a three-dimensional groove in Shp2.”
It was a huge task – and an unfamiliar one for
most biologists. Fortunately the FMP has expertise
in the computational and other types of screening needed to take it on. The job required converting the shapes of 2.7 million small compounds and plugging them into the PTP domain.
This process “from weeks to months” of processing time on banks of powerful computers. “All of
the computers in our labs were churning away on
this,” Jens says. He laughs. “Even my personal
laptop – at night, when I wasn’t using it, it was
plugged into the ‘farm’ and working through virtual docking experiments.”
As the computer was trying to plug the compounds into Shp2, Klaus also had it try to fit the
molecule to other proteins with PTP modules. It
20 A virtual puzzle
was important to try to find a compound that
docked onto Shp2 but not the wide range of
other proteins with similar structures.
The docking program suggested 2,271 potential
binding partners for Shp2. Klaus narrowed the list
down to the 843 most potent compounds, and
from those he extracted 235 that docked strongly
onto Shp2 but far more weakly – or not at all –
onto other PTP domains. Sixty of those were chosen for further study. He expanded the search;
the screening unit purchased other compounds
that were similar to the best hits. One of them
might have an even more potent effect on the
protein.
The FMP team now stepped up to take things
further. “A primary hit produced by a docking
program is still an optimistic guess,” says Ronald
Kuehne, a drug design expert from the FMP. “At
this point the hit has to be carefully validated by
additional biological tests and by adding structurally related compounds for structure activity
analysis (lead structure characterization), aimed at
understanding the biological and chemical properties of the compounds.”
Klaus and his colleagues performed two types of
tests. The first showed that 20 of the compounds
could block Shp2’s activity in the test tube. They
next checked for its effects on cells. Walter’s lab
has developed a cell culture system to tease out
Finding an inhibitor for Shp2 required converting the shapes of 2.7 million small
compounds and plugging them into the PTP domain, which required from weeks
to months of processing time on powerful computers.
information about the information pathway
stretching between Met, Shp2, and genes.
“Activating the Met pathway in these cells causes
them to crawl away from each other, a cell
migratory behavior we see in development and
cancer,” Walter says. “The signals pass through
Shp2. It gives you a simple test to find an
inhibitor: if the cells don’t migrate, you’ve found
one. If they crawl away from each other, you
keep looking.”
Eight of the 20 substances blocked the crawling
behavior. One of them, a compound called PHPS1
(name generated from the chemical structure),
had the strongest effects in both tests and
seemed to be a good starting point for further
chemical investigations. From a database of over
two million puzzle pieces, Klaus had narrowed in
on the one that provided the best fit.
One of the most difficult steps in the creation of
a new inhibitor or drug is optimization – taking a
substance that has an effect on a molecule and
making subtle changes that make it tens or hundreds of times stronger. The substances in pharmacological libraries have usually been developed
for other purposes – the core structure of PHPS1,
for example, is used in food coloring – and need
now to be improved to do other things in this
project. Doing so requires an interdisciplinary collaboration of chemists, drug designers and biologists, moving back and forth between the test
tube, experiments in cells, and finally animal systems.
“The first issue for the chemist is to look at the
features of a substance that allow it to bind and
to think of ways that will make it more potent,”
says Stefanie Grosskopf, a PhD student in Jörg’s
group. “Then you synthesize new versions of the
molecule and try them out.
“In the beginning you need to develop a general
method to synthesize this substance. Then you
compare what you have synthesized to the ver-
Walter Birchmeier's lab has developed a cell culture system to
find molecules that stimulate or inhibit cell migrations. A.
Untreated cells. B. When stimulated with a growth factor, the
cells migrate. C. When these cells are then treated with
PHPS1, the cells stop migrating.
A virtual puzzle 21
A diagram of PHPS1 (left). The right diagram
points out sites that bind to amino acids in
the target protein (circles) and regions that
might be modified to improve its activity as
an inhibitor of Shp2.
sion that is commercially available. In our case
there was no difference. Finally, the procedure
has to be optimized to achieve a fast and efficient way to synthesize libraries of small molecules that are slight variations of this starting substance. With my 4-step synthesis I prepared
around 80 derivatives based on PHPS1.”
That isn’t simple: it usually involves many steps of
synthesis that often take weeks or longer, using
chemical methods to snip parts off a substance
and add others. Each of the variants made by
Stefanie had to be tested again: Did they inhibit
Shp2’s activity, without interfering with other
molecules with a PTP domain? Some versions
passed the tests, performing better than the original form of PHPS1; Stefanie is still working on
the promising candidates and new derivatives of
them.
Part of that work involves studying the other puzzle piece, identifying exactly specific amino acids
in Shp2 that are crucial to the docking of PHPS1.
Klaus and his colleagues created versions of Shp2
with single mutations – like a test to see whether
spelling mistakes make a text incomprehensible.
This led to a roadmap that shows the chemists
which features of the protein are necessary for
binding – and it also gave Stefanie a better idea
22 A virtual puzzle
of what to do on her side. Even clearer images
are in the pipeline: the lab of Udo Heinemann at
the MDC has now obtained Shp2 in crystal form,
which will allow it to be used in tests that fit
inhibitors into the protein’s active site.
“PHPS1 needs to be developed further to become
an effective research tool and useful as a drug,”
Stefanie says. “We’ve already improved its efficiency dozens of times. The substance has several
characteristics that make it attractive as a drug
candidate: it is non-toxic, and it can slip through
the membrane to enter cells – it has to do that to
work. But it needs to be even more powerful to
become a candidate for drug development.”
Interest on the part of companies is there – especially because of the link between Shp2, the Met
pathway, and cancer.
“PHPS1 is the first compound that specifically
inhibits Shp2 without affecting other important,
closely related proteins,” Walter says. “That’s
especially promising because of our previous
work, showing that this molecule is probably the
crucial node in aberrant signals that lead to cancer. When we looked at the effects of PHPS1 on
cultures of tumor cells, we found that it blocks
processes such as growth and migration. This
gives us a good basis to move on and see if it is
PHPS1 has several characteristics that make it attractive as a drug candidate: it is
non-toxic and can slip through the membrane to enter cells. But it needs to be
more powerful to become a candidate for drug development.
effective in coping with tumors in living animals –
while leaving healthy biological processes in
place.”
The researchers enthusiastically agree that the
project is a good model of how the FMP and
MDC work together. “One great thing about this
campus is that clues about proteins that are
potentially relevant to disease can come from a
cell biologist such as Walter and then be taken
up by academic drug discovery,” Jörg says. “Each
step is a creative process that involves inventing
the right screening methods and making the
active and specific molecules that you need to
really prove you have a valid drug target.”
PHPS1 still has a long way to go before it might
be used as a cancer drug. Chris Eckert, a technician who is working on the project within the
groups of Walter and Jens, has personal experi-
ence moving compounds through the steps of
testing in cell cultures and animal models; he
used to do the same thing for the company
Oncotest. “But despite the improvements that we
have made in the primary hits, there are still lots
of open questions about their behavior.
Nevertheless, the best compound has made it
through several hurdles and is being used in early
animal testing.”
The chemists are now beginning the next round
of optimizing the compounds. In the meantime,
current versions of the molecules are being tested
in Walter’s lab to explore Shp2’s role in development and disease. Even if some of the products
lead to dead ends, one success could have a
huge payoff. It’s for moments like these, the
researchers agree, that such an interface facility
shines and reveals its purpose.
A virtual puzzle 23
Doorways to the brain
T
here is a wonderful picture of Paul Ehrlich,
the famous scientist of the turn of the last
century, at work in his office. He is making
notes in a file that is balanced on one knee. The
reason that he hasn‘t placed it on the desk in
front of him is obvious: there is no space. Its surface, like the two chairs in the background and
the shelves behind them, is piled high with
paperwork and more files. You need only read
the speech that Ehrlich gave upon receiving the
1908 Nobel Prize for Physiology or Medicine to
realize that there is no relation between the disorder in his office and the orderliness of his mind.
If a living system doesn’t behave the way you
expect, it is often a hint that something completely different is going on. Edwin Goldmann,
one of Erlich’s students, performed the experiment in reverse, introducing the dye directly into
the spinal fluid of mice. Now it was absorbed by
the brain and nervous system but did not enter
other tissues. The experiments revealed the existence of the blood-brain barrier, a system that
protects the brain from many infections and toxins. At the same time, it blocks the passage of
drugs or substances that might be helpful in
fighting disease. This is one of the things that
have brought it to the attention of Ingolf Blasig
and his lab at the FMP.
Ehrlich is best known for finding a cure for
syphilis, his insights into the immune system and
the activity of toxins, and the articulation of concepts that have become central to modern pharmacology. Along the way, he made a number of
serendipitious discoveries. By the late 19th century,
researchers knew that understanding human biology – and the causes of disease – would require a
deep investigation of cells and their chemistry. As
Ehrlich stated in his Nobel speech: “It is, I think, a
generally acknowledged and undisputed fact that
everything which happens in the body . . . must
ultimately be attributed to the cell alone; and furthermore, that the cells of different organs are
differentiated from each other in a specific way
and only perform their different functions by
means of this differentiation.”
The brain is not completely isolated. Nutrients
and other necessary substances are able to leave
the bloodstream to supply the cells of the central
nervous system. A century after the experiments
of Ehrlich and Goldmann, the discriminatory
activity of the blood-brain barrier is still not completely understood – unfortunate, because that is
a key to developing drugs for the treatment of
diseases of the nervous system.
To glimpse the structure and behavior of cells, scientists needed a way to stain them, a problem
which occupied Ehrlich and many other
researchers of his generation. He developed new
types of dyes and injected them into mice and
rats. A substance called trypan blue spread
through the bloodstream and was then taken up
by the body’s tissues. Curiously, however, the
stain did not enter the brain or spinal fluid.
Ehrlich attributed this to some unknown characteristic of brain cells or nerves, something about
their chemistry that rejected the dye.
Ehrlich comprehended this; he also believed that
if you could find something that entered cells,
you might be able to attach therapeutic substances to it for delivery. As he said, “Since what
happens in the cell is chiefly of a chemical nature
and since the configuration of chemical structures
lies beyond the limits of the eye’s perception, we
shall have to find other methods of investigation
for this. This approach is not only of great importance for a real understanding of the life processes, but also the basis for a truly rational use of
medicinal substances.”
Doorways to the brain 25
To form tight junctions, claudins have to interact with each other and other
proteins, but little is known about how they do so. Answering that question is
crucial to learning to manipulate the blood-brain barrier.
Ehrlich did not live long enough to witness the
development of tools necessary to accomplish
these goals; today’s scientists can bring a wide
range of methods to the questions he posed.
Ingolf’s lab is creatively using a spectrum of
chemical, physical, and biological techniques to
peer deeply into the cell, to the level of proteins
and other molecules that were invisible a hundred years ago. Their aim is the same: to learn to
open and close the doorways to the brain.
Ingolf says that the main components of the tight
junctions have been identified, but scientists
don't yet understand the process by which the
molecules seal the gaps between cells. Proteins
called claudins, which are lodged in the membranes of neighboring cells and latch onto each
other, play a central role. This was established ten
years ago by Shoichiro Tsukita’s group at Kyoto
University in Japan. The lab took cells that did not
form tight junctions and began adding single
components of the system. When they added
claudins, the cells began to connect themselves
to their neighbors in junction-like strands.
“To form the tight junctions, claudins have to
interact with each other and with other types of
proteins, and very little is known about how they
do so,” Ingolf says. “Answering that question is
crucial to learning how to manipulate the barrier.”
Nutrients and other substances slip through the
linings of blood vessels to reach cells. In most tissues, viruses, bacteria, and the blood cells that
hunt these parasites exploit the same route. But
the vessels that supply the brain are sealed by
tight junctions: proteins that bind cells to each
other in tangled fibers; under the microscope, the
cells seem to be fused to each other. This greatly
restricts what can fit through the gaps. The barrier helps protect the brain from invaders, but it
also means that very few drugs can enter the
tissue.
26 Doorways to the brain
Claudins are intricately woven through membranes in a way that leaves some regions of the
molecules outside the cell, where they can bind
to proteins on their neighbors. It is often impossible to purify proteins attached to membranes or
obtain them in crystal form, which might give
researchers a look at the details of their structure.
So Ingolf and his colleagues have resorted to
other methods to study the features that govern
their activity. “One approach we use is based on
the endothelial cells that line capillaries in the
brain,” Ingoolf says. “We grow these cells in a
single layer in culture dishes and study how they
behave when we make changes in claudins or
other tight junction proteins.“
Human cells contain the genes to make over 20
varieties of claudin proteins. Each tissue that
needs them produces only some of them, and a
claudin typically only recognizes its own type. This
permits the establishment of different kinds of
connections in various tissues and organs – otherwise, all blood vessels might be as hard to penetrate as those of the brain.
Ingolf Blasig, Victor Manuel Castro
Villela, Jörg Piontek, Jimmi Cording,
Anna Veshnyakova, Reiner Haseloff,
Barbara Eilemann
In 2003 a Japanese group used genetic engineering techniques to develop a strain of mouse without one of the molecules, claudin-5. It was an
important study, Ingolf says, because the newborn pups had defective blood-brain barriers.
“Small molecules such as drugs could now pass
through from the bloodstream,” he says. “This
suggests that manipulating claudin-5 might give
you control over the barrier.”
Accomplishing this would probably require a clear
picture of the protein. It was known that the two
ends of claudin-5 lay inside the cell, weaving
through the membrane in such a way that two
loops hung outside. “We also suspected that single copies of the molecule – like many membrane
proteins – might bind in groups of two or more
as its activity changed,” Ingolf says. “But whether
it really did so was unknown, as well as what
parts of the loops were important in establishing
tight junctions. Those questions were important
to answer if we hoped to relax the junctions and
open the barrier.”
Light microscopy offered a way to study whether
multiple copies of claudin-5 formed groups in the
membrane. In 2006 the lab created a version of
the protein with an extra module – a fluorescent
tag that gives off a signal when illuminated by a
laser. The method permitted the researchers to
track the protein in the cell; it also gave them a
way to observe its interactions using a method
called fluorescence resonance energy transfer, or
FRET. The technique is one specialty of Burkhard
Wiesner’s lab at the FMP, frequent collaborators
of Ingolf’s group.
Illuminating a fluorescent molecule with light
causes it to absorb energy and then radiate it
again. The amount of energy can be precisely
measured. If two fluorescent proteins dock onto
each other, each absorbs a bit of the other’s energy. This is the basis of FRET. The method allowed
Mutant phenotype
postdoc Jörg Piontek, student Nikolaj Zuleger and
other members of Ingolf’s group to detect binding between claudin-5 proteins in the cell membrane.
“Jörg is an excellent cell biologist and microscopist,” Ingolf says. “He is working under a
major grant that he obtained from the German
Research Council (DFG). He has his own students
and is virtually running his own lab, with new
grants in the pipeline. Nikolaj was a trainee in the
group, but I have rarely had a student who was
so good at the bench, with his hands. His work
with FRET was an important contribution to the
project. Now he’s gone on to do a PhD at
Oxford.”
The experiments showed that multiple claudin-5s
bind in the membrane of a cell, in addition to
helping build strands to neighbors. This creates a
kind of horizontal tightening, a bit like pulling a
drawstring to close a bag and keep something
from falling out. In this case, the closure of the
gaps keeps all but the smallest molecules from
passing into the membrane from outside.
“But the study didn’t reveal which features of
claudin5 permit it to dock onto other copies – or
might prevent it from doing so,” Ingo says.
“Since those gaps have to be closed to make
tight junctions, this is where we began focusing
our attention.”
Changing single amino acids in claudin-5 alters the location of
the molecule in the cell. Left: locations of the changes in one
of claudin-5's loops (red); the grey line represents a membrane.
Right: fluorescence microscope images showing the protein's
location in cells. Top row: claudin-5 normally sits on the cell
surface. Second row: Some alterations don't change this. Third
row: Other changes cause it to become distributed through
the cell cytoplasm. Fourth row: Different substitutions lock the
molecule to membranes inside the cell.
Cells assemble proteins as linear strings of amino
acid subunits, starting at what scientists refer to
as the head and moving to the tail. During this
process, proteins are attached to membranes and
folded into shapes that determine their functions.
In the case of most claudins, this leaves two loops
dangling outside the cell. The first loop helps
cinch together neighboring cells. Recently Ingolf’s
lab has been working on the second loop.
“Under the microscope we had observed that
these loops self-assemble into large clusters,” he
says. “That’s the behavior you would expect if
they are responsible for horizontal tightening.
"As a group leader you depend on your people; they take the lead in projects and
carry out the day-to-day work, so you have to give them something."
And there’s another strong hint that this loop is
important: in contrast to other parts of the molecule, it has barely changed over the course of
evolution. In the different forms of claudins
found in humans – as well as in different species
– the loop has basically remained the same.”
Which amino acids were responsible for the
chemical interactions that draw the loops together? To find out, the researchers began respelling
this part of the protein, creating mutants that
exhanged some letters for others. They added the
altered proteins to cultures of human cells that
normally do not produce claudin-5 or form tight
junctions. Some spellings drew the cells together;
others did not.
The same system offered a chance to find molecules that might block the behavior, and PhD student Lars Winkler was eager to perform the
experiment. Tsukita’s lab in Tokyo had already
found a molecule that binds to the loop of some
types of claudins, a toxin from bacteria called
CPE. The Japanese group had already shown that
treatments with CPE increased the passage of
small molecules into cells. It also worked in animals: after treatment, drugs could pass into the
rat intestine at a high rate, nearly 400 times the
rate of the drugs when coupled to current therapeutic “transporters.” But there wasn’t any evidence that CPE would have a similar effect on
the brain; it wasn’t known to bind to claudin-5.
A model showing how individual claudin-5 molecules link to each other in a step-wise way to help build tight junctions.
“Even so, Lars came in one day and told me he
wanted to work with the toxin,” Ingolf smiles.
“I told him it was too early – we still had a lot of
mutants to make. So we made a deal – if they
kept working on the mutants, I’d order the CPE
and let them go ahead. As a group leader you
depend on your people; they take the lead in
projects and carry out the day-to-day work, so
you have to give them something.”
CPE wasn’t easy to obtain. “Tsukita and his colleagues had the gene for the toxin, but they
weren’t giving it out to just anyone. Of course
that’s understandable – they were hoping to
obtain a structure of the toxin bound to the loop,
and other groups might beat them to the story.
We promised not to work on the crystal structure, and they sent it to us.”
The mutant studies were beginning to yield the
results that Ingolf had hoped for. The second
loop contains 16 amino acids. Using FRET, the scientists showed that nine of the changes had little
or no effect on the horizontal tightening in the
membranes. But changes in the other 7 letters
loosened things up, creating larger gaps.
More work with the microscope provided a possible explanation. “Claudin-5 is synthesized within
the cell, in thread-like structures called the endoplasmic reticulum,” Ingolf says. “After that it is
normally transported to the periphery of the cell
and inserted into the membrane. But many of the
interesting mutants were being held inside. If
they never reach the surface, they can’t contribute to the tight junctions. So one effect of the
mutants is to scramble codes in the protein that
act as sorting instructions, telling cells where to
put them.”
Lars was now ready to investigate how CPE interacts with claudins. First he wanted to see which
types of claudins the toxin binds to. The lab set
up an array of probes made up of different
claudin family members. They found out that CPE
binds to five of the types, but not claudin-5.
When Lars checked living cells, however, he discovered that the two molecules did interact.
What features determine whether CPE binds to a
particular claudin? The scientists studied differences in the molecules’ chemistry and discovered
a five-letter code in the middle of the loop that
seemed to be required. Two spelling changes in
the loop of claudin-5 caused a drop in its interactions with the toxin. Other types of changes to
the protein decreased the horizontal tightening
between claudins in the membrane – and gave
rise to more binding with CPE.
“This yielded information that we could combine
with to make a model of the loop’s structure,”
Ingolf says. “And that gave us hints about modifications you could make in CPE to control
Barbara Eilemann
claudin-5, some aspects of the junctions, and
potentially the blood-brain barrier.”
The group’s progress on claudin-5 has been possible because of the range of methods that have
been brought together, each revealing aspects of
the molecule that can be combined into a
structural picture. The interdisciplinary
approach of the group reflects Ingolf’s own
background.
“My mother was a cancer prophylaxis researcher
in the GDR,” he says. “I became interested in
biochemistry, but particularly the connections
between cell chemistry and diseases such as cancer. When it came time to work on my diploma
thesis, I proposed doing it here in Buch at the
Robert-Rössle-Clinic. My advisor tried to talk me
out of it; he said it was risky. At the time, most
biochemists focused on one enzyme or one drug,
but I was determined to find a way to do something related to disease.”
He chose the risky path, coming to Buch, and it
gave him a unique introduction to clinicians’
approach to research. That’s necessary to become
familiar with issues such as toxicity and the body’s
tolerance for foreign substances, he says. And it
can give you unexpected insights into a biological
process. One project, for example, revealed a link
to lysosomal storage disease: a defect in small
Highly reactive forms of oxygen have to be carefully managed by the body. They
disrupt the blood-brain barrier in neurodegenerative conditions such as Alzheimer's
and Parkinson's disease.
cellular structures called lysosomes that digest
and recycle molecules.
“I remember a chance meeting that I had with
pediatricians who were working on patients; they
didn’t know the mechanisms underlying the disease, and I wasn’t aware of the kinds of health
problems that patients experience. Combining
those perspectives can be very helpful to both
sides.”
One theme of the group has been to study how
cells cope with highly-reactive forms of oxygen
atoms. These energetic atoms drive chemical
processes throughout the body – but they have
to be carefully managed. Flaws in the control sys-
tem lead to a number of diseases, including heart
problems. They can also disrupt the blood-brain
barrier, which is disturbed in neurodegenerative
conditions such as Alzheimer’s and Parkinson’s
disease.
“We met with physicians who were actually
doing operations on hearts, getting a first-hand
look at structural changes in the tissue such as
problems with ventricles,” Ingolf says. “They
began to notice a disturbing trend where two to
three years after an operation, some of the children were suffering from memory and behavioral
problems. We looked at the situation and discovered that during surgery, the children’s bodies
were cooled – maybe they were suffering from
oxidative stress.”
The lab carried out an analysis of samples of
patient blood and discovered that this was likely
the case. They recommended that nitrous oxide
be added during the procedure, which has a protective effect on the blood-brain barrier. As a
result, the side effects suffered by the patients
disappeared.
“I’ve been lucky to have regular exchanges with
people who are not narrowly focused. That’s a bit
harder for today’s students, who are under quite
a bit of stress to obtain results and publish. I try
to push them to make these broader contacts,
and it’s a theme when we go off on doctoral
retreats.”
Ingolf cites a few professors that had an important influence on his career path: Albert
Wollenberger, who combined clinical and fundamental research while setting up major cardiovascular research activities in Buch. In the institute
for Cancer Research, he worked under Arnold
Graffi. “He used to come into the lab every
morning and ask you, ‘What did you learn from
your experiments today?’” Ingolf smiles. “Not a
bad question. So sometimes I come into the lab
and ask my students, ‘What did you learn from
your experiments today?’”
That reflects, he says, the larger interdisciplinary
attitude of the FMP. “Hartmut Oschkinat has
done a lot in this respect,” he says. “Over the
years he has helped us acquire a strong set of
methods that broaden our perspectives. Of
course many people arrive with a sharp focus on
a particular family of molecules, or some structural issue. That’s important, but we need to see
things more widely.”
A battery for the ear
"Each species lives in its own unique sensory world, to which other species may be
partially or even totally unaware. A whole series of specific devices alien to human
perception have emerged: biosonar, in bats; infrared detectors in snakes; sensitivity
to magnetic fields in birds... What an organism detects in its environment is only
part of what is around it, and that part differs in different organisms... Our perceptions are not direct recordings of the world around us; rather, they are constructed
internally, according to a set of innate rules. Colors, tones, tastes, smells do not
exist as such, outside of the brain."
Richard Axel, Vision of the Future, MIT World lectures
J
ust before a conductor steps up to the podium, an orchestra tunes up. The concert master usually takes an “A” from the oboist,
then passes it along to the string section. Each violinist has to tune four strings against a loud background of other people doing the same thing –
especially when the wind and brass sections join
in. Focusing on a single instrument is different
than the way a musician listens during a concert in
a church, when the aim is to hear all of those
sounds simultaneously, and to blend in rather than
stand out. And to the performer this sounds considerably different than to the birds flying up high
along the vaulted painted ceiling of the cathedral.
Tuning a cello, distinguishing the sounds that
make up speech, and appreciating a suite by
Bach start with the architecture of the inner ear.
Its structures respond to changes in air pressure
and convert them into electrical signals. That job
is handled by rows of cells with hair-like cilia that
wave in tune to vibrations. This alters the activity
of proteins in their membranes, causing the cells
to take in charged ions. There are two effects:
the sound is amplified, then turned into electrical
signals that are transmitted to nerves. They route
the signal on to hearing centers in the brain.
Some of those mutations affect channels and
other proteins that regulate the passage of ions
into and out of cells, which are the focus of
Thomas’ lab. His systematic work on the proteins
that regulate this process has led to various parts
of the body, including the kidney, muscle, the
brain – and the inner ear.
“Channels and other proteins govern the transmission of electrical impulses between cells,”
Thomas says. “That requires the passage of
charged atoms through membranes. And the ear
turns out to be a beautiful system to investigate
the molecules involved in this process.”
The lab's studies of channel proteins have
brought them to a tiny region of the inner ear
called the organ of Corti – named after the
Alfonso Corti, the 19th-century anatomist who
discovered and mapped it. Some of the most
crucial steps of hearing take place in this organ,
“Hearing depends on structures at many scales:
from the overall anatomy of the ear, to the cells
that make it up, and the molecules that govern
their functions,” says Thomas Jentsch, a group
leader jointly appointed by the FMP and MDC.
“This means that many things can disrupt the
whole system. So it’s no surprise that when you
examine people suffering from hereditary forms
of partial or complete deafness, you find mutations in nearly a hundred molecules.”
A battery for the ear 35
living animals. So in addition to studying these
processes at the level of the cell, with all the tools
of molecular biology and cellular biophysics, we
can see how they affect the much larger phenomenon of hearing.”
an intricate tissue that contains several types of
cells. Each has distinct functions in perceiving
sound, passing it along, and transforming it into
electrical impulses.
In these processes, membrane channels have a
central role to play; they control the passage of
positively charged potassium and negatively
charged chloride atoms into and out of cells.
They also participate in the takeup and release of
fluids. Both functions have an impact on hearing.
The cells of humans and mammals can produce
many different potassium and chloride channels,
among them various so-called CLC proteins, a
family of chloride channels that Thomas discovered and which remain an important focus of his
work. Their locations in the body and subtle differences in structure determine their functions.
“One surprise in our studies,” Thomas says, “was
that several different ion channels and transporters we studied for other reasons turned out
to be essential for hearing. In fact, two of these
are mutated in forms of inherited human deafness.” These ion transport proteins are found in
very specific locations, in often just one or a few
cell types of the inner ear.
“They contribute to hearing in a variety of ways,”
Thomas says. “One method used in our work is
to remove specific channel proteins or make
slight changes in them to see how that affects
cells and tissues. This allows us to understand
their role in the organism and in many cases also
provides us with mouse models for human disease. In the ear we have the fortunate situation
that we know what sounds go in, and we can
measure how the ‘output’ of cells changes in
36 A battery for the ear
Thomas is a lanky man with grey hair and a short
beard; on the day we meet he is wearing a black
t-shirt covered with mathematical formulas.
“Maxwell’s equations,” he explains. The formulas
describe the properties of electrical and magnetic
fields – yes, the nature of his work requires math
and a wide variety of other skills. That can be
challenging when new students enter the group,
Thomas admits, but it’s also a great opportunity.
The kinds of biological questions scientists are
asking now need to be approached in a highly
interdisciplinary way.
The work also prepares young scientists for a
variety of careers. Thomas cites some recent
examples: Raúl Estévez has gone on to establish
his own group as professor in Barcelona; Anselm
Zdebik, a physiologist who also did genetic
manipulations of mouse strains, is a lecturer at
the University College of London. Gesa Rickheit, a
PhD student who took the lead on recent studies
of the ear, now works for a company in Köln that
develops strains of mice for research in industry
and academia.
Unraveling the role of ion channels in the organ
of Corti required many types of expertise, including careful studies of the electrical properties of
cells, genetic work in mice, and an exquisite
preparation of tissue samples for study under the
microscope. For instance, several years ago,
Thomas’ group had identified a new potassium
channel of inner ear sensory cells and found that
it is mutated in human deafness. Generating
‘knock-out’ mice lacking this channel and other
mice carrying the exact mutation found in a deaf
patient allowed Thomas to clarify the underlying
pathology. The result is a mouse model that clin-
ics are using in pilot studies of gene therapy for
deafness. Genetic mouse models also brought
to light the fact that two different transport
molecules that couple the movement of chloride
to that of potassium are also essential for
hearing.
To introduce the latest project, Thomas sits in
front of a huge computer screen and gives a
short tour of the scala media. Tucked into the
cochlea – a structure in the inner ear that looks
like a snail's shell – this small, triangular structure
contains the organ of Corti, which acts as an
amplifier as well as the converter that transforms
sounds into electrical impulses.
Most of the triangle is a cavity filled with fluid.
The scala media acts as a resonance chamber,
together with two neighboring spaces that are
likewise filled with fluid (the scala tympani and
scala vestibuli). Sound vibrations are picked up by
rows of sensory hair cells located at the base of
the triangle, in the organ of Corti. The leg of the
triangle is formed by a thin layer of cells called
Reissner’s membrane, and the right leg is called
the stria vascularis. The fluid enclosed by these
three sides also serves as a battery, because it is
held at a positive voltage and has a high concentration of potassium.
Both of these properties provide the driving force
for positive charged potassium ions to enter hair
cells. They do so through ion channels located at
the top, which respond to the motion of the
hairs. This flow of positive charge creates electrical current and transforms mechanical vibrations
into changes of voltage. The ions leave the cell
again through their base – also requiring potassium channels. These were the molecules that
Thomas’ lab had found to be mutated in some
forms of human deafness.
Zooming in on a small region at the base of the
scala media, Thomas points out the hair cells.
They come in two different kinds, both with
“Reissner's membrane" to
the diagram, pointing to the
long pink stripe on the top
left side.
A microscope image and a diagram of the Organ of Corti. The
black circular arrow shows the path taken by potassium ions. A
high concentration of the ions in the center cavity provides the
driving force whereby they enter cells.
Generally, "electrically excitable" tissues like nerves and muscle undergo changes
in membrane voltage due to an inflow of sodium ions. In the organ of Corti, an
influx of potassium is responsible.
tooth-like cilia that stretch upward to follow the
turns of the snail-like cochlea. “These three parallel rows of cells are the outer hair cells,” he says.
“Vibrations caused by sound cause the cilia to
move, and the resulting change in ion flow and
voltage triggers a movement of motor proteins
that sit in their membrane. They cause the membrane of the entire cell to contract at a very fast
speed. This amplifies the sound and increases the
sensitivity of hearing by about 50 decibels.” The
vibrations are now picked up by a single row of
inner hair cells, which generate the electric voltages to be passed along to nerves and the brain.
Generally, “electrically excitable” tissues like
nerves and muscle undergo changes in membrane
voltage due to an inflow of sodium ions. In the
organ of Corti, however, changes in voltage in
hair cells are based on an influx of potassium. This
difference is important in the operation of the ear.
“The organ of Corti would have to be built differently if the voltage of hair cells were changed by
an influx of sodium,” Thomas says. “The sodium
would have to be removed by a ‘pump’ that is
directly fueled by chemical energy. The fuel would
have to be provided from the blood supply, so
the organ would need to be interlaced with
blood vessels. Those would interfere with vibrations – it would be like stuffing a drum with
wool – so nature has found another solution.”
Instead of active pumps that require fuel, the
flow of potassium is regulated by “passive”
mechanisms. This is due to the high concentration of potassium and the charge of the fluid of
the scala media at the top of the hair cells, and a
low concentration of potassium and zero voltage
at the bottom. In this situation, ions flow in from
the top and depart at the bottom – if the
channels are functioning properly.
“Neither step requires a pump,” Thomas says.
“Metabolic energy is needed to establish this difference between the voltage and concentration
of potassium, but this occurs at a different site,
the stria vascularis. The name of this tissue comes
from the fact that it is indeed penetrated by
blood vessels and it serves as the battery for the
compartment.”
So an understanding of the role of potassium has
given scientists a clear view of these early, crucial
stages of hearing: Ions are pumped through the
stria vascularis and give the fluid in the chamber
an unusually high potassium concentration and
positive voltage. “That fluid comes into contact
with the hair cells,” Thomas says. “As they
vibrate, they open channels to let the ions in. This
causes the inner hair cells to change their electrical voltage. These potassium ions leave the cells
through their base and then flow back into the
stria vascularis. Both functions only work if the
fluid has this particular composition and voltage.
What if that didn’t happen because, for example,
there were defects in ion channels in the stria
vascularis? This leads to deafness.”
Earlier studies of a particular chloride channel
called ClC-K showed that it only functions if it is
bound to another smaller protein called barttin.
Mutations in barttin cause a rare but severe
human disease called Bartter syndrome type IV, in
which patients have a severe salt and fluid loss
through the kidneys and are also deaf from birth
on. Microscope studies carried out by the lab had
shown that the ClC-K/barttin channel is present
in cells of the stria vascularis. They hypothesized
that a lack of barttin should not only lead to a
decrease of voltage and potassium in the scala
media, but also to the collapse of this fluid space.
“When ions are transported across cell layers,
they also drag water with them,” Thomas says.
“We therefore expected that the scala media
wouldn’t get filled with enough fluid if we
knocked out barttin in mice. The membrane
collapses, as if you were to break the head of
your drum.”
The multiple functions of the channels pose
another problem. “Knocking out one of these
molecules has effects on other tissues,” Thomas
says. “ClC-K/barttin chloride channels are crucial
for salt and fluid absorption in the kidney, as
known from patients with mutations in genes
encoding either of the proteins. When we eliminated barttin totally in mice, the animals died
shortly after birth.”
Since mice only begin to hear at about two
weeks of age, that makes it impossible to study
hearing in animals that completely lack the proteins. This fact – that genes in an adult tissue are
frequently needed for other functions in embryonic development, in other parts of the body – is a
frequent problem in using knockout animals. The
solution is to develop conditional knockout
control of that molecule,” says Thomas. “Then
when you mate the mouse strain bearing the
scissors with another strain that has targets
around barttin, you get some mice that have
both tools. The aim is to get an animal in which
barttin functions normally throughout the body –
except in the cells that produce Cre.”
strains in which mice lack a molecule only in a
specific tissue. The method involves building an
artificial gene which is under the control of
another molecule.
To remove barttin only from the ear, it had to be
put under control of a gene used in the ear, but
not the kidney. To accomplish this, Gesa had to
use two tools, developed in different strains of
mice. First she needed a pair of “scissors” that
could cut out a region of DNA. Researchers have
developed a molecule called Cre that can do this.
It docks onto sequences called loxP sites, cuts out
whatever information lies between them, and
glues the broken ends of DNA back together. The
loxP sites tell scissors where to make the cuts. So
the lab’s first task was to develop a strain of
mouse with an artificial barttin gene that had an
extra bit of code: loxP sites. The sequences were
placed around a crucial module of the molecule;
if it gets cut out, the severely shortened barttin
protein no longer docks onto channel proteins,
and they no longer function.
Now the trick was to get a pair of scissors that
are made by ear cells, but not in the kidney. This
would avoid the early death of animals due to
salt and fluid loss.
Finding a way to control the Cre scissors turned
out to be one of the hardest parts of the project.
“The way this is done is to find another gene
that is only active in the ear, and put Cre under
First Gesa tried to link Cre to several genes
thought to be active only in the head. The results
were frustrating: Either barttin stayed active in
the ear, or it was lost in both the organ of Corti
and the kidney – leading to a loss of fluid, and
the mice died before they normally became capable of hearing. Neither experiment said anything
about the channels’ role in hearing.
Finally she found a strain with Cre under the control of a molecule called Sox10. This combination
worked: Cre removed barttin only in the inner
ear, but not in kidney. Without barttin, the ClC-K
proteins could not transport ions – in fact, without their partner, these proteins were not even
produced by the cells. The scientists were now
ready to study the channel’s effects on hearing.
Gesa’s mice were subjected to a variety of hearing tests. Electrodes were placed on the animals’
skulls to measure the brain stem’s response to
sound. A series of “click tests” showed that mice
lacking barttin suffered a hearing loss of about 60
dB, compared to littermates which had the gene.
Thomas and Gesa suspected that this was due to
a drop in the scala media. “If that were the case,
we expected to see the same type of collapse of
Reissner’s membrane as with other mice in which
ion transport by the stria vascularis had been disrupted.” A study of the mice, however, showed
that the membrane remained intact.
This didn’t mean that everything was going well
in the scala media. The scientists measured a significant drop in its electrical charge. The battery
of the ear was running low, and there wasn’t
enough “current” running in to recharge it. After
several weeks, Thomas says, this led to a degeneration of the outer hair cells. That happened first
in regions of the cochlea responsible for registering high frequencies, and took longer in cells that
picked up low frequencies.
As the outer cells are lost, the inner hair cells
remain undisturbed. Yet they do not generate a
strong enough signal because the mechanical
amplification by outer hair cells is lost.
Thomas says you would expect to find a similar
type of disturbance if you disrupted the system at
places other than the stria vascularis: for example,
if you interfered with channels that in cells
handing over ions to the stria vascularis for
secretion. Other groups have carried out these
experiments, and they also lead to mice with
impaired hearing.
But hearing begins in the outlands of the nervous
system; if a sound can’t be perceived in the first
place, it can’t be interpreted. The investigations
of Thomas’ lab into the organ of Corti are helping lay bare the mechanics of these first stages of
the reception of sound and its transformation
into nerve impulses. And his studies of the subtle
ways that cells in the ear regulate the passage of
ions is shedding light on similar processes in
other parts of the body, and how they become
disrupted during disease.
The work doesn’t yet explain why most people
experience partial deafness as they age, or why a
symphony sounds different to bats than to
humans. The answer to this second question lies
partly with evolution, which has fine-tuned the
ear’s hair cells to respond to different frequencies
in various species. Bats can hear sounds up to
100,000 Herz (most human ears are insensitive to
sounds above about 20,000 Hz); they use these
very high pitches in a form of biosonar that lets
them navigate in the dark. The rest of the answer
has to do with structures in the brain that interpret sounds as positional information, language,
or music.
Hubs, networks, and
partners that moonlight
T
he old video recorder was on its last legs.
It had begun to eat films: from time to
time the magnetic band became snarled,
and you could no longer remove a cassette. Now
my daughter’s favorite movie was stuck inside
and nothing would make it come out. Naively I
decided to take the machine apart – if I remembered where all the parts belonged, it ought to
be possible to reassemble them. After unscrewing
the cover and removing dozens of pieces – with
the film still buried deep inside – my daughter
and I realized it was hopeless. When we tried to
put it back together, there were five or six components that didn’t seem to fit anywhere. An
hour later we went out to buy a DVD recorder.
“We’re curious about the way binding partners
recognize these sequences,” Christian says.
“What structural features allow them to dock
there at all? And if a molecule can bind to one,
why doesn’t it bind to all of them?”
Proteins have such complex surfaces that a single
molecule may have dozens of potential binding
sites for partners. Without a direct look at a particular interface, it is often difficult to say exactly
how two proteins dock onto each other. In some
cases, the PRS may not really be responsible for
The protein machines that operate within a cell
are unimaginably small and much more delicate
than any of the machines we use on a daily basis.
They often contain dozens of molecules that have
to be assembled and sometimes repaired, but
there is no expert (or even frustrated parent)
around to do the job. Instead, proteins dock onto
each other piece by piece, in a process governed
by the arrangement of their atoms and the laws
of chemistry and physics. And unlike the video
recorder, where components remain in place
(hopefully) once they have been installed, the
cell’s machines are highly dynamic. As new pieces
snap into place, they cause subtle rearrangements of the modules that have already been
assembled.
Christian Freund, Eberhard Krause and their colleagues at the FMP are trying to understand the
self-assembly of molecular machines by looking
at a pattern found in hundreds of proteins. The
molecules they are interested in have docking
sites called proline-rich sequences, or PRS,
because they contain unusually high amounts of
the amino acid proline. Many proteins have multiple PRS scattered over extended regions, which
means that a single molecule may offer several
docking sites for other proteins.
an interaction. To know, you would have to crack
open machines – but here the dynamic behavior
of the components poses a problem. If you
remove something, there’s no guarantee that its
internal arrangement will continue to reflect the
normal positions of the pieces.
This makes the questions that Christian is posing
extremely complicated, requiring collaborations
with other groups and a wide range of technologies. It’s worthwhile, he says, because of the
Hubs, networks, and partners that moonlight 43
potential payoff: an elaboration of principles that
explain the construction of a much wider range
of machines. And maybe new ways to control
them through drugs or other molecules.
to a messengerRNA molecule. When the pieces
are in place, the spliceosome unwinds the RNA
and begins its cut-and-paste job. At each stage of
assembly and splicing, the components undergo
rearrangements that are essential in completing
the job.
In search of answers, the scientists decided to
carry out a careful study of an assembly of proteins that contains PRSs. The machine is the
spliceosome, which plays an important role in
transforming genetic information into proteins in
eukaryotic cells (the branch of life that includes
humans, animals, and plants). Genes encode RNA
molecules that are used as templates to make
proteins. Before this happens, eukaryotic RNAs
usually undergo splicing, a process which
removes part of their information. This job is handled by a huge machine called the spliceosome,
which docks onto the molecule, cuts out some of
its regions, and pastes the RNA back together.
Now the molecule can be used to make proteins.
“The spliceosome contains an unusual number of
proline-rich sequences and the domains that bind
to them, called PRS recognition domains, or
PRDs,” Christian says. “Unlike docking sites in
many other proteins, their interactions are fairly
weak, and not too specific. What this means is
that PRDs can often bind to sequences in several
molecules, even if they have slightly different
chemistries and shapes, rather than always finding one and only one partner.”
In humans, the spliceosome consists of over 100
proteins and small RNA molecules organized in
groups called small nuclear ribonuclear particles
(snRNPs). They dock onto each other in a stepwise manner. Along the way, the machine binds
44 Hubs, networks, and partners that moonlight
This information has been obtained through studies in the test tube, in which molecules are confronted with each other to see if they are able to
bind and how strongly they do so. But such
experiments have not revealed how important
PRS-PRD docking really is in the spliceosome.
Components might be binding at other regions.
Christian decided to focus on a spliceosome protein called CD2BP2. “It’s an interesting molecule
because it acts as an ‘adaptor’ within a particular
"We could detect the presence or absence of a molecule. But in many cases the
quantity of a protein present in the cell determines how it functions, and this was
very difficult to determine."
snRNP – it brings partners together,” he says. “It
mainly does so by its GYF domain, which has a
fold that recognizes proline-rich sequences. It
uses this surface to bind to a spliceosome protein
called SMB/B’. On the opposite surface of the
GYF domain is a second site that binds to another protein, called U5-15K.”
Experiments have shown that the GYF domain
recognizes PRS from different molecules. But U515K can’t bind here; it only docks onto the second site. “This shows that there are subtle differences, which means that the PRS-recognition
domain might bind to a different set of proteins,”
Christian says. “As the activity of the machine
changes and its components shift positions, different PRS might be used to assemble snRNPs.
The fact that CD2BP2 has these different facets
made us think it might be a good starting point
to answer more fundamental questions about the
dynamics of spliceosome assembly.”
Michael Kofler, a PhD student in Christian’s lab,
started by building a version of the GYF domain
that could be used as “bait”. The goal was to
expose the GYF module to other molecules and
see which ones could be “pulled down.”
Christian, Eberhard and his colleagues used mass
spectrometry in combination with techniques that
label all cellular components with stable isotopes
– a method called SILAC – to determine the identity of CD2BP2’s binding partners.
absence of a molecule. But in many cases the
quantity of a particular protein present in the cell
determines how it functions.”
New mass spectrometry-based methods such as
SILAC are finally providing a solution. Researchers
grow cells in an environment whose amino acids
contain isotopes – “heavy” versions of atoms. As
new proteins are built, they incorporate the modified amino acids. In the mass spectrometer, these
proteins give a different “isotope signature” than
proteins built using normal amino acids. This
offers scientists a way to compare the amounts of
specific proteins made by cells at different times,
or in different contexts.
For example, the researchers could extract all the
molecules that directly or indirectly bind to the
GYF domain from cellular extracts. Then they
could block the PRD docking sites with another
molecule, an inhibitor, and perform the experiment again. These two conditions should give different results: in the first, GYF should be docked
Michael Schümann,
Sabine Lange
Eberhard’s specialty is studying the protein population within cells, using methods like mass spectrometry. The aim is to show how in a variety of
contexts, different types of cells activate and
silence genes as they carry out their functions.
Proteins play a huge role in all the cell’s activities,
so it is crucial to know when a cell synthesizes
them – and in what amounts.
“Until recently this question of quantitation was
very difficult,” Eberhard says. “We could identify
proteins on the basis of their effects on cell signaling and we could detect the presence or
A model of the assembly of spliceosomes: A. The components of these large molecular machines are held in close proximity by interactions
between PRSs (yellow) and the PRDs that recognize them (blue, green, and red). B. When the machine has been assembled, the interactions
have a variety of functions, such as delivering additional components. Some of the sites remain accessible to other modules of the machine
(a), whereas others are hidden inside subcomplexes of proteins (b).
to molecules via their proline-rich sequences and
the U5-15K binding site; in the second, all the
partnering mediated via the PRS site should be
abolished . It was important to measure the difference quantitatively, Eberhard says, so here was
a case that the SILAC method could step in and
contribute.
The study revealed that GYF can bind to a large
number of spliceosome components. Most of the
contacts happened at proline-rich sites. When the
scientists changed the chemical spelling of these
regions, or used inhibitors to block access to
them, few of the proteins would bind. They also
discovered that both CD2BP2 and a protein called
FBP21 are able to bind to the same PRS in
SMB/B’. That’s interesting, Christian says, because
SMB/B’ might act as a PRS hub that allows simultaneous interactions with various binding partners. In collaboration with Michael Schaefer’s
group at Charite/Leipzig University, they confirmed that SMB/B’ indeed acts as a “bridge” for
the colocalization of CD2BP2 and FBP21 in living
cells.
“The picture that these experiments reveal is that
PRSs and the domains which bind to them create
flexible networks of interactions within the
machines,” he says. “Multiple proteins may
simultaneously interact with PRS hubs, or compete to bind with them, depending on the
numerous constraints imposed by other components of the machine.” Evidence for this scenario
came from experiments carried out in Reinhard
Lührmann’s lab at the Max Planck Institute in
Göttingen with whom the group collaborated. In
test tube studies, the team showed that adding a
high number of molecules that bind to PRS sites
blocks the assembly of the spliceosome at an
early stage and inhibits splicing.
“This implies that the dynamic networks steered
by PRS recognition events are important during
the assembly of the machine, but probably not
for its actual enzymatic functions,” Christian says.
“That allowed us to make another prediction:
GYF probably doesn’t dock onto many molecules
that actually carry out the splicing reactions. And
in fact, that’s what our experiments showed.”
Another question addressed in the study has to
do with the method. The spliceosome has been
the object of countless studies over the years,
using a variety of methods that attempt to
answer similar questions: how are the snRNP submodules assembled, and then how do they come
together to make the complete machine? The
strategy of Christian and Eberhard has been to
start with a single domain and observe its interactions with other parts of the machine. How do
those results fit in with what has already been
discovered?
“It’s a good fit,” Christian says. “Several complete snRNPs formed around the GYF domain –
including the one known to contain CD2BP2. We
think that means that the method accurately
reflects the assembly of some submodules.”
Christian says his lab takes a bottom-up perspective to questions about life. “We usually start
with a pattern or structure, such as proline-rich
sites, and work our way up. First you see what
the region binds to and study the details of individual interactions. Then you try to define its role
in machines and get an idea of how it helps in
their assembly and functions. Finally, we hope
these studies will show us basic principles about
machines that can be extended to other molecules and processes.”
Doing so requires using such a wide range of
techniques that mastering them all is virtually
impossible for a single group. One solution is
partnerships, such as Christian’s collaboration
with Eberhard’s lab. Such projects bring together
people with different interests and backgrounds –
combinations of perspectives that often help in
approaching complex biological questions.
As a PhD student, Christian worked in a group
that emphasized nuclear magnetic resonance,
one of the major techniques for structural biology, at the Max Planck Institute for Biochemistry in
Martinsried. This was followed by a postdoctoral
position in biochemistry at the University of
Zürich and a three-year stint as a postdoc in the
United States, with joint appointments at Harvard
Medical School and the Dana Farber Cancer
Institute. The work brought him into contact with
the intersection between structural biology and
fundamental questions in immunology. For example, he is an active participant in a network funded by the German Ministry for Education and
Research (BMBF), whose purpose is to develop
the basic structure and harmonic frame are given
by the original basso continuo. It reminds me of
one of Darwin’s quotes: ‘There is grandeur in this
view of life. From so simple a beginning, endless
forms most beautiful and most wonderful have
been, and are being evolved.’” If you ever get the
chance to attend a conference at the FMP, you
may be lucky enough to hear Christian perform
with Bernd Reif, another FMP group leader specializing in protein structures and complexes, who
has a fine baritone voice.
Eberhard has other hobbies: he is an avid reader
of history, particularly of the last two centuries.
And as often as possible – weather permitting –
he packs up his windsurfing equipment and
heads to the Baltic Sea. “You can’t do science all
day, every day, and only that,” he says. “It helps
you keep some balance in your everyday life.”
innovative therapies based on molecular and cellular processes.
Christian’s main focus within such networks is to
find ways to analyse and perturb interactions
between proteins that lead to disease. Toward
that goal, he says, it is essential to develop new
tools to study those interactions, such as small
molecular probes. That’s the subject of another
networking grant which was approved for funding by the German Research Council in 2007.
Those are tough questions requiring a lot of dedication; still, Christian finds time for things other
than science. He is a passionate musician, talented at the piano. “What fascinates me about the
great composers is their capacitiy of coding nonverbal forms of communication, that result in the
perception of beauty”, Christian says. “Take a
piece by Johann Sebastian Bach – for example,
the Goldberg variations, and you find a striking
simplicity of the aria, the theme, which contrasts
the complexity of individual variations. However,
Most of Eberhard’s career has kept him in Berlin,
where he has worked on protein chemistry since
the days before German reunification. His colleague Michael Bienert (whose work is described
in the story “A construction kit”) likewise worked
on protein chemistry during the GDR regime.
After making a short detour to physical chemistry,
Eberhard wrote a dissertation on protein chemistry and then became a group leader in the pharmaceutical industry, working on insulin. He was
responsible for getting new versions of the molecule into clinical trials.
At the time, the major source of the hormone
used in treating diabetes was obtained from pigs,
but the molecule was subtly different from
human insulin, leading to immune reactions or
other side effects for long-term users. Eberhard
and his colleagues were trying to make the molecule more “human” using chemical methods.
That work became unnecessary with new genetic
techniques that could directly produce human
insulin in cells and model organisms.
The study of proteins has also undergone huge
changes. “Mass spectrometry began as a chemical method that wasn’t very well suited to the
These studies give researchers a method to zoom in on specific patterns that
contribute to binding, and to assess their importance in a given interaction.
study of large polymeric organic molecules such
as proteins,” he says. “But with the development
of new soft ionization techniques, mass spectrometry became more ‘biological’ and has
opened up new horizons in protein research.
Based on this technology, it was suddenly possible to directly identify the proteins found in cells
and take apart complex machines. This led to the
creation of an entirely new field: ‘proteomics’, or
the study of the global activity of cellular proteins.”
Keeping up with the technology has been a challenge – one that Eberhard was eager to face. His
strong background in analytical chemistry gave
him the necessary skills. In the late 1990s he
decided to swap protein and peptide chemistry
for proteomics and established a powerful mass
spectrometry group at the FMP. That brought him
into contact with Christian, whose work has
steadily moved from single interactions to a view
of dynamic machines in the context of the whole
proteome.
Recently the partners have been collaborating to
understand a protein called Tsg101. It has a
domain called UEV which binds to proline-rich
sequences in other proteins. “This molecule has
received a lot of attention lately because it has
several important functions in the cell, and also
plays a role in cancer and HIV infections,”
Christian says.
One of Tsg101’s healthy functions is to participate
in a sort of cellular postal system. It docks onto
proteins with PRSs and helps pack them into
small membrane bubbles called vesicles that are
then delivered to specific locations in the cell. This
explains its role in AIDS: during an infection,
viruses causes the cell to mass-produce their components, which are also wrapped in a membrane
and released so that they can infect other cells.
Some of the viral molecules also contain PRSs,
which allow Tsg101 to dock on. Without the protein, this doesn’t happen, and the virus becomes
less dangerous.
to Tsg101 than its normal cellular partners. This
gives it an edge at competing for the protein’s
binding sites. If you change the spelling of PTAP in
HIV proteins, you stop them from binding to
Tsg101 at all. That also means that a careful
study of HIV might help researchers identify the
characteristics of PTAP that make it so good at
binding. In the search for drugs that would inhibit
Tsg101, this information would be good to have.
But to identify those characteristics, it would be
necessary to compare many different versions of
PTAP. And for that, it would be good to have
complete list of Tsg101’s UEV binding partners.
One reason for the ruthless efficiency of HIV in
taking over cells is that viruses evolve very quickly.
This has probably sculpted the PRS in HIV – a
motif called PTAP – to be much better at binding
Many remained to be found. The researchers
used the strategy they had developed for CD2BP2
to extract Tsg101 from cells with its partners
attached. Once again, Eberhard’s group used
An analysis of the functions of other molecules that bind to Tsg101 (center) suggests that the protein plays a role in a wide variety of
mportant cellular processes. In this diagram, the binding partners are sorted into six functional groups (the colored fields encircling Tsg101).
mass spectrometry in combination with SILAC
method to find the best binders.
“What we found suggested that Tsg101 is
involved in a number of cellular processes with a
wide variety of functions,” says Andreas
Schlundt, a PhD student in Christian’s lab who
pursued the project. “Those potentially include
transporting molecules, passing along signals,
and determining which messenger RNAs are used
to make proteins. Another function has to do
with ridding the cell of proteins that are unnecessary or worn out. Tsg101 helps link them to a
molecule called ubiquitin, which modifies them
and marks them for destruction.”
Contact with this latter machine happens via a
protein adaptor called TAL, which contains the
PTAP motif. Interestingly, Christian says, when
TAL docks on, it also tells cells to break down
Tgs101. That determines how many copies of the
protein are available at any one time, which plays
an important role in how the cell (or HIV) can use
it. But TAL doesn’t always dock on, and this
might be due to secondary binding sites that help
it do so. If those were covered up by other mole-
cules, or unavailable for other reasons, the contact might not take place.
The real importance of these studies, Christian
says, is that they give researchers a method to
zoom in on specific patterns that contribute to
binding, and to assess how important each
sequence or structure is to a given interaction.
And as so many stories in this report show, that’s
a valuable tool to have, especially when it comes
to proteins linked to the molecular machines that
are essential for life. Discovering the characteristics that allow partnerships between proteins is
one of the most fundamental questions in today’s
biology. To accomplish that, partnerships between
labs are essential.
A molecular
construction kit
I
f you’re driving across campus and see a car
with a canoe tied to the roof, it’s sure to
belong to Michael Beyermann of the FMP.
The early summer weather has been turbulent –
sunny mornings often giving way to afternoon
showers – but as soon as the skies clear, Michael
will be ready.
In his office over a cup of tea, he pulls out a
detailed map of Berlin and the neighboring
regions and describes water routes that he knows
well. For a short tour, he recommends the slowmoving Alte Spree. If you have more time, his finger traces a route from Hangelsberg to the nearby Dämeritzsee, which you can paddle across. He
knows the bends of the rivers, the camping sites,
and the places to park your car.
Michael is equally familiar with another landscape: the rugged surfaces of cells. If you could
shrink to microscopic size and float on the fat
molecules that make up membranes, you would
find yourself in a jungle-like terrain of molecules.
Some proteins stand bush-like on the surface,
with a rope-like tail anchoring them in place; others extend loops or strands. Many of them pass in
and out of the membrane like a loosely sewn
thread. Some have regions that hang below the
surface, in the cell’s cytoplasm.
It is usually impossible to predict exactly how the
cell will fold it into a knot-like shape that exposes
some of its amino acid building blocks, hides others inside, or folds intricately within the membrane to create a passageway.
“The chemical spelling of a protein and various
types of experiments often tell us which parts of
the molecule lie inside, which cross the membrane, and which segments are outside the cell,”
Michael says. “We might be able to say, for
example, that twenty amino acids extend outwards and create a loop. But without detailed
structural information about that region, it’s hard
to say what it will bind to or how it will behave.”
It is usually impossible to purify or obtain crystals
from these membrane-spanning molecules, which
could give researchers a detailed look at their
structures. So Michael is using another strategy:
creating artificial versions of a protein’s domains
and mounting them on a platform. This tiny construction kit is providing new insights into features of molecules that may explain how the
Michael says that all of these parts of a molecule
play a crucial role in its functions. “Their composition and shape determine what other molecules a
protein can dock onto,” he says. “The regions
outside can bind to proteins on neighboring cells,
free-floating molecules, drugs, or viruses. Many
of them create signals that tell a cell how to specialize or behave. Membrane regions pass those
signals into the cell. Moreover the regions that
pass through the membrane may create passageways that ions or molecules can pass through.”
The recipes of membrane proteins can be read
from the genome, which encodes them, but in
many cases this string-like chemical code is all the
information that scientists have about a molecule.
A molecular construction kit 53
regions function; it may also yield strategies to
control them.
The human genome encodes more than 2000 Gprotein coupled receptors, or GPCRs, and much
of what we know about the world depends on
them. The membrane proteins of this family
respond to light, smells, or other sensory stimuli.
Many are triggered by hormones or neurotransmitters. Their name comes from the fact that
their intracellular regions bind to G proteins.
These molecules often act as amplifiers, responding to a single stimulus by activating other proteins over and over, like a single person sending
the same e-mail hundreds of times. The result is a
cascade of signaling that prompts changes in the
behavior of the cell. This process is so important
that disturbances often lead to disease; defects in
GPCRs or G protein signaling have been linked to
a range of serious health problems including diabetes, cardiovascular disease, and cancer.
“We’d like to understand how peptide ligands –
small proteins or other molecules made of amino
acids – bind to GPCRs,” Michael says. “The goal
54 A molecular construction kit
is to discover how various molecules provoke different responses on the part of the receptor
when they bind. This is a structural question, but
we don’t have structures for the peptide receptors or the domains of these proteins that lie outside the cell. That’s particularly important because
most drugs on the market target GPCRs. Not
having a clear understanding of the binding
process is a big obstacle to making the drugs
more potent, eliminating side effects, and
improving them in other ways.”
Most drugs are usually small substances that bind
to pockets tucked into the surface of the target
protein or other sites. “But many biological
processes are the result of interactions between
proteins, or peptides and proteins,” Michael says.
“This causes the problem of specificity for small
molecules; drugs often hit more targets than you
want. You might be able to get around this by
developing peptides or small proteins that work
as a drug, instead of using other kinds of substances. But here the problem is that the body
usually takes such molecules apart fairly quickly.”
For nearly two decades, Michael’s group has been
working on a GPCR called the corticotropinreleasing factor receptor, or CRFR. Corticotropin
releasing factor (CRF) is a hormone secreted by
the pituitary gland, located at the base of the
brain, and it helps the body respond to stress. The
hormone and its receptor are probably hard at
work when Michael has been paddling his canoe.
“And when a crazy swan attacks,” he laughs.
Years of working on CRF receptors have given
him an intimate familiarity with the molecule’s
extracellular domains. If you were to meet the
protein while traveling along the membrane, he
says, you would encounter a long, string-like tail,
surrounded by three loops. But the critical details
are lacking.
“We don’t know the shapes of the loops, or how
they interact with each other,” he says. “One reason that various molecules can bind to the CRF
receptor may be that they interact with more
than one of the domains in different ways. The
variations influence what signals get sent and
how the cell responds. But in most cases we
don’t know with certainty where or how the
ligands bind.”
A way to find out might be to create different
versions of CRF ligands by introducing mutations
in the regions found to be crucial for receptor
activation, changing their chemical spellings letter-by-letter. Particular changes lead to a selective
activation of specific signaling pathways, showing
that one region near the head of the ligand
seems to play the most critical role in the way it
docks onto its receptor.
Mutter and Stéphane Vuilleumier, who mounted
three receptor loops onto a template.
“That was a great effort at the time,” Michael
says. “What was missing was a way to synthesize
the tail and mount it with precision onto the template. We confronted the same challenge when
we decided to take on this project.”
The first task faced by the group was to find a
simple way to create high quantities of the individual segments of the CRFR. They made the
loops artificially with chemical methods, attaching
individual amino acids to each other in the right
order. “This can be done effectively with short
sequences such as the loops, which consist of
about 20 amino acids,” Michael says. “But
CRFR1’s extracellular tail is four times as long, so
assembling it from scratch and mass-producing it
was going to be an enormous task. We tried, but
in the end turned to a ‘biological’ solution. We
Jan Oliver Jost
But these results haven’t answered the lab’s questions about the characteristics that permit the
receptor to differentiate between various signals.
And they haven’t allowed the researchers to predict whether CRF receptor will bind to a new
molecule – such as a drug – and how it will
respond.
“The major hurdle to get the CRF receptor in
hand is that it is very difficult to make membrane
proteins like this one using cellular systems,”
Michael says. To get around this problem,
Michael and his colleagues have adopted another
strategy: They produce the loops and the long
external tail of the CRF receptor synthetically and
then mount the pieces on a base. Instead of
inserting the pieces into membranes, the scientists plug them onto a peptide template. This
method was proposed 20 years ago by Manfred
added a gene that encoded the sequence to bacteria. These cells made large quantities of the
protein, which we extracted, folded and purified.”
The next challenge was to build the peptide template to which the segments could be attached.
The liquid nature of membranes usually allows
the proteins that float there to move flexibly –
which means that loops and other structures
might rearrange themselves or bind to each other
in a variety of ways as conditions change. If the
base were too stiff, this might not happen.
The solution was to create a platform made of
small clusters of amino acids that had specific
Dirk Schwarzer, Michael Beyermann
characteristics. Some, for example, help the template dissolve in water – a necessary step in working with the molecule – and others assist in plugging it consecutively the single loops into it. More
importantly, Michael says, the receptor domains
had to be placed close to each other but
mounted flexibly enough to provide space for
unpredictable interaction between them.
Finally, PhD student Stephan Pritz and technician
Annerose Klose needed to physically attach the
loops and tails to the base – and to plug them
into precise places. To attach the tail they turned
to an enzyme from bacteria that recognizes
amino acids at the ends of peptide and protein
chains and glues them together – in this case the
The CRF receptor "mimic" made by
Michael's lab. The black bar is the template; attached to it are three loops that
have been built synthetically. The longer
green tail was produced in cells, then
extracted and plugged into the template.
N-tail – on the platform in a particular location.
They solved the problem with the loops by
adding a small extra sequence that allowed it to
be attached through chemical methods.
important step, because it has been extremely
difficult to obtain engineered proteins that have
all the subtle features of receptors in human or
animal cells.”
The lab now had a large molecule with all the
right pieces. Would it behave like the external
domains of CRF receptor? They tested their artificial receptor – Michael calls it a mimic – against
two of the receptor’s binding partners, a peptide
called sauvagine and urocortin 1. The two molecules bind to the natural form of the receptor at
different strengths. Most interestingly, Michael
says, they behave the same way when they
encounter the mimic.
So making mimics of CRF receptor requires a mixture of chemical synthesis and cell-based systems.
Michael’s group has perfected techniques that are
widely needed by scientists at the FMP and
throughout the biology community. Many other
types of experiments depend on the creation of
small artificial proteins or peptide. So for many
“This proved that we had a tool kit that could be
used to study details of the binding process,”
Michael says. “We could start making various
changes in the loops and the tail and watch how
they affected the partners’ ability to bind. That’s
what we’re doing now. It’s allowing us to pinpoint which specific points of the protein
sequences and structures are crucial.”
The study also reveals that the approach should
be equally useful in studying other membrane
proteins. “This opens the door on a new generation of artificial protein mimics – even if the molecules are very complex,” he says. “That’s an
years the group has brought valuable expertise to
collaborative projects with other labs, and has
thus been confronted with a broad range of biological and pharmacological projects. That’s also
the case in the lab of one of Michael’s neighbors,
a new group leader named Dirk Schwarzer.
“Dirk is particularly interested in producing large
molecules that can’t be built synthetically with
normal methods; nor can they usually be created
through the types of cellular systems that are
available,” Michael says. “So he’s also developing
an original recipe of techniques. Making one of
these molecules may require producing part of
the protein in cells, other parts in the chemistry
lab, and then putting them together. His focus is
more toward producing modified proteins than
mimics. Those efforts are tremendously useful as
tools for studying the role of specific modifications on the function of proteins. “
Michael has been building artificial proteins
throughout his career, since the days before the
FMP existed. Along with several other current
members of the group, he worked in the Institute
of Drug Research at the Academy of Sciences of
the G.D.R., here on the Berlin-Buch campus.
“In the G.D.R. the institute had a different orientation,” he says. “We were what was called an
‘Academy-Industry Complex’; the academic institutes were most frequently considered an
‘extended workbench’ of industry. We were
expected to work on applied problems and provide specific services. The main focus of my work
then was to develop new ‘mixed’ methods of
protein synthesis and peptide synthesis.”
After the fall of the Berlin wall, a careful evaluation of the campus was made, with the aim of
restructuring the institute and bringing it into a
unified national system. Some of the laboratories
and infrastructure were retained; others had to
go. Michael says the transition was not easy, but
his boss, peptide chemist Michael Bienert, did a
great deal to smooth the way.
One change was to choose a focus for his own
group. “After the ‘Wende’ we began orienting
ourselves more toward pharmacological themes,
with a greater input from biology. We could have
gone the ‘combinatorial chemistry’ route taken by
many groups, searching for inhibitors through
random screening, using chemistry to optimize
the hits and then, starting with those, spin off
compounds.” (This is the approach taken by Jens
von Kries’ screening unit and Jörg Rademann’s
group, described in “A virtual puzzle.”) “Instead,
we decided to maintain our focus on polypeptides. And the group has developed a position for
itself with this direction.
“CRFR was one of the first molecules we took
on. Twelve or thirteen years ago we started
focusing on the external domains, and that was
about the time we started to think of building a
mimic. It was unclear how long that would take.”
He smiles. “After a decade, all the pieces have
come together. Now the question is how fruitful
this approach will be, and how widely it can be
extended to other membrane proteins.”
For a full account of all 2007/2008
publications, grants, collaborations,
patents, innovations, teaching activities
well as a series of image film please refer
to the Data CD enclosed at the back of
the report.
Reports from
research groups
Members of the group
Structural Biology
Protein Structure
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Linda Ball*
Rui M Brito*
Ludwig Krabben *
Parveen Choudhary-Mohr *
Victoria Ann Davies,
Jana Körner*/**
Tobias Werther*
Barth-Jan Rossum
Alba Silipo*
Group Leader:
Hartmut Oschkinat
H
artmut Oschkinat studied chemistry in Frankfurt and
obtained his PhD in the group of Horst Kessler. During
this time, he visited the lab of Ray Freeman in Oxford. As
a post-doc he worked with Geoffrey Bodenhausen in Lausanne
and at the Max-Planck-Institute for Biochemistry, first in the
group of Angela Gronenborn and Marius Clore, later independently. In 1992, he became group leader at the European
Molecular Biology Laboratory (EMBL) in Heidelberg. He joined
the FMP in 1997 and was appointed professor of Structural
Chemistry at the Freie Universität Berlin.
The group applies solid-state and solution NMR to the
structural investigation of biological processes, of biological processes and aiming in the long runs at a characterization of protein structures in a biologically relevantnative-close environment. A major focus is in the further
development of the solid-state NMR technology, and in
applications to membrane protein systems or heterogeneous protein complexes.
Overview of work and results in 2007/2008:
Everyone who has looked at a cell through a microscope
has experienced the thrill of seeing life’s inner space. The
study of this inner space at higher resolution is limited by
a gap in current structural biology between the global
information delivered by laser scanning microscopy or
electron microscopy (EM) using ‘systems-like’ samples,
and the techniques that yield high-resolution structural
information such as X-ray crystallography or NMR on
highly purified preparations. Potentially, solid-state NMR
can be applied to similar samples, providing portions can
be labelled with magnetically active nuclei such as 13C and
15
N. This may be accomplished by reconstituting functional systems and by increasing signal-to-noise by a factor of
20-100 through the application of dynamic nuclear polarisation (DNP) which involves irradiation of the sample with
microwaves at a temperature around 95 Kelvin. Recently,
such a spectrometer was installed at the FMP, and the
technology We expect that MAS solid-state NMR will be
mainly applied to samples which are at least in part reconstituted from individual components, enabling in this way
the introduction of NMR-suitable isotopes such as 13C and
15
N. Examples are synaptic vesicles (Takamori et al., 2006)
or model membrane systems with a restricted number of
protein components.
Apart from such challenging investigations, we extrapolate that a wide area of yet unconquered ground in struc62 Structural Biology
tural biology will be amenable to solid-state NMR investigations, including a number of ‘old’ structural problems
representing heterogeneous, multi-component systems.
Along these lines, membrane protein systems in native
lipids are an obvious target of solid-state NMR. In our laboratory, we investigate an ABC transporter system, outer
membrane protein G, and G-protein-coupled receptors.
Furthermore, a wide variety of biological functions is associated with the appearance of heterogeneous, ‘dynamic’
complexes that are very difficult to prepare in pure states
by in-vitro methods. A paradigm example is the small heat
shock protein (sHSP) αB-crystallin which acts as a chaperone, being part of the cellular protection system against
stress. Dysfunctions and mutations in αB are associated
with the occurrence of cataracts in the eye lens, cardiomyopathies, multiple sclerosis and Alzheimer’s disease. The
chaperone-like function of αB-crystallin is intimately connected to its ability to form polydisperse oligomers. An
example is the small heat shock protein alphaB-crystallin
which forms oligomeric assemblies of 26-32 subunits. We
have determined the structure of the dimer (Fig. 1) which
is the basic building block of the oligomeric complex,
using the full-length protein construct.
In addition to the solid-state NMR studies, we investigated solution NMR structural investigation of a chaperone
for the family of low density lipoprotein receptors
(LDLRRP’s) by solution NMR. is made. The LDL receptorR
have mainly been characterized for its function in systemic
cholesterol homeostasis. It rapidly bindsbinds cholesterolrich LDL particles and triggers their internalisation. The
folding of LDLRs is a difficult task, partly because of the
very high number of intramolecular disulfide bonds present in the complement-type repeat (CR) and epidermal
growth factor (EGF) domains, as well as the very complex
packing of six contiguous YWTD repeats into a six-bladed
β-propeller structure functionally interacting with the Cterminal adjacent EGF-domain [8, 9]., and Iin mammalian
cells, the folding of this β-propeller/EGF-motive in the
endoplasmic reticulum (ER) is assisted by mesoderm development (MESD) which is a 224 amino acid mouse protein
that acts as a molecular chaperone for receptors of the
low density lipoprotein receptor (LDLR) family. We derived
an NMR based model for the structure of the highly conserved core region of MESD corresponding to residues 45
– 184 and provide in vivo evidence that this domain is
essential and sufficient for MESD function. The model predicts that MESD consists of a central β-α-β-β-α-β-fold
domain, with an N-terminal flexible extension terminating
in an α-helix. NMR-studies onto the internal dynamics of
MESD provide evidence that the N-terminal helix loosely
interacts with the β-sheet of the core domain (Fig. 2).
Dr. Elizabeth Dowler
Dr. Vivien Lange**
Nils Cremer *
Feng Ge (doctoral student)**
Johanna Becker (doctoral student)*/**
Matthias Hiller (doctoral student)**
Nestor Kamdem (doctoral student) */**
Britta Kunert (doctoral student) *
Sascha Lange (doctoral student) */**
Arne Linden (doctoral student) */**
Stefan Markovic (doctoral student) */**
Jan Hendrik Holtmann (doctoral student) **
Christian Köhler (doctoral student)**
Anne Wartenberg, (doctoral student) */**
Janet Zapke (doctoral student)*/**
Dr. Annette Diehl (technical assistant)
Lieselotte Handel (technical assistant)**
Martina Leidert (technical assistant)**
Natalja Erdmann (technical assistant)
Karola Marsch (technical assistant)**
In vitro-binding studies identified the receptor motive recognized by MESD and we derived in vivo functional evidence for the relevance of these contacts for MESD assisted LDLR folding.
Thi Bich Thao (technical assistant)*
Daniel Olal (doctoral student)*/**
Arndt Pechstein (doctoral student) */**
Dr. Silke Radetzki (technical assistant)**
Kristina Rehbein, (technical assistant)**
Andreas Ziegler (student)*
Johanna Meyer (student)*
Katja Riemann (student)*/**
Selected Publications
Ramirez-Espain X, Ruiz L, Martin-Malpartida P, Oschkinat H,
Macias MJ. (2007) Structural Characterization of a New Binding
Motif and a Novel Binding Mode in Group 2 WW Domains. J
Mol Biol. 373(5): 1255-68.
Podust LM, von Kries JP, Eddine AN, Kim Y, Yermalitskaya LV,
Kuehne R, Ouellet H, Warrier T, Alteköster M, Lee JS,
Rademann J, Oschkinat H, Kaufmann SH, Waterman MR
(2007) Small Molecule Scaffolds for CYP51 Inhibitors Identified
by High Throughput Screening and Defined by X-Ray
Crystallography. Antimicrob Agents Chemother. 51(11):
3915-23.
Fiedler S, Knocke C, Vogt J, Oschkinat H, Diehl A (2007)
Production of 2H-, 13C-, and 15N-Labeled OmpG via High Cell
Density Fermentation. GEN, 27(9).
Figure 1. Solid-state NMR structure of the α-crystallin domain
dimer solved from αB-crystallin oligomers using the full length protein (Uniprot ID P02511). Its architecture (top) comprises a prolineand phenylalanine-rich N-terminal segment (~60 residues), the
conserved α-crystallin domain (~90 residues), and a C-terminal
region (~25 residues), which contains the conserved IXI/V motif.
HR1 represents the heterogeneous region 1 connecting the N-terminal region with the α-crystallin domain. Disease related mutations are indicated (D140N, R120G). A: α-crystallin domain dimer as
ribbon chart, cataract and cardiomyopathy causing mutation sites
are indicated. B: Stereo-view of the ten lowest energy structures
are shown superimposed.
A
Scholz I, Jehle S, Schmieder P, Hiller M, Eisenmenger F,
Oschkinat H, van Rossum B-J (2007) J-deconvolution using
maximum entropy reconstruction applied to 13C-13C solid-state
cross-polarization magic-angle-spinning NMR of proteins. J Am
Chem Soc. 129(21): 6682-3.
Jehle S, van Rossum BJ, Stout JR, Noguchi SR, Falber K,
Rehbein K, Oschkinat H, Klevit RE, Rajagopal P. (2008) Alpha-Bcrystallin: A hybrid solid-solution state NMR investigation reveals
structural aspects of the heterogeneous oligomer. J Mol Biol,
385(5): 1481-97.
Becker J, Ferguson N, Flinders J, van Rossum JB, Fersht AR,
Oschkinat H (2008) A Sequential Assignment Procedure for
Proteins that have Intermediate Line Widths in MAS NMR
Spectra: Amyloid Fibrils of Human CA150.WW2. ChemBioChem
9: 1946 – 1952.
Hiller M, Higman VA, Jehle S, van Rossum BJ, Kühlbrandt W,
Oschkinat H (2008) [2,3-(13)C]-labeling of aromatic residuesgetting a head start in the magic-angle-spinning NMR assignment of membrane proteins. J Am Chem Soc 130(2): 408-9.
Kohler C, Andersen OM, Diehl A, Krause G, Schmieder P,
Oschkinat H (2007) The solution structure of the core of
mesoderm development (MESD), a chaperone for members of
the LDLR-family. J Struct Funct Genomics. 7(3-4), 131-8
B
C
Internal and external collaborations
Internal: Gerd Krause (Modelling), Ronald Kühne (Molecular
Modeling), Jörg Rademann (Medicinal Chemistry), Bernd Reif
(Solid-state NMR), Thomas Jentsch (Physiology and Pathology
of Ion Transport)
External: Alan Fersht (LMB Cambridge), Rachel Klevit, Ponni
Rajagopal (University of Washington, Seattle), Werner
Kühlbrandt (MPI für Biophysik, Frankfurt), Larissa M. Podust,
Michael Waterman (Vanderbilt University, Nashville).
A) NMR structure of MESD, employing a fully automated structure
calculation without NOE assignments. B) Chemical shift changes on
the core domain induced by the presence of the N-terminal helix. C)
Structure of the core domain with the N-terminal helix using
assigned constraints and residual dipolar couplings.
*part of period reported
**part time
yellow Position funded externally (3rd-party funds) for at least part of the reporting period.
Structural Biology 63
Structural Biology
Solution NMR
Group Leader:
Peter Schmieder
P
eter Schmieder trained as a chemist at the University of
Frankfurt, where he did his diploma work in Prof. Kessler’s
group in 1988. Together with the Kessler lab he moved to
Munich in 1989 where he obtained a Ph.D. on method development in solution state NMR. After three years as a postdoc with
Prof. Wagner at Harvard Medical School where he did protein
NMR, he joined the FMP in 1995 and became group leader of
the solution state NMR group in 1997.
In order to address questions of biological and pharmacological importance, the group Solution NMR spectroscopy
uses the full repertoire of solution state NMR techniques
in conjunction with a variety of labeling patterns. Such
questions range from the development of new techniques
for solution state NMR to the elucidation of the constitution and configuration of biological active peptides and to
the determination of the three-dimensional structure of
peptides and proteins.
In continuation of the work on photochromic proteins,
the group focused on investigating the structure and
dynamics of the chromophor binding pocket of phytochrome proteins. This work also led to the initiation of
structural investigations of blue-light receptors that harbour a LOV domain. We continued our work on small
photoswitchable peptides designed to interfere with protein-protein-interaction domains. In addition, we started
to collaborate with the Charité to investigate the dynamics of the binding interface of MHC molecules type I,
while concluding our work on antimicrobial peptides with
the design of a non-petidic molecule with antimicrobial
activity comparable to that of the original peptides.
In the field of phytochrome research the understanding of
the mechanism of light reception by the chromophore
was boosted with the publication of two X-ray-structures
in 2008 that gave detailed information on the two thermostable photostates of phytochromes, albeit from two
entirely different phytochromes harboring different chromophores. We were able to determine independently the
structure of both thermostable formes of the chromophore in the same protein by NMR (Figure 1) and in
addition show that the chromophore is not embedded in
its binding pocket in a rigid manner but rather exhibits a
rather pronounced mobility. This led to the conclusion
that light absorption is converted into signal transduction
not by a structural rearrangement of the chromophore
64 Structural Biology
but rather by reorganisation of the hydrogen bonding network around the chromophore. This in turn leads to a
rearrangement of amino acid side chains, thus transducing the signal to the rest of the protein. Our work was further supported by the investigation of phycocyanin, which
may serve as a model system for phytochrome proteins
since it harbors the same chromophore albeit in a completely different structural context.
Another class of photochromic proteins is the blue-light
receptors that usually contain flavin molecules as chromophores. An example are proteins harboring a LOV
domains. In collaboration with Wolfgang Gärtner of the
MPI in Mülheim we started to investigate the structure
and the structural rearrangements of a blue-light absorbing protein from Bacillus subtilis, named YtvA. It contains
a LOV and a STAS domain and the interaction between
those two domains and its alteration upon light absorption are not known yet. Thus it is impossible to explain
how the minute changes taking place in the LOV domain
are transduced to the STAS domain and how it is affected
by the changes in the LOV domain. The expression of the
protein has been established and appropriate labeling has
been performed (Figure 2).
The work with photoswitchable ligands for protein-protein interactions domains turned out to be more problematic than expected, difficulties with the solubility of the
peptides and their mobility in various solvents prevented
the determination of structures so far. It was, however,
possible to synthesize active and switchable peptides (AG
Beyermann) that will be further characterized by NMRspectroscopy and calorimetry.
NMR spectroscopic investigations of loaded MHC molecules of class I turned out to be relatively promising.
Labeling of the complexes, which are large by NMR standards, was possible and investigations of the dynamic
behavior of the proteins and in particular the peptide
started with initial resonance assignments and studies of
free beta-2-microglobulin. Since the production of the
complexes is fairly complicated, optimization of the
expression of the proteins and an expression of the peptides is currently under way.
Last but not least the NMR facility itself grew with the
installation of new spectrometers and work implementing
a library of puls programs and data sets that will ease the
recording of even highly complex NMR experiments continued.
Matthias Dorn (doctoral student)**
Janina Hahn (doctoral student)**
Tolga Helmbrecht (doctoral student)**
Marcel Jurk (doctoral student)*/**
Marco Röben (doctoral student) **
Sabine Seedorff (doctoral student)**
Monika Beerbaum (technical assistant)**
Brigitte Schlegel (technical assistant)
Eva Klein (student)*/**
Martin Zieger (student)**
Hahn J, Kühne R, Schmieder P (2007) 15N solution-state NMR
study of a-C-phycocyanin. Implications for the structure of the
chromophore binding pocket of the cyanobacterial phytochrome
Cph1. ChemBioChem 8: 524-2249-2255.
Oschkinat H, van Rossum BR (2007) J-deconvolution using
maximum entropy reconstruction applied to 13C-13C solid state
CP MAS NMR of proteins. J. Am. Chem. Soc. 129: 6682-6683.
Appelt C, Schrey AK, Söderhäll JA, Schmieder P (2007)
Design of antimicrobial compounds based on peptide structures.
Bioorg. Med. Chem. Lett. 17: 2334-2337.
Hahn J, Strauss HM, Schmieder P (2008) Heteronuclear NMR
Investigation on the Structure and Dynamics of the
Chromophore Binding Pocket of the Cyanobacterial
Phytochrome Cph1. J. Am. Chem. Soc. 130: 11170-11178.
Coin I, Beerbaum M, Schmieder P, Bienert M, Beyermann
M (2008) Solid-Phase Synthesis of a Cyclodepsipeptide:
Cotransin. Org. Lett. 10: 3857-3860.
Figure 1. Structure of the chromophore in both thermostable states
of the photocycle of phytochrome Cph1. The NOESY spectra were
used to determine the conformation and also to draw conclusions
about the mobility of the chromophore while embedded in the binding pocket.
Appelt C, Wessolowski A, Dathe M, Schmieder P (2008)
Structures of cyclic, antimicrobial peptides in a membrane-mimicking environment define requirements for activity. J. Pept. Sci.
14: 524-527.
Coin I, Schmieder P, Bienert M, Beyermann M (2008) The
depsipeptide technique applied to peptide segment condensation: Scope and limitations J. Pept. Sci. 14: 299-306.
Hupfer M, Glöß S, Schmieder P, Grossart HP (2008) Methods
for Detection and Quantification of Polyphosphate and
Polyphosphate Accumulating Microorganisms in Aquatic
Sediments. Int. Rev. Hydrobiol. 93: 1-30.
FMP authors in bold, group members underlined
Internal collaborations at the FMP: Anne Diehl, Ronald Kühne,
Gerd Krause, Bernd Reif, Michael Beyermann
External collaborations: Jon Hughes (University Giessen), Tilman
Lamparter (University Karlsruhe), Peter Hildebrandt (TU Berlin),
Karola Rück-Braun (TU Berlin), Roderich Süssmuth (TU Berlin),
Wolfgang Gärtner (MPI for Bioanorganic Chemistry), Andreas
Ziegler and Barbara Uchanska-Ziegler (Charité Berlin)
Figure 2. 1H-15N-Correlation (TROSY) of the YtvA protein. The protein
was labeled with 15N and 2H at non-exchangeable sites. The good dispersion of the signals and the stability of the protein indicate that a
resonance assignment and an extraction of structural information will
be feasible.
**part time
Structural Biology
Structural Bioinformatics
Group Leader:
Gerd Krause
G
erd Krause studied chemistry at the University in Leipzig
and graduated in 1982 with a PhD in biochemistry at
the Martin Luther University Halle. After working at a
research unit in the pharmaceutical industry in Magdeburg, he
took up a research position at the Institute of Drug Research in
Berlin in 1986. Five years later, he worked as a visiting scientist
at the Drug Design Centre in the lab of Garland Marshall at
Washington University in St. Louis, MO, USA before joining the
structural biology research section at the FMP in 1992 where he
is leader of the group of Structural Bioinformatics. His major
interests are the relationships between sequence-structure-function of protein-protein- and ligand-protein interactions in order
to find new approaches for pharmacological intervention.
The group focuses on sequence-structure analysis of proteins using structural bioinformatics combined with experimental functional studies of changed sequence(s) to
reveal sequence- and structure-function relationships of
proteins. Its main aim is the rational discovery of molecular mechanisms and sites for protein-protein interactions
and protein-ligand interactions. Locations for potential
pharmacological intervention are narrowed down to the
amino acid and atom level by predicting functional sensitive residues or atoms that are subsequently experimentally evaluated, such as by side directed mutagenesis or peptide mapping.
Intramolecular determinants of the activation
mechanisms of 7TM receptors
Within the family of G-protein-coupled receptors (GPCRs)
with seven transmembrane helices, (TMHs) the thyrotropin receptor (TSHR) belongs to the subfamily of glycoprotein hormone (GPH) receptors (GPHRs).The project
aims to understand the structural consequences of the
molecular activation mechanism of TSHR in order to find
new ways for pharmacological intervention.
Amino acid side-chain substitutions that modify the receptor phenotypes are of great importance for the elucidation
of structural-functional properties of the wild-type receptor. Utilizing the huge amount of functional data available
from both naturally occurring and designed mutations in
the TSHR and GPHRs, we developed a web accessible
resource system for sequence-structure-function-analysis
(www.ssfa-GPHR.de) at glycoprotein hormone receptors.
Our normalization of the functional data from ~1000
published mutations allows for a comparison by semiquantitative analysis and thus the discrimination of molecular and structural determinants (Fig. 2) (Kleinau et al.
Mol. Endo. 2007).
TSHR is characterized by a high level of basal activity.
Dysfunction causes several diseases. We were therefore
interested in mutations that decrease the basal activity on
signaling of the TSHR. Mutations that lower basal activity
always had a suppressive influence on TSH induced signaling and on constitutively activating mutations (CAMs).
A
B
Interaction sites of junctional proteins
Junctional proteins such as the tetraspan transmembrane
claudins connect and seal the contact sites within tight
junctions (TJ). We aim to understand the intermolecular
mechanism at the paracellular cleft between two cells.
Claudins seem to build cell- cell contacts via interactions
between the two extracellular loops (ECLs), which are
exposed to the opposing membrane. We therefore studied structure–function relationships of the extracellular
loops of claudines. This led to differentiation into two
groups, designated as classic claudins (subtypes 1-10, 14,
15, 17, 19) and non-classic claudins (subtypes 11-13, 16,
18, 20-24), according to their degree of sequence similarity. Molecular homology modelling studies led us to the
hypothesis that ECL2 of claudin-5 show a helix-turn –helix
motif (Fig.1) for interacting with each other from two different cells (Krause et al. 2008). This homology model was
experimentally confirmed in collaboration with I Blasig,
FMP (Piontek et al. FASEB J. 2008).
Figure 1. Junctional protein claudin-5: Confirmed homology-model
of the extracellular loop 2 (ECL2) . A Helix-Turn-Helix structural motif
is based on the sequential homologous structural fragment out of
the proteine structure 2BDV (PDB Databank). A) Monomer, exactly
those residues that are stabilising the loop structure, are showing
fold defects upon site directed mutations. B) Intermolecular dimer
model at the paracellular cleft; exactly those aromatic residues that
are forming the interactions, are showing effects on trans-interactions between two cells upon mutations.
Dr. Sebastian Müller
Dr. Gunnar Kleinau
Dr. Catherine Worth*
Ann-Karin Haas (doctoral student)*/**
Christian Schillinger (doctoral student) */**
Paul Grzesik (doctoral student) */**
Franziska Winkler (student) */**
Daniel Techen (student) */**
Kleinau G, Claus M, Jaeschke H, Mueller S, Neumann S,
Paschke R, Krause G (2007) Contacts between extracellular loop
two and transmembrane helix six determine basal and hormone
induced activity of the thyroid stimulating hormone receptor J
Biol Chem 282(1): 518-25.
Figure 2. Mutation data resource for Glycoprotein Hormone
Receptor, synopsis of Sequence structure function Analysis
(www.ssfa-GPHR.de): Sequence – Function – Effects of hormones
(dashed arrows) and mutations (bold arrows) give rise to different
phenotypes that represent levels of various activation states Ro-R*,
e.g. constitutively active mutants (CAMs). The introduced percentage
values of mutant phenotypic data enable the filtering to discriminate
the phenotypes according to the difference D (gray arrows) between
the respective activation states. Structure – Output tools allow to
assign and to map spatial locations for similar and different functionalities as 2D- (table) and/or 3D- (Model) outputs. Example: mutations
decreasing simultaneously the Gas and Gaq mediated signalling are
clustered in the transmembrane core of the TSHR serpentine domain
(red balls). Mutations that are selectively suppressing only Gaq activation are localized in the ECL3 (orange balls).
Our results suggest that the conformation of a basally
active GPCR is directly related to the complete active
receptor conformation (Kleinau et al. Cell. Mol. Life. Sci.
2008).
We utilized constitutive active pathogenic mutants
(CAMs) to analyze conformational changes that are necessary for the TSHR receptor activation. Our first hypothesis was that full signalling occurs via multiple extracellular
signal propagation events. Additive and even synergistic
effects of combinations of CAMs in the ECLs provided evidence for cooperative signal triggering at the extracellular
portion of TSHR (Kleinau et al. FASEB J. 2008). Secondly,
we focused on a pathogenic CAM I568V in ECL2 of TSHR,
which was suggested by our molecular model to be
embedded in an environment of hydrophobic residues
provided by transmembrane helix bundle. Double and
reciprocal double mutations identified a potential interaction partner at TMH6 and confirmed a dynamic interface
between TMH6 and ECL2 critical for signalling (Kleinau et
al. JBC 2007).
We predicted an extended hormone binding site of the
human TSHR. Using a multidisciplinary strategy in cooperation with R. Paschke, University of Leipzig, our assumption was confirmed by narrowing down distinctive acidic
residues in the hinge region that are involved in binding
basic residues of bovine TSH (Müller S et al. JBC. 2008).
In cooperation with S. Neumann and M. Gershengorn
(NIH Bethesda, USA ) a first low molecular weight (weak)
antagonist for the human TSH was developed (Neuman S
et al. 2008).
Kleinau G, Brehm M, Wiedemann U, Labudde D, Leser U,
Krause G (2007) Implications for molecular mechanisms of glycoprotein hormone receptors using a new Sequence-StructureFunction Analysis resource Mol Endocrin 21(2): 574-80.
Kleinau G, Jaeschke H, Mueller S, Raaka B, Neumann S, Paschke
R, Krause G (2008) Evidence for cooperative signal triggering at
the extracellular loops of the TSH receptor. FASEB J 22 (8): 2798808.
Kleinau G, Jaeschke H, Mueller S, Worth CL, Paschke R, Krause
G (2008) Molecular and structural effects of inverse agonistic
mutations on signaling of the thyrotropin receptor – a basally
active GPCR. Cell Mol Life Sci 65(22): 3664-76.
Krause G, Winkler L, Mueller SL, Haseloff RF, Piontek J,
Blasig IE (2008) Structure and function of claudins. Biochim
Biophys Acta. 2008 Mar; 1778(3):631-45
Mueller S, Kleinau G, Jaeschke H, Paschke R, Krause G (2008)
Extented hormone binding site of the human TSHR: Distinctive
acidic residues in the hinge region are involved in bovine TSH
binding and receptor activation. J Biol Chem 283 (26):18048-55.
Neumann S, Kleinau G, Costanzi S, Moore S, Jiang JK, Raaka
BM, Thomas CJ, Krause G, Gershengorn MC. (2008) A Low
Molecular Weight Antagonist for the Human Thyrotropin
Receptor with Therapeutic Potential for Hyperthyroidism.
Endocrin 49(12):5945-50.
Piontek J, Winkler L, Wolburg H, Mueller SL, Zuleger N, Piehl
C, Wiesner B, Krause G, Blasig I (2008) Formation of tight
junctions: Determinants of homophilic interactions between
strand forming classic Claudins FASEB J. 22(1):146-58.
Worth CL and Blundell TL (2008) Satisfaction of hydrogen-bonding potential influences the conservation of polar sidechains.
Proteins [Epub ahead of print].
The research group collaborates closely within the FMP as well
as with universities to ensure a perfect interplay between theoretically derived models and experimental proof.
FMP-groups:
I. Blasig, J Piontek: Interactions of tight junction proteins
P. Schmieder: Structures of tight junction proteins
R. Schülein: GPCR signalling mechanisms
H. Oschkinat, J. Rademann: Interference of protein-protein
interaction
Siems WE: Naturetic peptides
External:
S. Neumann, M. Gershengorn NIH, Bethesda, USA
V. Haucke: Freie Universität Berlin
M. Fromm: Charité Universitätsmedizin Berlin
R. Paschke: Uni Leipzig
H. Biebermann: Charité Universitätsmedizin Berlin
**part time
yellow Position funded
externally (3rd-party funds)
for at least part of the
reporting period.
Structural Biology
a
b
Drug Design
Group Leader:
Ronald Kühne
R
onald Kuehne studied biochemistry at the Martin-Luther
University in Halle- Wittenberg, Germany, and graduated
with a PhD in biochemistry from this University in 1980.
Following his work in the Molecular Modelling Group at the
Institute of Drug Research, he is now head of the Drug Design
Group at the FMP Berlin. His research focuses on modelling
protein-ligand interactions using molecular dynamics simulations and automated docking procedures.
The Drug Design group focuses on the design of low
molecular weight ligands which bind to target proteins
like G-protein coupled receptors, proteins encoded by the
major histocompatibility complex (MHC), and proteins
involved in protein-protein interactions. Within this
research area the group uses the full repertoire of in-silico
methods for the docking of small molecules and peptide
ligands into binding pockets, and to develop and optimize
lead structures derived from screening hits. In connection
with the ChemBioNet initiative, the group has enhanced
its research activities in computational chemistry and
library design.
Design of G-protein coupled receptor modulators
A key area of our research is the model-based design of
ligands for G-protein coupled receptors (GPCR). Our main
topics are GPCR of the human reproductive axis and the
brain-gut-peptides receptor cluster. Compounds targeting
these receptors are used for the treatment of obesity, sexhormone dependent disorders, and human fertility. The
GPCRs are modelled using known X-ray structures as templates and refined in molecular dynamics simulations.
These models are exploited in virtual screening, lead optimisation, and pharmacophore mapping to develop new
receptor antagonists (Fig.1). Group members are included
in ten patents that were filed by our cooperation partner
(Bayer Schering Pharma AG). In order to optimize peptide
ligands of GPCR`s, we developed a new machine learning
approach based on a topology preserving neural network.
Proline-mediated protein-protein interaction
ProlineRichMotif (PRM) recognition domains (PRD) are
highly abundant. Found in many multicomponent signalling complexes, PRDs recognise proline residues by
means of surface accessible stacked aromatic amino acid
Figure 1. Homology model of the melanin-concentrating receptor 1
(MCHR1) with the antagonist bound in the binding pocket. Carbon
atoms in the antagonist are colored in blue, while those of the receptor are colored in green. Schematic representation (a) and closeup of
the binding pocket (b). Selected residues are denoted using the
Ballesteros-Weinstein numbering scheme.
Open binding site
Closed binding site
Figure 2. Model of the non-receptive state of MHC (red) studied
using molecular dynamics simulations.The receptive MHC is shown in
green. The residues involved in stabilization of the closed form are
indicated as sticks. Stabilizing H-bonds are indicated as dashed lines.
residues. PRDs are involved in the modulation of
cytoskeleton dynamics, activation of T-cells, and replication of viruses. We designed an organic building block
which mimics vicinal prolines in a poly-proline II (PPII) helix.
We found that all of the diproline motifs within the proline-rich ligands of VASP-EVH1 and Fyn-SH3 could be
replaced by our scaffold without significant loss of binding affinity. This is the first example for the successful
replacement of a proline recognition motif by a lowmolecular weight building block. The project is funded by
the DFG (FKZ KU 845/2-1)
Design of MHC-loading enhancers (MLE)
Class I and class II MHC molecules are proteins encoded
by the major histocompatibility complex (MHC). They
function as peptide receptors that display antigens on the
cell surface for surveillance by T-cells. Upon recognition,
these antigens can trigger the destruction of the cell – a
quality that made them the focus of experimental tumour
immune therapies. While exogenously added peptides
can activate tumour specific T-cells very efficiently, their
efficacy is severely reduced by the low number of MHC
molecules accessible for loading. Using a combined strategy of screening and modeling we found small molecules
that are able to generate peptide-receptive MHC molecules. These small molecules open the binding site of
human class II MHC molecules by specific interactions
with a defined pocket (see fig. 2). The project is funded by
the BMBF (FKZ 01GU0514).
Dr. Michael Lisurek
Dr. Bernd Rupp
Dr. Federica Morandi*
Dr. Anna Schrey
Dr. Jörg Wichard
Nuria Cirauqui (doctoral student)*
Robert Opitz (doctoral student) */**
Dr. Frank Eisenmenger (system administration)
Stefan Hübel (technical assistant)
Design of capture compounds
Capture Compound Mass Spectrometry (CCMS) technology uses multifunctional small molecules to recognize,
capture and isolate proteins. We established models for
binding and cross-linking modes of several classes of capture compounds. From these results, we derived protein
surface properties crucial for a successful capture event, as
well as information about the influence of conformational changes in the proteins associated with ligand binding
during capturing. This project is funded by the caprotec
bioanalytics GmbH.
Appelt C, Schrey AK, Söderhäll JA, Schmieder P (2007)
Design of antimicrobial compounds based on peptide structures.
Bioorg Med Chem Lett 17, 2334-2337.
Computational chemistry within the ChemBioNet
initiative
The FMP manages the German initiative of the
ChemBioNet. Its aim is the biological characterization of
low molecular weight ligands by high throughput screening. We developed a new library design strategy to
enriche the screening compound library with bioactive
compounds. This program package was used to design
the ChemBioNet library. Further a program package for
automated analysis and data report of screening data was
developed. All packages are implemented in Pipeline Pilot.
Internal collaborations
The special expertise of the Drug Design group in the modelling
of protein-ligand interaction, the homology modelling of proteins (e.g. G- protein coupled receptors), NMR structure calculation, ligand design, and design of screening libraries has led to
numerous internal collaborations. Main topics are the design of
small ligands to modulate poly-proline mediated protein-protein
interactions (AGs Oschkinat, Freund, Beyermann), library design
and structure-activity relationships of screening results (AGs
Rademann, v. Kries) and the design of MHC-loading enhancers
(Freund, v. Kries).
External collaborations
The special expertise of the Drug Design group led to external
collaborations with academic and industrial partners. External
academic collaborators are Prof. Schmalz (Univ. Cologne,
synthesis of proline recognition motif mimetics), Dr. Grötzinger
(Charite, peptide ligand optimization using learning machines),
Dr. Rötzschke (MDC, MHC loading enhancer), Prof. Schäfer (Univ.
Leipzig, TRP-channel blocker), Prof. Monge (Univ. Navarra, MCH1
receptor antagonists), Prof. Meyer (TU München, progesteron
receptor). Common projects with industrial partners were
processed with the Bayer Schering Pharma AG, the caprotec
bioanalytics GmbH, the Jerini AG, and the EMC microcollections
GmbH.
Chevelkov V, Faelber K, Schrey A, Rehbein K, Diehl A, Reif
B (2007) Differential line broadening in MAS solid-state NMR
due to dynamic interference. J Am Chem Soc 129, 1019510200.
Hahn J, Kühne R, Schmieder P (2007) Solution-State (15)N
NMR Spectroscopic Study of alpha-C-Phycocyanin: Implications
for the Structure of the Chromophore-Binding Pocket of the
Cyanobacterial Phytochrome Cph1. Chembiochem 8, 2249-55.
Podust LM, von Kries JP, Eddine AN, Kim Y, Yermalitskaya
LV, Kühne R, Ouellet H, Warrier T, Alteköster M, Lee J,
Rademann J, Oschkinat H, Kaufmann SHE, Waterman MR
(2007) Small-Molecule Scaffolds for CYP51 Inhibitors Identified
by High-Throughput Screening and Defined by X-Ray
Crystallography. Antimicrob Agents Chemother 51, 3915-3923.
Oschkinat H, van Rossum BJ (2007) J-deconvolution using
maximum entropy reconstruction applied to 13C-13C solid-state
cross-polarization magic-angle-spinning NMR of proteins. J Am
Chem Soc 129, 6682-6683.
Zimmermann J, Kühne R, Sylvester M, Freund C (2007)
Redox-regulated conformational changes in an SH3 domain.
Biochemistry 46 6971-6977
Gupta S, Höpner S, Rupp B, Günther S, Dickhaut K,
Agarwal N, Cardoso MC, Kühne R, Wiesmüller K, Jung G,
Falk K & Rötzschke O (2008) Anchor side chains of short peptide fragments trigger ligand-exchange of class II MHC molecules. PLoS ONE 3, e1814.
Ueffing N, Keil E, Freund C, Kühne R, Schulze-Osthoff K &
Schmitz I (2008)Mutational analyses of c-FLIPR, the only murine
short FLIP isoform, reveal requirements for DISC recruitment Cell
Death Differ 15(4): 621-4.
Schmidt MF, Isidro-Llobet A, Lisurek M, El-Dahshan A, Tan
J, Hilgenfeld R, Rademann J (2008) Sensitized detection of
inhibitory fragments and iterative development of non-peptidic
protease inhibitors by dynamic ligation screening. Angew Chem
Int Ed. 47(17): 3275 – 3278.
Lisurek M, Simgen B, Antes I, Bernhardt R (2008) Theoretical
and experimental evaluation of a CYP106A2 low homology
model and production of mutants with changed activity and
selectivity of hydroxylation. Chembiochem. 9(9):1439-49.
Wichard JD, Cammann H, Stephan C, Tolxdorff T (2008)
Classification models for early detection of prostate cancer. J
Biomed Biotechnol 2008:218097.
Rivera G, Bocanegra-García V, Galiano S, Cirauqui N, Ceras
J, Pérez S, Aldana I, Monge A (2008) Melanin-concentrating
hormone receptor 1 antagonists: a new perspective for the
pharmacologic treatment of obesity. Curr Med Chem.
15(10):1025-43.
**part time
reporting period.
Structural Biology
Solid-State NMR
Group Leader:
Bernd Reif
B
ernd Reif obtained his diploma degree in physics and
biochemistry from the Universität Bayreuth and his
PhD-degree in chemistry from the Universität Frankfurt
in 1998. After his postdoctoral years at the Massachusetts
Institute of Technology (1998-1999), he returned to Germany
as Emmy Noether Research Group Leader (TU München,
2000-2002). Since 2003 he has been working as research
group leader at the FMP Berlin, a position he held first in
affiliation with the Charité Universitätsmedizin (2004) and
later with the Humboldt Universität zu Berlin (2007).
characterize the time scale and amplitude of slow motional processes in the solid-state. We found that motional
correlation times can be as high as 500 ns. Solid-state
NMR is ideally suited to characterize motional processes.
In solution-state NMR, relaxation is mostly caused by overall tumbling. Rotational diffusion is, however, absent in
the solid-state. Conformational fluctuations can therefore
be sampled in the solid-state with high accuracy (Agarwal
et al., 2008). For a similar reason, high-resolution deuterium NMR spectra can be obtained in the solid-state. We
started to apply these techniques to Alzheimer’s disease
ωr = 8270 Hz
Elucidation of the Structure and Dynamics of
Proteins in the Solid-State
15
N Chemical Shift (ppm)
We use Nuclear Magnetic Resonance (NMR) in order to
characterize biomolecular systems which are at the interface between solution and solid. In this context, we focus
on membrane proteins and amyloidogenic peptides and
proteins. By nature, structural information of these systems is difficult to obtain by means of X-ray crystallography or standard solution-state NMR methods. We address
these systems by applying a combination of modern solution-state and solid-state NMR methods. This requires the
development of especially adapted NMR techniques. So
far, about 20 proteins are known for which a correlation
between aggregation and disease is established. The most
prominent examples are Alzheimer’s disease (AD), the
prion diseases (BSE, CfJ) and Huntington’s disease.
However, little is know about the mechanism which leads
to aggregation, as well as about the structure of the amyloid fibrils. We would like to gain further insight into the
structure of oligomeric intermediate states which are
associated with protofibril formation. In addition, we are
interested in characterizing dynamic chemical exchange
processes between the soluble and aggregated state of
the respective proteins.
H Chemical Shift (ppm)
1
N Chemical Shift (ppm)
ωr = 24000 Hz
Perdeuteration of a protein in the solid-state enables a resolution in the proton dimension which is comparable to
the resolution achievable in solution-state NMR spectroscopy of medium size proteins (Chevelkov et al., 2006).
The spectral quality allows us to include a proton dimension in resonance assignment experiments which in turn
increases the reliability of the assignment process (Linser
et al., 2007). Using dipole, CSA cross-correlated relaxation
experiments (Chevelkov et al., 2007), we were able to
15
Overview of work and results in 2007/ 2008:
H Chemical Shift (ppm)
1
H,15N correlation spectrum recorded for the α-spectrin SH3 domain
in the crystalline-state. The individual peaks are split according to the
one-bond scalar coupling 1JNH between the proton and the nitrogen. A differential intensity on the multiplet components is indicative
for slow motional processes in the backbone of the protein.
1
Dr. Veniamin Chevelkov*
Dr. Katja Faelber
Dr. Mangesh Joshi
Dr. Juan Miguel Lopez del Amo *
Vipin Agarwal (doctoral student) */**
Sam Asami (doctoral student) *
Muralidhar Dasari (doctoral student)*/**
Tomas Jacso (doctoral student) */**
Rasmus Linser (doctoral student)*/**
Andi Mainz (doctoral student)*
Kerstin Steinhagen (technical assistant)*
Uwe Fink (technical assistant)
Stefan Bibow (student) */**
(AD) ß-amyloid peptide. The disease is characterized by
deposition of plaques of this peptide in the brains of AD
patients. Solid-state NMR is therefore the method of
choice for providing structural information at atomic resolution. It is the goal of these studies to obtain a better
understanding of the mechanisms which result in fibril
formation and aggregation. In addition to structural investigations, we employ small molecules which modulate the
neurotoxic behaviour of the peptide to obtain a relation
between structure and the malfunction. Furthermore, the
interactions between β-amyloid and small heat shock proteins (sHSP) which are found to colocalize in the plaques
of AD patients are invesitgated. Membrane protein projects involve NMR investigations of the maltose ATP binding cassette transporter, and the E.Coli multidrug resistance transporter EmrE. Addition of the ligand tetraphenylphosphonium TPP+ yields a characteristic shift of
the carboxylic carbon resonance of E14 which is identified
using amino-acid selective experiments (Agarwal et al.,
2007).
Agarwal V, Fink U, Schuldiner S, Reif B (2007) MAS SolidState NMR Studies on the Multidrug Transporer EmrE. BBA –
Biomembranes 1768: 3036-3043.
Chevelkov V, Faelber K, Schrey A, Rehbein K, Diehl A, Reif
B (2007) Differential Line Broadening in MAS solid-state NMR
due to Dynamic Interference J. Am. Chem. Soc. 129: 1019510200.
Agarwal V, Xue Y, Reif B, Skrynnikov NR (2008) Protein sidechain dynamics as observed by solution- and solid-state NMR: a
similarity revealed J. Am. Chem. Soc. 130: 16611-16621.
Linser R, Fink U, Reif B (2008) Proton-detected Scalar Coupling
based Assignment Strategies in MAS Solid-State NMR
Spectroscopy applied to Perdeuterated Proteins J. Magn. Reson.
193 (1): 89-93.
Internal
Dr. Michael Beyermann
Dr. Christian Freund
Dr. Sandro Keller
Prof. Hartmut Oschkinat
External
Prof. Gerd Multhaup (FU Berlin)
Prof. Erwin Schneider (HU Berlin)
Prof. Shimon Schuldiner (Hebrew University, Jerusalem)
Prof. Nikolai Skrynnikov (Purdue University)
**part time
Structural Biology
Protein Engineering
Group Leader:
Christian Freund
C
hristian Freund studied chemistry in Düsseldorf and
München before obtaining his PhD at the Max-PlanckInstitute for Biochemistry under the guidance of Tad
Holak. Research as a post-doc in Zürich with Andreas
Plückthun, and with Gerhard Wagner and Ellis Reinherz in
Boston was followed by establishing his own research group
at the FMP/FU Berlin in 2000.
The research group focuses on structural and functional
properties of intracellular scaffolding proteins. NMR spectroscopy and protein engineering is complemented by
biochemical and cellular techniques to decipher protein
function in the context of the cellular proteome.
One major focus of the year 2007 was to achieve a vigorous understanding of the redox properties of the N-terminal hSH3 domain of the T cell scaffolding protein ADAP.
We developed an NMR method to determine redox
potentials for the formation of intramolecular disulfide
bonds. We could show that the measured redox potential
for the two neighbouring cysteines in hSH3-N is compatible with a model whereby cellular activation leads to the
formation of an oxidized or lipid-modified protein with
altered functional properties. The structures of the
reduced and oxidized forms of the protein were solved by
NMR spectroscopy and display significant differences in
their RT loop, potentially leading to a modified binding
behaviour in regard to a yet to be identified interaction
partner (Figure 1).
Based on these results we developed a more general
method to determine the redox potential of disulfide
bond formation by NMR. The relative peak intensities of
resonances that differ between reduced and oxidized
forms of a protein are measured at varying concentrations
of reduced to oxidized glutathione. The redox potential
can then be derived in many instances by data fitting and
application of the Nernst equation (Figure 2). We validated our approach for human thioredoxin, the ADAP hSH3N domain and the Tnk-1 SH3 domain.
In another project, we investigated the role of CD2BP2, a
protein originally identified as binding partner of the T cell
adhesion molecule CD2, in T cells. Knock-down of protein
levels to ~50 % did not lead to any significant change of
in the production of cytokines as for example interleukin2. We conclude that CD2BP2 is not a stochiometric master regulator of cytokines in major T cell populations.
CD2BP2 contains a GYF domain, a small domain that
binds to proline-rich sequences. It is contained in a limited number of proteins and can be divided into two major
subfamilies, the CD2BP2- and SMY2-type GYF domains.
While we had previously defined the binding profile for
this domain by phage display and peptide SPOT analysis,
we more systematically analysed the protein complexes
that are mediated by GYF domain interactions.
Combining GYF domain based pull down experiments
with SILAC/MS and epitope-specific inhibition allowed us
to delineate the probable functional context these proteins are involved in. It was confirmed that CD2BP2 is part
of the so-called U5 snRNP (small nuclear ribonucleoprotein particle) and that it probably is contained in other
complexes involved in splicing. In comparison, SYM2-type
GYF domain containing proteins are mostly cytoplasmic in
their localization and are probably involved in processes
that control mRNA stability and transport.
In a collaborative research program we were investigating
MHC-peptide interactions. Primarily, we developed a protocol for refolding of the MHC:peptide complex from
E.coli. The alpha- and beta-chains of the HLA-DR1 MHC
molecule are expressed separately so that individual
labelling by isotopes is now possible. E. coli derived mate-
Figure 1. Superposition of the structures of the two forms of the
hSH3N domain shown as backbone cartoons. Regions of the protein
with significant changes are color coded (oxidized: orange; reduced:
green) and comprise residues 13-19, 34-43 and 79-81. The backbone r.m.s.d. for these regions is 2.39 Å in average. Structurally conserved regions between the two forms are depicted in light grey,
regions which are not well defined in the NMR ensembles (residues
20-25 and 58-64) are shown in dark grey. The side-chains of the
well-defined aromatic residues and cysteines are shown, hydrogen
atoms are omitted for clarity. The inset shows the conformation of
the eight-membered ring in the oxidized form.
Dr. Kirill Piotukh
Dr. Jana Sticht*
Nele Alder-Behrens*
Sabine Geithner*
Bernhard Meineke (doctoral student)*
Andreas Schlundt (doctoral student)**
Marc Sylvester (doctoral student)**
Michael Kofler ((doctoral student)*/**
Matthias Heinze (doctoral student)*/**
Gesa Albert (doctoral student)*/**
Daniela Kosslick (doctoral student)*/**
Roland Lehmann (doctoral student)*/**
Miriam Rose Ash (research assistant) *
Katharina Thiemke (technical assistant)
Kathrin Motzny (technical assistant) **
Markus Heuser (student) **
Cindy Büssow (apprentice) *
rial maintains full binding capacity, but is more homogenous than the corresponding protein expressed in insect
cells. It is therefore well suited for NMR and crystallographic studies.
Furthermore, we initiated a project that aims at detecting
MHC peptide interactions by Xenon-NMR. CyrptophaneA molecules that are able to capture 129-Xenon can be
coupled via a flexible linker to the hemagglutinin-peptide
(HA). Binding of the latter to MHC should result in a
chemical shift change of the caged xenon signal. Utilizing
hyperpolarized xenon dramatically increases sensitivity
and potentially allows us to detect this interaction in the
nanomolar range.
Heinze M, Kofler M, Freund C (2007) Investigating the functional role of CD2BP2 in T cells. Int Immunol 19: 1313-1318.
Piotukh K, Kosslick D, Zimmermann J, Krause E, Freund C
(2007) Reversible disulfide bond formation of intracellular proteins probed by NMR spectroscopy. Free Radic Biol Med 43: 12631270.
Zimmermann J, Kühne R, Sylvester M, Freund C (2007)
Redox-regulated conformational changes in an SH3 domain.
Biochem 46: 6971-6977.
Rathert P, Zhang X, Freund C, Cheng X, Jeltsch A (2008) Analysis
of the substrate specificity of the dim-5 histone lysine methyltransferase using peptide arrays. Chem & Biochem 15 (1): 5-11.
Ueffing N, Keil E, Freund C, Kühne R, Schulze-Osthoff K,
Schmitz I (2008) Mutational analyses of c-FLIPR, the only murine
short FLIP isoform, reveal requirements for DISC recruitment. Cell
Death Diff 15 (4): 773-782.
Uryga-Polowy V, Kosslick D, Freund C, Rademann J. (2008)
Resin-bound aminofluorescein for C-terminal labeling of peptides:
high-affinity polarization probes binding to polyproline-specific
GYF domains. ChemBiochem 9 (15): 2452-2462.
Internal: with the groups of Eberhard Krause (Mass
Spectrometry), Jörg Rademann (Medicinal Chemistry) and
Ronald Kühne (Molecular Modeling)
External: with A. Jeltsch (Jacob University Bremen), Ingo Schmitz
(Universität Düsseldorf)
L. Mitschang (Physikalisch-Technische Bundesanstalt), Olaf
Rötzschke (MDC, Singapur Immunology network)
Figure 2. A. NMR chemical shifts of the NH-resonances of T30 and
K39 of human thioredoxin at reducing (red) and oxidizing (blue) conditions. B. GSH/GSSG titration curves for these two resonances as
followed by changes in NMR signal intensity. Red and blue curves
represent resonance heights in the reduced and oxidized state, correspondingly. Redox potentials were calculated using the Nernst equation from the ratio of concentrations of reduced (GSH) and oxidized
(GSSG) glutathione. Experimental data were fitted to a modified
Logistic function.
**part time
Structural Biology
Group Leader:
Philipp Selenko
In-cell NMR
O
riginally from Austria Philipp Selenko obtained his PhD
from the European Molecular Biology Laboratory
(EMBL) in Heidelberg (2002) and left Germany for his
postdoc at Harvard Medical School (2003-2008). He was
awarded an EMBO fellowship (2003-2004), a Human Frontiers
in Science (HFSP) fellowship (2004-2006) as well as a Max
Kade fellowship granted by the Austrian Academy of Science
(2006-2007). In 2007, he received the Charles King Trust Prize
by the Bank of America. In 2007, he returned to Germany on
a Emmy Noether fellowship by the Deutsche Forschungs
Gemeinschaft (DFG) to work as a research group leader in the
“Structural Biology” section of the FMP.
We employ high-resolution NMR spectroscopy to study
proteins inside live cells. The overall aim of our group is to
develop tools for observing biomolecules in their native
cellular environments and at atomic resolution. In particular, we analyze the structural and functional in vivo properties of proteins that do not display ordered three-dimensional conformations in vitro. Because many of these
‘intrinsically disordered proteins (IDPs)’ play important
roles in human neurodegenerative disorders (amyloid diseases), we focus on elucidating their native in vivo characteristics inside live cells.
culture models for in-cell NMR measurements in cells of
higher organisms.
Current projects in the lab include in vitro-, cell-free-, and
in-cell NMR analyses of human alpha-synuclein (aSyn),
one of the key players in Parkinson’s disease. We are particularly interested in aSyn’s role in oxidative stress related
cellular responses. We hope to elucidate whether aSyn
plays a direct role in cellular stress response pathways that
lead to apoptosis and neural cell death. Our goal is to
directly visualize conformational alterations of aSyn in cells
that have been exposed to known stress-promoting conditions. As these are known to result in aSyn aggregation,
we hope to gain insight into early structural events during
this pathological transition.
At the same time, we employ NMR spectroscopy to study
post-translational protein modifications of aSyn-, and of
other folded and intrinsically unfolded proteins. These
real-time NMR measurements enable mechanistic insights
into multiple, simultaneous modification events that are
not restricted to protein phosphorylation reactions only.
Here, our goal is to establish in-cell NMR spectroscopy as
a unique tool for non-invasive, quantitative analyses of
intracellular PTM activities, both under physiological-, and
pathological conditions.
In addition to studying structural aspects of IDPs in cellular environments, we employ in-cell NMR techniques to
delineate functional in vivo aspects of proteins with
respect to their post-translational modification (PTM)
behaviour. Here, our emphasis is to provide both mechanistic insights into different types of modification reactions (phosphorylation, acetylation, methylation etc.) as
well as to analyze signal/response behaviours of entire
modification networks inside cells. Since aberrant signalling pathways are often implicated in complex human
diseases such as cancer for example, we use in vivo NMR
spectroscopy to develop novel diagnostic tools for network medicine.
Our lab has been operational since early 2008, when we
had completed our move from Boston to Berlin. Over the
past year we have established a state-of-the-art NMR laboratory dedicated exclusively to studying the cellular in
vivo behaviour of proteins by in-cell NMR spectroscopy.
Besides Xenopus laevis oocytes, which constitute our primary eukaryotic model systems for in-cell NMR analyses,
we are in the process of establishing mammalian tissue
74 Signal Transduction/Molecular Genetics
Figure 1. The African clawed frog Xenopus laevis serves as a wellestablished model system in the areas of cell-, and developmental
biology. Xenopus oocytes can be manipulated by microinjection in
order to deposit defined quantities of biomolecules inside these cells.
We employ this approach to deliver NMR isotope labelled proteins
into the otherwise native, unlabeled intracellular environment.
Panels to the right display a close-up view of a stained cryo-section
of a single Xenopus oocyte. The high degree of macromolecular
crowding of the cells’ cytoplasm is readily appreciated. Effectively all
biomolecules inside a live cell experience an environment that is vastly different compared to an isolated in vitro experimental setup.
Dr. Stamatios Liokatis*
Dr. Silvain Tourel*
Dr. Michaela Herzig*
Beata Bekei (doctoral student)*/**
Silvia Verzini (doctoral student)*/**
Tim Thongwichian (doctoral student)*/**
Marleen van Rossum (technical assistant)**
Selenko P (2008) The Structural Biology of IDPs inside Cells, in:
Instrumental Analysis of Intrinsically Disordered Proteins:
Assesing Structure andConformation (John Wiley & Sons Inc).
Selenko P, Wagner G (2007) Looking into live cells with in-cell
NMR spectroscopy. Journal of Structural Biology 158(2): 244253.
Selenko P, Frueh DP, Elsaesser S, Haas W, Gygi S, Wagner G
(2008) In situ observation of protein phosphorylation by highresolution NMR spectroscopy. Nature Structural and Molecular
Biology 15: 321-329.
Figure 2. Aberrant signalling networks cause a multitude of human
diseases. We employ in-cell NMR spectroscopy to quantitatively
annotate derailed modification activities that lead to pathological
conditions like uncontrolled cell growth, erratic apoptotic behaviour,
or unrestrained cell cycle progression.
FMP Integrated Project grant (with D. Schwarzer and E.
Krause) 2008.
Ongoing collaborations with the laboratories of G. Wagner
(Harvard Medical School), R. Sprangers (MPI, Tuebingen) and W.
Fischle (MPI, Goettingen), E. Wanker + T. Sommer (MDC, Berlin),
C. Hackenberger (FU, Berlin), R. Linding (ICR, London)
**part time
Signal Transduction/Molecular Genetics 75
Protein Trafficking
Ingrid Ridelis Rivas (doctoral student) */**
Antje Schmidt (doctoral student)*/**
Katharina Schulz (doctoral student)**
Susanne Vogelbein (doctoral student)*/**
Carolin Westendorf (doctoral student)*/**
Claudia Rutz (technical assistant)**
Jan Wolkenhauer (student)*/**
Gisela Papsdorf (technical assistant)
Group Leader:
Ralf Schülein
R
alf Schülein studied biology at the University of Würzburg and
completed his graduate work with a PhD thesis on E. coli
toxin transport in the laboratory of Werner Goebel
(Department of Microbiology, University of Würzburg). He then
joined the laboratory of Walter Rosenthal (Department of
Pharmacology, University of Gießen) as a postdoc and started his
work on the trafficking mechanisms of G protein-coupled receptors (GPCRs). Together with Walter Rosenthal, he moved to the
FMP in 1997. He received his “Habilitation” in pharmacology
and toxicology at the Charité Universitätsmedizin Berlin in 2002.
Membrane proteins must have a correct subcellular location to exert their normal function. To allow correct protein
sorting, cells possess a complex transport system, the
secretory pathway (Fig. 1.) Transport starts with the insertion of the proteins into the membrane of the endoplasmic
reticulum (ER), a process mediated by the protein-conducting translocon complex. Folding of the proteins is checked
by a quality control system (QCS) and only correctly folded
forms are allowed to enter the vesicular transport via the
ER/Golgi intermediate compartment (ERGIC) and the Golgi
apparatus to the plasma membrane.
Mutations in the genes of membrane proteins frequently
lead to misfolded proteins failing to pass the QCS. These
proteins are retained, degraded and may cause inherited
disorders such as nephrogenic diabetes insipidus (NDI;
mutant vasopressin V2 receptors, V2R).
The Protein Trafficking group is interested in the quality
control mechanisms of mutant GPCRs in order to identify
new drugs influencing folding and transport processes.
Moreover, we study the the ER insertion mechanisms of
GPCRs and develop new methods for the subcellular
localization of membrane proteins.
1. Quality control of GPCRs in the early secretory
pathway
It was previously thought that quality control of membrane proteins is restricted to the ER.
However, when we took folding defective NDI-causing
V2Rs as a model, we could show that mutant receptors
may also be trapped in the ERGIC and that these receptors can be rescued by some amphipathic peptides such as
penetratin and KLAL (1). The peptide-effect is associated
with an increase in cytosolic Ca2+ resulting most likely from
ionophoric properties of the peptides (1).
Plasma membrane
Tr
Golgi
ERGIC
mRNA
Nucleus
Ri
ER
Figure 1. Transport of integral membran proteins along the secretory
pathway. In the beginning, proteins are integrated into the ER membrane by the translocon complex (Tr). Correctly folded proteins are
transported via the ERGIC and the Golgi apparatus to the plasma
membrane. Misfolded proteins are retained in the early secretory
pathway by the QCS and finally degraded (not shown).
Whereas these peptidic inhibitors are promising tools to
study the QCS in post-ER compartments, their therapeutic potential is limited. Taking GFP-tagged NDI-causing
V2Rs as a model, we now established screening algorithms
for small molecules facilitating V2R folding and/or transport using automated microscopy in live cells (cooperation
with the Cellular Imaging group, B. Wiesner). Initially,
nuclei and plasma membranes of the cells are stained by
Hoechst 33258 and Trypan blue respectively (Fig. 2A, left
and central panel). By subtracting the resulting masks, the
intracellular region can be defined (Fig. 2A, right panel). If
these stains are applied to compound-treated cells
expressing an NDI-causing V2R, the system can determine
automatically if the substance is able to move the receptor to the cell surface. Using this technique, we have
recently screened the ChemBioNet library of the FMP
(cooperation with the Screening Unit, J.-P. von Kries). We
have found a novel compound (IR-23933) facilitating
transport of a mutant V2R not only in microscopical but
also in cell surface biotinylation assays in a dose-dependent manner (Fig. 2B) (unpublished).
2. ER insertion mechanisms
The ER insertion of GPCRs and other membrane proteins
is mediated by two different types of signal sequences.
The majority of the proteins contain signal sequences that
form part of the mature protein. A smaller group, however, possesses additional N-terminal signal peptides that are
A
Nucleus stain
Hoechst 33258
Membrane stain
Trypan Blue
Subtraction
1. Nucleus mask
2. Membrane mask
3. Intracellular mask
B
V2R L336T mutant
0
25
50
µM IR-23933
α-Biotin
α-Pan-cadherin
Figure 1. Screening for substances increasing cell surface transport
of mutant V2Rs. (A) Staining procedures used for automated
microscopy. Nuclei and plasma membranes of cells are stained by
Hoechst 33258 and Trypan blue respectively, and an intracellular
mask is calculated by subtraction. If these stains are applied to compound-treated cells expressing an NDI-causing V2R, it can be determined automatically if the substance is able to move the receptor to
the cell surface. Using this technique, we have screened the
ChemBioNet library and identified a substance (IR-23933) facilitating
transport of the V2R mutant L336T. (B) Compound IR-23933 facilitates transport of the V2R mutant L336T in a cell surface biotinylation
assay. Stably transfected HEK 293 expressing the GFP-tagged receptor mutant were treated with IR-23933 (25 and 50 µM) or left
untreated (-). Plasma membrane proteins were labelled with SulfoNHS-Biotin, precipitated and cell surface receptors were detected by
immunoblotting using an anti-GFP antibody (upper panel). As a loading control, Pan-Cadherin was detected (lower panel).
cleaved off during the insertion process. We studied why
some receptors require cleavable signal peptides and have
previously shown that they serve functions as different as
N-tail translocation over the ER membrane (endothelin B
receptor, ETBR) or regulation of receptor expression (corticotropin-releasing factor receptors type 1 and 2a). More
recently, we have shown that in the case of the ETBR, the
signal peptide alone is unable to open the protein-conducting translocon at the ER membrane. For an efficient
gating process, the 26 amino acids are required that follow the signal peptide (2).
Oueslati M, Hermosilla R, SchönenbergerE, Oorschot V,
Beyermann M, Wiesner B, Schmidt A, Klumperman J,
Rosenthal W and Schülein R (2007) Rescue of a nephrogenic
diabetes insipidus-causing vasopressin V2 receptor mutant by
cell-penetrating peptides. J Biol Chem 282: 20676-20685.
Alken M, Schmidt A, Rutz C, Furkert J, Kleinau G,
Rosenthal W and Schülein R (2009) The sequence after the
signal peptide of the G protein-coupled endothelin B receptor is
required for efficient translocon gating at the endoplasmic
reticulum membrane. Mol Pharmacol 75: 801-811.
Schmidt A, Wiesner B, Weißhart K, Schulz K, Furkert J,
Lamprecht B, Rosenthal W and Schülein R (2009) Use of
Kaede fusions to visualize recycling of G protein-coupled
receptors. Traffic 10: 2-15.
External collaborations
Nikos Tsopanoglou, University of Patras, Patras,
Greece
Marlys Koschinsky, Queen’s University,
Kingston, ON, Canada
Ramanujan Hegde, National Institutes of Health (NIH), Bethesda,
USA
Ricardo Hermosilla, Charité – Universitätsmedizin
Berlin, Campus Benjamin Franklin, Molecuar Pharmacology and
Cell Biology
Joachim Jankowski, Charité – Universitätsmedizin
Berlin, Campus Benjamin Franklin, Institute for Clinical
Pharmacology
Klaus Weißhart, Carl Zeiss MicroImaging GmbH, Jena
Structural Bioinformatics (G. Krause)
Peptide Synthesis (M. Beyermann)
Screening Unit (J. P. von Kries)
Cellular Imaging (B. Wiesner)
3. Development of new methods for subcellular localization of proteins in live cells.
Transport studies of proteins between subcellular compartments would be greatly facilitated if the fluorescence
of a marker fusion protein could be switched once the
protein has reached a particular compartment.
We could now show that the photoconvertible Kaede protein represents an ideal fluorescent tag for GPCR trafficking studies (3). Neither the pharmacological nor the trafficking properties of three model GPCRs were affected by
C terminal Kaede fusions. Moreover, Kaede does not
oligomerize when fused to a membrane protein. Thus, the
Kaede technology may represent a powerful tool for real
time protein trafficking studies in general.
**part time
Anchored Signalling
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Vedrana Tabor *
Sabine Friedl *
Frank Götz *
Solveig Grossmann*
Christian Hundsrucker*
Marta Szaszak *
Pavel Nedvetzky *
Carolyn Vargas
Group Leader:
Enno Klußmann
E
Enno Klußmann studied genetics at the University of
London, UK, and biology at the Philipps-University
Marburg. He received his PhD from the University of
Marburg and his habilitation in pharmacology and toxicology
from the Charité, Berlin. He worked as a postdoctoral fellow in
the Department of Gastroenterology and the Institute of
Pharmacology at the Charité before joining the FMP where he
became group leader in 2003.
ease characterised by a massive loss of water. AVP mediates activation of PKA which in turn phosphorylates AQP2
eliciting the redistribution. We have shown that an AKAP
which we had previously identified, AKAP18δ, tethers
PKA and a PDE of the PDE4 family, PDE4D, to AQP2-bearing vesicles. Functionally, PDE4D controls vesicular, AKAPtethered PKA activity, and thereby most likely the intracellular localization of AQP2 and thus water reabsorption in
renal principal cells (Stefan et al., 2007).
A role of myosin Vb and Rab11-FIP2 in the
aquaporin-2 shuttle
Protein kinase A (PKA) is a ubiquitous serine/threonine
kinase that controls a large variety of cellular functions.
PKA consists of a dimer of regulatory RI or RII subunits and
two catalytic subunits, each of which is bound to an R
subunit. Binding of cAMP to the R subunits induces a conformational change, which causes release and thus activation of the catalytic subunits. They phosphorylate their
substrates in close proximity. Specificity of PKA action is
achieved by controlling its cellular localization through A
kinase anchoring proteins (AKAPs). AKAPs bind PKA
through an amphipathic α-helical structure (RII-binding
domain) which interacts directly with the dimerization and
docking (D/D) domain of regulatory subunit dimers.
Besides PKA, AKAPs directly bind other signalling proteins
such as other protein kinases, protein phosphatases,
phosphodiesterases (PDEs), GTPases, adaptor proteins and
substrate proteins of PKA through unique interacting
domains. Thus AKAPs coordinate multi-protein signalling
complexes establishing compartmentalized signalling. Our
aim is to elucidate the role and the molecular mechanisms
of compartmentalized cAMP/PKA signalling in controlling
vasopressin-mediated water reabsorption in renal principal cells and cardiac myocyte contractility.
Compartmentalization of cAMP-dependent signalling by phosphodiesterase-4D is involved in the
regulation of vasopressin-mediated water reabsorption in renal principal cells
Antidiuretc hormone (vasopressin, AVP) regulates water
reabsorption from primary urine by inducing the redistribution of the water channel aquaporin-2 (AQP2) from
intracellular vesicles into the plasma membrane of renal
collecting duct principal cells. Increases in the plasma AVP
levels are associated with water retention in chronic heart
failure, whereas defects in the regulation of the AQP2
redistribution cause nephrogenic diabetes insipidus, a dis-
The motor protein myosin Vb transports vesicles along the
F-actin cytoskeleton. We have shown that the small G
protein Rab11 is the receptor for myosin Vb on AQP2bearing vesicles. According to our data, the complex consisting of myosin Vb and Rab11, and in addition, the
Rab11-interacting protein, Rab11FIP2 transports AQP2bearing vesicles in response to AVP challenge through the
recycling compartment to the plasma membrane
(Nedvetsky et al., 2007).
Microtubules are needed for the perinuclear
positioning of aquaporin-2 after its endocytic
retrieval in renal principal cells
In addition to an involvement of the F-actin cytoskeleton,
we observed that microtubules play a role in the transport
of AQP2. Microtubule-dependent transport of AQP2 is
predominantly responsible for trafficking and localization
of AQP2 inside the cell after its internalization but not for
the exocytic transport of the water channel. Dynein is the
microtubule-associated motor protein that apparently
transports AQP2-bearing vesicles along microtubules
(Vossenkämper et al., 2007).
AKAP18δ-based complex regulates Ca2+ re-uptake
into sarcoplasmic reticulum of cardiac myocytes
In cardiac myocytes, β-adrenergic receptors mediate activation of the cAMP/PKA-dependent signalling pathway that
regulates heart rate and contractility. In cardiac myocytes,
AKAP18δ forms the basis of a multi-protein complex
including PKA and phospholamban (PLN). This complex
regulates the activity of the sarcoplasmic reticulum Ca2+ATPase (SERCA2). We have shown that AKAP18δ acts as a
scaffold that coordinates PKA phosphorylation of PLN.
Displacement of PKA from the complex interferes with the
phosphorylation of PLN. This prevents the subsequent dissociation of PLN from SERCA2, thereby lowering Ca2+ re-
Dr. Anne Höner
Dr. Evelina Grantcharova*
Jessica Tröger (doctoral student)*/**
Gesa Schäfer (doctoral student)*/**
Claudia Noack (doctoral student)*/**
Jana Bogum (doctoral student) */**
Verena Ezerski (doctoral student)*
Philipp Skroblin (doctoral student)**
Katja Santamaria (doctoral student) */**
Frank Christian (doctoral student)**
Sven Beulshausen (doctoral student) */**
Viola Popara (doctoral student) */**
Andrea Geelhaar (technical assistant)
Beate Eisermann (technical assistant) *
Michael Gomoll, (technical assistant)*
Jürgen Malkewitz (technical assistant)*
uptake into the sarcoplasmic reticulum induced by adrenergic stimuli (Lygren et al., 2007).
We have initiated a chemical biology programme for the
identification of small molecule disruptors of AKAPdependent protein-protein interactions. Such molecules
will be utilized to elucidate cellular functions of the interactions. In the long term, the identification of compounds
modulating the function of AKAPs may lead to a new
class of drugs for the treatment of a variety of diseases
(e.g. renal and cardiovascular diseases). We have received
substantial funding for the initiation of the programme
from the EU and the BMBF.
In collaboration with the groups of Miles Houslay and
George Baillie, University of Glasgow, Scotland, we have
characterized several direct protein-protein interactions of
PDEs of the PDE4 family.
AKAP18δ
RIIα
merge
Sylvia Niquet (technical assistant) *
Hendrikje Göttert (student)*/**
Mareike Boltzen (student) */**
Tordis Borowski (student)*/**
Anita Neumann (apprentice)
Benjamin Leibrandt (apprentice)
Andrey Christian da Costa Gonsalves*
Oskar Petrucci, (student) */**
Stefan E, Wiesner B, Baillie GS, Mollajew R, Henn V, Lorenz
D, Furkert J, Santamaria K, Nedvetsky P, Hundsrucker C,
Beyermann M, Krause E, Pohl P, Gall I, MacIntyre AN,
Bachmann S, Houslay MD, Rosenthal W, Klussmann E (2007)
Compartmentalization of cAMP-dependent signaling by phosphodiesterase-4D is involved in the regulation of vasopressinmediated water reabsorption in renal principal cells. J Am Soc
Nephrol 1: 199-212.
Lygren B, Carlson C, Santamaria K, Lissandron V, McSorley T,
Litzenberg J, Lorenz D, Wiesner B, Rosenthal W, Zaccolo M,
Tasken K, and Klussmann E (2007) AKAP complex regulates
Ca2+ re-uptake into heart sarcoplasmic reticulum. EMBO Rep 8:
1061-1067.
Nedvetsky PI, Stefan E, Frische S, Santamaria K, Wiesner B,
Valenti G, Hammer III JA, Nielsen S, Goldenring JR, Rosenthal
W, Klussmann E (2007) A role of myosin Vb and Rab11-FIP2 in
the aquaporin-2 shuttle. Traffic, 8: 110-23.
Hundsrucker C, Klussmann E (2008) Direct AKAP-mediated
protein-protein interactions as potential drug targets. Handb Exp
Pharmacol. 186: 483-503.
Internal
B. Wiesner, FMP
AKAP18δ
Serca2a
merge
M. Beyermann, FMP
E. Krause, FMP
B. Reif, FMP
G. Krause, FMP
AKAP18δ
PLB
merge
J. P. von Kries, FMP
Walter Rosenthal, FMP
External
V. Groß, MDC
Serca2a
RIIα
merge
M. Bergmann, MDC and Asklepios Klinik St. Georg,Hamburg
S. Bachmann, Charité-Universitätsmedizin Berlin
F. W. Herberg, Universität Kassel
Biaffin GmbH und Co KG, Kassel
Biolog Life Science Institute, Bremen
AKAP18δ and regulatory RII α subunits of PKA colocalize with phospholamban (PLB) and SERCA2 in cardiac myocytes (adapted from
Lygren et al., EMBO Rep.8, 1061-1067, 2007).
M. Houslay, University of Glasgow, Scotland
K. Tasken, University of Oslo, Norway
M. Zaccolo, University of Glasgow, Scotland
D. M. Cooper, University of Cambridge, England
**part time
Cellular Imaging
Group Leader:
Burkhard Wiesner
Application of new caged compounds
Protein-protein interaction
Caged compounds are photolabile inactive derivatives of
biological active substances, from which the active biomolecule, e. g. an intracellular transmitter, is rapidly freed
by UV or Infrared (two-photon process) light. Our group
was able to demonstrate the application of novel caged
substances such as caged cAMP, caged cGMP, caged
progesterone and used them successfully to study cyclic
nucleotide-gated ion channels in living cells.
Fluorescence resonance energy transfer (FRET) is the nonradiative transfer of photon energy from a donor fluorophore to an acceptor fluorophore whereby both are
located within close proximity of 1-10 nm. Another
method for the investigation of protein-protein interactions is fluorescence lifetime imaging microscopy (FLIM). A
third method is fluorescence correlation spectroscopy
(FCS). Using all of these biophysical methods (FRET, FLIM
and FCS) we will examine whether G protein-coupled
receptors form dimers and/or monomeric proteins at the
plasma membrane of living cells.
Motility of proteins in cellular structures
Fluorescence recovery after photobleaching (FRAP) is a
popular method that utilizes changes in the recovery of
fluorescence after local bleaching events to measure the
dynamics of 2D or 3D molecular mobility e. g. diffusion or
transport of fluorescence-labelled molecules in membranes or inside living cells. In most cases it is sufficient to
determine the accurate half- and final recovery time to
describe the differences between proteins or protein
mutants. We modify the data analysis in order to (i) determine quantitatively the immobile fraction, (ii) calculate
protein interactions, and (iii) determine the binding affinity of different proteins over time.
The group’s experience in microscopic techniques and single-cell
techniques has led to good collaborations with many FMP
groups. Thus, the group is developing into a core facility, while
being an equal research partner.
Internal cooperations include the following
Synthetic Organic Biochemistry: applications of new
caged compound.
Protein Trafficking: studies of co-localisation, protein-protein
interactions, translocations of proteins.
Anchored Signalling: protein-protein interactions, translocation
of proteins, intracellular ion concentrations.
Molecular cell physiology: protein-protein-interactions.
Peptide synthesis: Cellular uptake of peptides.
Peptide Transport: cellular uptake of peptides.
Biochemical Neurobiology: colocalisation studies.
Medicinal Chemistry: cellular uptake of peptides.
Screenng Unit: automated microscope as screening technology
for different processes.
Cellular uptake of substances
One of the main goals of research at the FMP is the modulation of protein functions. This requires the intracellular
delivery of interacting molecules. Some research groups
focus on the elucidation of the structural requirements of
peptides as uptake-promoting and targeting tools for
attached cargos and lipid-based carriers. Using confocal
microscopy we investigated the cellular uptake of different fluorophore-tagged substances (e. g. peptides, nucleosides, pro-nucleotides). We looked at the cellular distribution of different substances, the differences of the cellular uptake at different cell types, and the kinetics of the
cellular uptake. We were able to quantify differences in
uptake caused by alterations in pro-nucleotide structure.
Such results will greatly aid the development of tissueselective drugs.
External
Prof Dr. Andreas Hermann, Molecular Biophysics and Cell
Biology, Humboldt University of Berlin, Germany: protein-protein
interaction.
Prof. Dr. Uwe Vinkemeier, School of Biomedical Science,
University of Nottingham, UK: motility of proteins in cellular
structures.
Prof. Dr. Roger A. Johnson, Physiology and Biophysics, Stony
Brook University New York; USA: Cellular uptake of compounds.
Prof. Dr. Thomas Walter, Cardiovascular Physiology, University of
Hull, UK: protein-protein interactions.
Prof. Dr. U.B. Kaupp, Molecular Sensory Systems, Caesar
Research Center Bonn, Germany: applications of new caged
compounds.
Dr. Dorothea Lorenz
Katja Lautz (doctoral student) */**
Anke Teichmann (doctoral student) */**
Jenny Eichhorst (technical assistant)
Brunhilde Oczko (technical assistant) *
Martina Ringling (technical assistat) **
Sascha Lange (student) */**
Stefan E, Wiesner B, Baillie GS, Mollajew R, Henn V, Lorenz
D, Furkert J, Santamaria K, Nedvetsky P, Hundsrucker C,
Beyermann M, Krause E, Pohl P, Gall I, MacIntyre AN,
Bachmann S, Houslay MD, Rosenthal W, Klussmann E (2007)
Compartmentalization of cAMP-dependent signalling by phosphodiesterase-4D is involved in the regulation of vasopresinmediated water reabsorption in renal principal cells. J Am Soc
Nephrol 18: 199-212.
Nedvetsky P, Stefan E, Frische S, Santamaria K, Wiesner B,
Valenti G, Hammer III AJ, Nielsen S, Goldenring RJ, Rosenthal
W, Klussmann E (2007) A role of myosin Vb and Rab11-FIP2 in
the aquaporin-2 shuttle. Traffic 8: 110-123.
(2008) Aberrant expression of Notch1 interferes with the B-lymphoid phenotype of neoplastic B cells in classical Hodgkin lymphoma Leukemia 22(8): 1587-1594.
Schwieger I, Lautz K, Krause E, Rosenthal W, Wiesner B,
Hermosilla R (2008) Derlin-1 and p97/VCP Mediate the ERAssociated Degradation of Human V2 Vasopressin Receptors.
Mol Pharmacol73: 697-708.
Sun X, Wiesner B, Lorenz D, Papsdorf G, Pankow K, Wang
P, Dietrich N, Siems WE, Maul B (2008) Interactions of
angiotensin-converting enzyme (ACE) with membrane-bound
carboxypeptidase M (CPM) – a new function of ACE Biol Chem
389: 1477-1485.
Zeisig R, Koklic T, Wiesner B, Fichtner I and Sentjurc M (2007)
Increase in fluidity in the membrane of MT3 breast cancer cells
correlates with the enhanced cell adhesion in vitro an increased
lung metastasis in NOD/SCID mice. Arch Biochem Biophys 459:
98-106.
Oueslati M, Hermosilla R, Schönenberger E, Oorschot V,
Beyermann M, Wiesner B, Schmidt A, Klumpermann J,
Rosenthal W, Schülein R (2007) Rescue of a nephrogenic diabetes insipidus-causing vasopressin V2 receptor mutant by cellpenetrating peptides. J Biol Chem 282 (28): 20676-20685.
Vossenkaemper A, Nedvetsky PI, Wiesner B, Furkert J,
Rosenthal W, Klussmann E (2007) Microtubules are needed
for the perinuclear positioning of aquaporin-2 after its endocytic
retrieval in renal principal cells. Am J Physiol Cell Physiol 293:
C1129-1138.
Lygren B, Carlson CR, Santamaria K, Lissandron V, McSorley T,
Litzenburg J, Lorenz D, Wiesner B, Rosenthal W, Zaccolom
M, Taskén K, Klussmann E (2007) AKAP complex regulates
Ca2+ re-uptake into heart sracoplasmatic reticulum. EMBO
reports 8: 1061-1067.
Plant TD, Zöllner C, Kepura F, Mousa SS, Eichhorst J, Schaefer
M, Furkert J, Stein J, Oksche A (2007) Endothelin potentiates
TRPV1 via ET1 receptor-mediated activation of protein kinase. C
Mol Pain 14: 3-35
Piontek J, Winkler L, Wolburg H, Müller SL, Zuleger N, Piehl
C, Wiesner B, Krause G, Blasig IE (2008) First homophilic
interaction determinants between strand-forming claudins identified. FASEB 22: 146-158.
Bit-Avragim N, Rohr S, Rudolph F, Van Der Ven P, Fürst D,
Eichhorst J, Wiesner B, Abdelilah-Seyfried S (2008) Nuclear
localization of the zebrafish tight junction protein nagie oko.
Dev Dyn 237: 83-90.
Hagen V, Dekowski B, Kotzur N, Lechler R, Wiesner B,
Briand B, Beyermann M (2008) {7[Bis(carboxymethyl)amino]coumarin-4-yl}methoxycarbonyl
Derivatives for Photorelease of Carboxylic Acids,
Alcohols/Phenols, Thioalcohols/ Thiophenols, and Amines.
Chemistry 14: 1621-1627.
Jundt F, Acikgöz Ö, Kwon S-H, Anagnostopoulos I, Wiesner B,
Mathas S, Lim HY, Hummel M, Stein S, Reichardt HM, Dörken B
**part time
reporting period.
Molecular Cell Physiology
Group Leader:
Ingolf E. Blasig
I
ngolf Blasig studied biology and biochemistry in Leipzig from
1970-74. His diploma thesis was on cancer research at the
Robert-Rössle-Hospital in Berlin, his dissertation dealt with the
pharmacology of myocardial infartion at the Academy of
Sciences (1984). He obtained his venia legendi for investigations
on myocardial dysfunction at the University of Halle in 1992.
From 1993-95, he was awarded project leader at the NIH, USA.
Since 1992 he has been head of the independent research
group for Molecular Cell Physiology at the FMP and is teaching
at the universities in Potsdam and Berlin.
mechanism and regulation are unknown. For the first
time, we demonstrated self-association of claudin-5 in
cell-cell contacts of intact cells using fluorescence resonance energy transfer (FRET), a prerequisite for explaining
the strand formation mechanism. In addition, we showed
that the second extracellular loop of claudin-5 (Piontek et
al., 2008) is involved in the sealing of the extracellular
clefts of the BBB. Functional investigations are in progress
to verify the effect of mutations in claudins on the BBB as
well as to develop small molecules (in collaboration with
J. v. Kries, Screening Unit, FMP) to modulate the barrier
tightness for pharmacological use. Another approach is to
modulate the BBB by specific peptides that affect the
extracellular loops of claudins (Winkler et al., in press).
Scaffolding function of ZO-1
The group focuses on the elucidation of structure, function, and manipulation of cell-cell contacts. The major
objective is to explore tight junctions (TJ) in barrier-forming endothelial and epithelial cells under normal and
pathological conditions to disclose the neuropathophysiological mechanisms that underlie stroke, lesional epilepsy,
and other conditions with the aim of finding better therapies. In addition, the development of new strategies
specifically of modulating the blood-brain barrier (BBB)
may lead to improved drug delivery. The tightness of the
BBB is mainly determined by transmembrane proteins
which constantly seal the intercellular cleft. We concentrate on the oligomerisation, scaffolding, and regulation
of TJ proteins.
Oligomerisation of claudins
In the BBB, claudin-5 tightens the barrier for pharmacologically relevant molecules with a molecular weight
smaller than 800 Da. However, the molecular interaction
Studies of ZO-1 (scaffolding cell contact and cytoskeleton
proteins) demonstrated that the SH3-domain, hingeregion, and GuK-domain interact as a common functional and self-associating unit. We found that occludin’s
cytosolic coiled-coil domain interacts with the unit and
leads to dimerisation. We devised a general dimerisation
concept of transmembrane TJ proteins via dimerising ZO1. We identified potential regulator proteins that preferentially bind to the hinge region of ZO-1, suggesting that
this region acts as adaptor for the regulation of cell-cell
contacts.
Redox-sensitivity of occludin
Occludin is a specific TJ protein of unknown function
which is affected by oxidative stress. We found that its
coiled coil-domain forms dimers which are redox-sensitive
and depend on the sulfhydryl concentration of the environment in low-millimolar range. The incubation of cells
that contain full-length occludin with sulfhydryl reagents
prevented dimerization. Mutation of a cystein in the
Scheme of the interaction possibilities
between tight junction
forming claudins as,
for example, observed
for claudin-5 and -3.
Homophilic and heterophilic cis- and
trans-interactions, i.e.
association between
two opposing cells
and along one cell
membrane,
respectively.
Dr. Reiner Haseloff
Dr. Christine Rückert
Dr. Jörg Piontek
Victor Manuel Castro Villela
(doctoral student)**
Jimmi Cording (doctoral student) **/*
Dörte Lohrberg (doctoral student)**
Christian Piehl (doctoral student)**
Juliane Walter (doctoral student)**
Ariane Wenzel, (doctoral student)**
Lars Winkler (doctoral student)**/*
Jingjing Zhang (doctoral student)*
Barbara Eilemann (technical assistant)
Sandra Richter (student)*/**
Peter Schläger (student)*/**
Claudia Semprich (student)*/**
coiled-coil domain did not show dimer formation either.
This demonstrates, for the first time, that a disulfide bridge
is involved in the dimerization of occludin. We concluded
that a redox-dependent dimerization of occludin may play
a regulatory role in the TJ assembly under physiological
and pathological conditions (Walter et al., in press).
Stress responses in brain capillary endothelial cells
(BCEC)
Applying proteomic approaches we aimed at identifying
proteins and corresponding signalling pathways altered in
BCEC under pathological conditions. Numerous proteins
were found that change expression as a result of hypoxia
(Haqqani et al., 2007). Application of free radicals to
BCEC led to a significant increase in the expression of peroxiredoxin. There are signs that this protein, involved in
the elimination of peroxides formed under stress conditions, may play a role in the endogeneous defence against
cellular injury in neuroinflammatory diseases such as multiple sclerosis (Schreibelt et al., 2008).
Outlook
We plan to reconstitute functional TJ at the protein, cellular, and animal levels. We will develop a common model
of TJ assembly showing how claudins form higher
oligomers that promote strand formation and regulate the
BBB. Regulatory aspects will be studied further such as the
direct influence of the extracellular loops of TJ-proteins.
Moreover, protein-protein binding assays will be developed in order to screen for pharmacologically relevant
agents in the FMP compound library. This way, new therapeutic approaches might be developed that allow us to
improve the delivery of neuropharmacologica agents that
overcome the BBB in therapy resistant brain diseases.
Gerd Krause
Eberhard Krause
Sebastian Müller
Lars von Kries
Michael Beyermann
Burkhard Wiesner
Michael Schümann
Katharina Metzner (student)*/**
Juliane Neumann (student)*/**
Susanne Fritzsche (student)*/**
Corinna Gagell (student) */**
Claudia Gehring (student) */**
Vivian Knöbel (student) */**
Lohrberg D, Krause E, Schümann M, Piontek J, Winkler L,
Blasig IE, Haseloff RF (2009) A strategy for enrichment of
claudins based on claudin affinity to Clostridium perfringens
enterotoxin, BMC Mol Biol, in press.
Winkler L, Gehring C, Wenzel A, Müller SL, Piehl C, Krause
G, Blasig IE, Piontek J (2009) Molecular determinants of the
interaction between Clostridium perfringens enterotoxin and
claudins. J Biol Chem, in press.
Walter JK, Rückert C, Voss SL, Müller J, Piontek J, Gast K,
Blasig IE (2009) The oligomerization of the coiled coil-domain
of occludin is redox-sensitive. Ann. New York Acad. Sci., in press
Krause G, Winkler L, Piehl C, Zuleger N, Blasig IE, Piontek
J, Müller SL (2008) Structure and function of extracellular
claudin domains. Ann. New York Acad. Sci. in press
Schroeter ML, Abdul-Khaliq H, Krebs M, Diefenbacher A, Blasig
IE (2008) NSE is unaltered whereas S100B is elevated in serum
of patients with schizophrenia – Original research and metaanalysis. Psychiatry Res, in press
Piontek J, Winkler L, Wolburg L, Muller SL, Zuleger N, Piehl
C, Wiesner B, Krause G, and Blasig IE (2008) Formation of
tight junction: determinants of homophilic interaction between
classic claudins FASEB J 22:146-158.
Krause G, Winkler L, Mueller SL, Haseloff RF, Piontek J and
Blasig IE (2008) Structure and function of claudins. Biochim
Biophys Acta 1778: 631-645.
Schreibelt G., van Horssen J, Haseloff RF, Reijerkerk A, Van der
Pol SJ, Nieuwenhuizen O, Krause E, Blasig IE, Dijkstra CD,
Ronken E, De Vries HE (2008) Protective effects of peroxiredoxin-1
at the injured blood-brain barrier. Free Rad Biol Med 45: 256-264.
Schroeter ML, Abdul-Khaliq H, Krebs M, Diefenbacher A, Blasig
IE (2008) Serum markers support disease-specific glial pathology
in major depression. J Affect Disord 111: 271-280
Schreibelt G, Kooij G, Reijerkerk A, van Doorn R, Gringhuis SI,
van der Pol SMA, Weksler BB, Romero IA, Couraud PO, Piontek
J, Blasig IE, Dijkstra CD, Ronken E, de Vries HE (2007) Reactive
oxygen species alter brain endothelial tight junction dynamics via
RhoA, PI3 kinase, and PKB signaling. FASEB J. 21: 3666-3676.
Haqqani AS, Kelly J, Baumann E, Haseloff RF, Blasig IE and
Stanimirovic DB (2007) Protein markers of ischemic insult in
brain endothelial cells identified using 2D gel electrophoresis and
ICAT-based quantitative proteomics: Comparison of in vitro and
in vivo models J Proteome Res 6: 226-239
Hartwig Wolburg, Universität Tübingen
Salim Seyfried, MDC Berlin
Klaus Gast, Universität Potsdam
Hans-Peter Hahn, MDC Berlin
Matthias Schroeter, Universität Leipzig
Otmar Huber, FU Berlin
Elga de Vries, Amsterdam
Walter Hunziker, Singapur
Jerrold Turner, Chicago
**part time
yellow Position funded externally (3rd-party funds) for at
least part of the reporting
period.
Biochemical Neurobiology
Group Leader:
Wolf-Eberhard Siems
W
olf-Eberhard Siems studied chemistry at the
University Rostock, Germany, and graduated with a
PhD in 1972. Following his work as a research assistant at the Institute of Artificial Insemination (IfKB) Berlin and
further postgraduate studies in biochemistry (“Natural Scientists
in Medicine” Programme at the Academy of Advanced Medical
Training Berlin), in 1980 he was appointed as scientist at the
Institute of Drug Research. Since 1992 he has been working as
head of the Biochemical Neurobiology Group at the FMP.
Our group investigates the biochemical, pharmaceutical,
and molecular aspects of membrane-bound peptidases.
We focus on angiotensin-converting enzymes (ACE as
well as ACE2), neutral endopeptidase (neprilysin/NEP) and
some related peptidases. ACE and esp. neprilysin cleave a
very broad spectrum of physiologically important peptides. As a consequence, they play essential roles in various body functions. In 2007 and 2008 we focused on the
biochemical and functional properties of neprilysin in relation to obesity, neuronal disorders and heart diseases.
1. Natriuretic peptides (ANP, BNP, and CNP) are cyclic
vasoactive peptide hormones of great diagnostic and therapeutic relevance. Degradation by neprilysin is postulated
as their main catabolic pathway. Nevertheless, some natriuretic peptides such as the human B-type natriuretic peptide (BNP) show resistance to recombinant neprilysin. We
compared the degradation of various mature, truncated,
and recombinant natriuretic peptides by neprilysin.
Degradation was clearly dependent on the length of the
N- or C-terminus but also on distinct sequence differences
within the essential loop structure of the natriuretic peptides. Based on these findings, we developed a model for
the interaction of neprilysin and natriuretic peptides (see
Fig. 1) that permits new insights into the mode of action
and prediction of substrates of neprilysin (Collaboration
with the groups of Gerd Krause, FMP, and Thomas
Walther, Univ. Hull, UK).
2. In the period under review we were interested in the
impact of a neprilysin deficiency in mice (NEP-/-) (Fig. 2).
In co-operation with the Charité (Berlin) and the University
of Hull (UK) we observed that NEP-/- mice develop mature
obesity. NMR-spectroscopy studies showed that the higher body weight in NEP-/- mice is exclusively due to an
accumulation of fat. As often observed in polygenetic
human obesity, NEP-/- mice were characterized by higher
blood glucose levels and a significantly impaired glucose
tolerance. The key role of neprilysin in determining body
mass was confirmed by pharmacological approaches. In
wild-type mice, the neprilysin inhibitor candoxatril (Pfizer)
increased body weight due to a stimulation of food
intake. Peripheral rather than central NEP is the control
switch for appetite control, since candoxatril cannot cross
the blood-brain barrier. Thus, lack in NEP activity, mediated genetically or pharmacologically, leads to a gain in
body weight and fat-accumulation. The described obesity
model is thus an ideal tool for research on development,
molecular mechanisms, diagnosis and therapy of the pandemic obesity.
Neprilysin is well accepted as one of the key enzymes in
initial human amyloid-β peptide (Aβ) degradation. We
confirmed that endogenous Aβ concentration is prominent in the brain of NEP-knockout mice at all investigated
time points (co-operation with J. Furkert, FMP). However,
immunohistochemistry with monoclonal antibodies
detecting murine Aβ did not identify any Aβ deposits even
in old NEP knockout mice (co-operation with the
University of Leipzig). Studies on learning and memory
(co-operation with Universities of Hull, Magdeburg and
HU-Berlin) surprisingly revealed that the ability to learn
was not reduced in old NEP-deficient mice but had significantly improved, and sustained learning and memory in
the aged mice was in parallel with improved long-term
potentiation (LTP) in brain slices of the hippocampus and
lateral amygdala. Our data implies a beneficial effect of
pharmacological inhibition of central NEP on learning and
memory in mice by accumulation of peptides others then
Aβ, but also degradable by NEP. Due to degradation studies and peptide measurements in the brain of both genotypes, we identified GLP-1 and galanin as two neuropeptide candidates involved in the improved learning in aged
NEP-deficient mice. The existence of peptides that
improve learning and memory in older individuals when
targeted by NEP might open a promising avenue into the
treatment of neurodegenerative diseases.
Nils Dietrich (doctoral student)*/**
Kristin Pankow (doctoral student)*
Xiaoou Sun, (doctoral student) */**
Bettina Kahlich (technical assistant)
Stephanie Führl (technical assistant)**
Matthias Münzer, (technical assistant) *
Tordis Borowski (student) */**
Anja Schwiebs (student) */**
Sommer WH, Rimondini R, Marquitz M, Lidström J, Siems
WE, Bader M, Heilig (2007) Plasticity and impact of the central renin-angiotensin system during development of ethanol
dependence. M J Mol Med 85(10): 1089-97.
Pankow K, Wang Y, Gembardt F, Krause E, Sun X,
Krause G, Schultheiss HP, Siems WE, Walther T. (2007)
Successive action of meprin A and neprilysin catabolizes Btype natriuretic peptide. Circ Res 101(9): 875-82.
Walther T, Tschöpe C, Sterner-Kock A, Westermann D,
Heringer-Walther S, Riad A, Nikolic A, Wang Y, Ebermann L,
Siems WE, Bader M, Shakibaei M, Schultheiss HP, Dörner A.
(2007) Accelerated mitochondrial adenosine diphosphate/adenosine triphosphate transport improves hypertension-induced heart disease. Circulation 115(3): 333-44.
Figure 1. Model for the interaction of a natriuretic peptide (exemplarily shown for CNP) in the interior cave of NEP
Sun X, Wiesner B, Lorenz D, Papsdorf G, Pankow K,
Wang P, Dietrich N, Siems WE, Maul B. (2008) Interaction
of angiotensin-converting enzyme (ACE) with membranebound carboxypeptidase M (CPM) – a new function of ACE.
Biol Chem 389(12): 1477-85.
Sun X, Becker M, Pankow K, Krause E, Ringling M,
Beyermann M, Maul B, Walther T, Siems WE (2008)
Catabolic attacks of membrane-bound angiotensin-converting
enzyme on the N-terminal part of species-specific amyloidbeta peptides. Eur J Pharmacol 588(1): 18-25.
Maul B, von Bohlen und Halbach O, Becker A, Sterner-Kock
A, Voigt JP, Siems WE, Grecksch G, Walther T. (2008)
Impaired spatial memory and altered dendritic spine morphology in angiotensin II type 2 receptor-deficient mice. J Mol Med
86(5): 563-71.
Figure 2. Young NEP-knockout mouse
Walther T, Albrecht D, Becker M, Schubert M, Kouznetsova E,
Wiesner B, Maul B, Schliebs R, Grecksch G, Furkert J,
Sterner-Kock A, Schultheiss HP, Becker A, Siems WE (2009):
Improved learning and memory in aged mice deficient in
amyloid beta-degrading neutral endopeptidase PLoS One
4(2):e4590.
Internal
Dr. J. Furkert
Dr. G. Krause
Dr. E. Krause
Dr. B. Wiesner
Dr. J.P. von Kries
External
Prof. T. Walther, The University of Hull (UK)
Prof. M. Bader, MDC Berlin-Buch, Koordinationsbereich
Hypertonie
Prof. G. Grecksch, Otto v. Guericke Uni Magdeburg
Prof. R. Schliebs, Univ. Leipzig,
**part time
Physiology and Pathology of
Ion Transport
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Luiza Bengtsson
Pawel Fidzinski*
Ioana Neagoe
Gaia Novarino
Carsten Pfeffer
Vanessa Plans
Guillermo Spitzmaul
Tobias Stauber
Lena Wartosch **
Stefanie Weinert
Department Leader:
Thomas Jentsch
T
homas Jentsch obtained his Ph.D. in physics from the Freie
Universität Berlin and the Fritz-Haber-Institut of the MaxPlanck-Gesellschaft in 1982, and his medical doctorate
also from the FU Berlin in 1984. After his doctoral and postdoctoral work at the Institut für Klinische Physiologie, FU, he moved
to the USA in 1986, where he worked as a postdoctoral fellow
with Harvey Lodish at the Whitehead Institute for Biomedical
Research (MIT). Upon his return to Germany in 1988, he became
research group leader at the ZMNH, Hamburg University. In
1993, he was appointed full professor and director of the
Institute for Molecular Neuropathology at the ZMNH. From
1995-1998 as well as again from 2001-2003 he was also director of the ZMNH. In 2006, Prof. Jentsch moved to Berlin to
become a full professor at the Charité, head of research group
Physiology and Pathology of Ion Transport at the FMP (LeibnizInstitut für Molekulare Pharmakologie) and MDC (MaxDelbrück-Centrum für Molekulare Medizin). Since 2008 he is
also Principal Investigator of Neurocure.
We aim to understand ion transport processes from the
molecular level (structure-function analysis) to the subcellular and cellular level (e. g. role in endosomes) up to the
level of the organism. The latter aspects are largely tackled by investigating the phenotypes of respective knockout and knock-in mice, and by analyzing corresponding
human diseases. We focus on three molecular classes of
ion transport proteins: CLC chloride channels and transporters, KCNQ potassium channels, KCC potassium-chloride cotransporters, and are starting new projects on other
channel families. Two of our most important research
areas concern the role of vesicular pH and chloride in the
endosomal/lysosomal system, and the regulation of neuronal cytoplasmic chloride and its impact on neuronal
function and development. As the ion transport proteins
under study are expressed in a wide range of tissues, we
analyze their function in many organs (brain, inner ear,
eye, kidney, pancreas, bone, testis, etc.).
CLC chloride channels and transporters
Proteins of the CLC gene family, discovered by us in 1990,
reside in the plasma membrane and intracellular vesicles.
Surprisingly, several (or all) vesicular CLCs are Cl-/H+86 Signal Transduction/Molecular Genetics
exchangers. We have generated KO mouse models for
most CLCs and have identified corresponding human diseases, yielding insights into their diverse physiological
functions. We have also identified two ancillary β-subunits
(barttin and Ostm1), mutations in which also cause
human disease. Our recent inner-ear specific deletion of
barttin in mice clarified the patahological mechanism
underlying deafness in human Bartter syndrome type IV.
Current projects focus on the role of vesicular CLCs in
determining endosomal/lysosomal pH and Cl- and the
impact on vesicular function and trafficking; structure/function analysis to understand the difference
between Cl- channels and Cl-/H+-exchangers; conditional
and multiple KO mice to unravel the importance of CLCs
for various cellular functions and their redundancy; investigation of CLC trafficking and identification of sorting
signals; identification and role of associated proteins.
KCNQ potassium channels
We cloned and characterized the K+ channels KCNQ2-5,
have shown that mutations in KCNQ2 and 3 cause neonatal epilepsy and mutations in KCNQ4 a form of dominant
deafness (DFNA2). KCNQ2-5 mediate highly regulated‚
M-type currents that are important for the regulation of
neuronal excitability. We have recently generated mouse
models for KCNQ4 and KCNQ5 (other mouse models are
in progress). Both KCNQ4 KO mice, as well as mice carrying a dominant negative mutation we have previously
identified in human deafness, develop deafness (the dominant negative with a slower time course). This is due to a
selective degeneration of sensory outer hair cells. We have
generated several KCNQ mouse models which we are
investigating with respect to the inner ear, central and
peripheral nervous system.
KCC K-Cl cotransporter
We have disrupted all four isoforms of electroneutral K-Cl
cotransporters in mice, leading to interesting pathologies
that include deafness, renal tubular acidosis, high blood
pressure, and degeneration of the central and peripheral
nervous system. We also explored the role of KCC1 and
KCC3 in regulating the volume of red blood cells and their
impact on the pathology of sickle cell disease. K-Cl
cotransport, in particular KCC2, plays a crucial role in
establishing the inhibitory response to GABA and glycine,
with the early excitatory response believed to be important for neuronal development. Current projects focus on
the function of KCC3 and KCC4 in non-neuronal tissues
and on the role of KCC2 and KCC3 in certain types of
Dr. Dietmar Zimmer (Research
Coordinator)*
Eun-Yeong Bergsdorf (doctoral student)*
Gwendolyn Billig (doctoral student)*/**
Matthias Heidenreich
(doctoral student)*/**
Sabrina Jabs (doctoral student) */**
Lilja Leisle (doctoral student)*/**
Kristin Natho (doctoral student)*/**
Patricia Preston (doctoral student)*/**
Patricia Seja (doctoral student)*/**
Anyess von Bock (technical assistant)
Alexander Fast (technical assistant)
Inga Freyert (technical assistant)*
Petra Göritz (technical assistant)
Nicole Krönke (technical assistant)
Ina Lauterbach (technical assistant)
Rainer Leben (technical assistant)
Janet Liebold (technical assistant) **
Ruth Pareja (technical assistant)*
Patrick Seidler (technical assistant)*
Stephanie Wernick (technical assistant)*
Silke Zillmann (technical assistant)
Verena Perneczky (student) */**
Cathleen Rohleder (student) */**
Florian Wagner (student) */**
All members except for the head of the group and Petra Göritz are employed by the MDC but funded equally by both institutes.
neurons, using conditional mouse models. To complement
these studies on transporters that lower intracellular chloride in neurons, we studied the effect on neuronal
excitability of NKCC1, a transporter that raises intraneuronal chloride. NKCC1 KO mice showed decreased neuronal excitability and network activity.
Barttin and CIC-K are expressed in the stria
vascularis of the cochlea.
Selected Publilcations
Rust M.B., Alper S.L., Rudhard Y., Shmukler B.E., Vicente R.,
Brugnara C., Trudel M., Jentsch T.J., Hübner C.A. (2007).
Disruption of erythroid KCl-cotransporters alters erythrocyte volume and partially rescues erythrocyte dehydration in SAD mice. J
Clin Invest 117: 1708-1717.
Blanz J., Schweizer M., Auberson M., Maier H., Muenscher A.,
Hübner C.A., Jentsch T.J. (2007). Leukoencephalopathy upon
disruption of the chloride channel ClC-2. J Neurosci 27: 65816589.
Zdebik A.A., Zifarelli G., Bergsdorf E.-Y., Soliani, P., Scheel O.,
Jentsch T.J., Pusch M. (2008). Determinants of anion-protoncoupling in mammalian endosomal CLC proteins. J Biol Chem
283: 4219-4227.
Maritzen T., Keating D.J., Neagoe I., Zdebik A.A., Jentsch T.J.
(2008). Role of the vesicular chloride transporter ClC-3 in neuroendocrine tissue. J Neurosci 28: 10587-10598.
Rickheit G., Maier H., Strenzke N., Andreescu C.E., De Zeeuw
C.I., Zdebik A.A., Jentsch T.J. (2008). Endocochlear potential
depends on chloride channels: mechanism underlying deafness
in Bartter syndrome IV. EMBO J 27: 2907-2917.
Barttin
KCNQ1
Pfeffer C.K., Stein V., Keating D.J., Maier H., Rinke I., Rudhard
Y., Hentschke M., Rune G., Jentsch T.J., Hübner C.A. (2009).
NKCC1-Dependent GABAergic Excitation Drives Synaptic
Network Maturation During Early Hippocampal Development. J
Neurosci 29: 3419-3430.
Bergsdorf, E.-Y., Zdebik A.A., Jentsch T.J. (2009). Residues
important for nitrate/proton coupling in plant and mammalian
CLC transporters. J Biol Chem 284: 11184-11193.
*part of period reported **part time
yellow Position funded externally (3rd-party funds) for at least part of the reporting
period.
We are collaborating with several FMP groups. This includes the
group of J. Rademann (synthesis of fluorescent Cl indicators), V.
Hagen (caged compounds), B. Wiesner (FRET and FLIM
microscopy), E. Krause (mass spectroscopical analysis of binding
partners), and the group of Hartmut Oschkinat (NMR analysis of
cytoplasmic protein domains). Our diverse mouse models and
expertise in various organ systems significantly bolster the
strength of the FMP in systems biology and pharmacology. As
our group belongs to both the FMP and the MDC (joint appointment), we increase the synergy between both institutions. We
were recently awarded a grant by the Leibniz-Society (Pakt für
Innovation) in which we collaborate with Gary Lewin (MDC) and
Dietmar Schmitz (Charité) in the analysis of KCNQ mouse models. Finally, we are a founding member of Neurocure, a ‘cluster
of excellence’ that includes a large number of groups from universities and the MDC. The FMP is additionally contributing to
Neurocure by providing support to the Junior Group of Andrew
Plested, who has obtained laboratory space within our department. The interaction with his group, which is working on glutamate receptor ion channels, is very fruitful.
Cytokine Signalling
Group Leader:
Klaus-Peter Knobeloch
K
laus-Peter Knobeloch received his diploma in biology in
1994 (Julius-Maximillian University Würzburg) and his
PhD-degree in 2000 (Institute for Immunology/ Virology
University of Würzburg & Leibniz-Institut für Molekulare
Pharmakologie Berlin). After a two year period as scientific
director for target validation/identification at Genpat77
Pharmacogenomics AG in 2003 he became research group
leader at the FMP.
Our group aims to understand the molecular and biological function of distinct components of the Ubiquitin- and
Ubiquitin like systems within the context of the whole
organism. Therefore we generate and analyze conditional
knock-out and knock-in models with a special focus on
the ISG15 modification system and Ubiquitin specific proteases.
Interferon stimulated gene 15 (ISG15)
It is well established that posttranslational modification by
ubiquitin serves as a basic mechanism to control a wide
range of cellular functions. Analogous to ubiquitin also
other proteins with structural similarity- so called Ubiquitin
like proteins (UBL) – can be covalently attached to target
proteins and modify their function. Examples are SUMO,
NEDD8 or FAT10. Interferon-stimulated gene 15
(ISG15/UCRP) was the first UBL described and represents
one of the genes most strongly upregulated upon IFN
induction. ISG15 is conjugated to a wide variety of target
proteins and “Isgylation” is mediated by the activity of E1,
E2 and E3 ligases. The conjugation represents a reversible
process and ubiquitin specific protease 18 (USP18/UBP43)
– which was originally described as an ubiquitin deconjugating enzyme – was reported to be an ISG15 specific
isopeptidase. Using knockout animals generated in our
lab, we investigate the function of the ISG15 modification
system. We were able to show that while ISG15 is dispensable for antiviral activity against VSV and LCMV it serves
as a critical molecule in the defence against Influenza and
Herpes infections. The functional role of the interplay
between ISG modification and the ubiquitin system on
the ligase and deconjugation level is currently under investigation.
Ubiquitin specific poteases (USPs)
In analogy to phosphatases in protein phosphorylation
ubiquitination of proteins is counteracted by the activity
of Ubiquitin deconjugating enzymes (DUBs). Although
much progress has been made in characterizing enzymes
that link ubiquitin to proteins, the understanding of deubiquitinating enzymes is just beginning to evolve. The
human genome contains more than 80 different DUBs,
most of which belong to the family of Ubiquitin specific
proteases (USPs).
The Ubiquitin isopeptidase UBPY (USP8) represents a particular interesting member of deubiquitinating enzymes as
the molecule is growth regulated and contains a structural motif for SH3 domain binding. Using conditional mutagenesis we generated mice that allow the time and cell
specific inactivation of UBPY in the context of the whole
organism. We were able to show that lack of UBPy results
in embryonic lethality, whereas its induced inactivation in
adults causes fatal liver failure. The defect is accompanied
by a strong reduction or absence of several growth factor
receptor tyrosine kinases (RTKs) like epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor (cmet) and ERBB3. Consequently embryonic fibroblasts
undergo growth arrest upon induced deletion of UBPy.
UBPy deficient cells exhibit aberrantly enlarged early
endosomes colocalizing with enhanced ubiquitination and
have reduced levels of HRS and STAM2. Collectively our
results demonstrate that UBPy is essential for receptor
tyrosine kinase stability and to maintain proper endosomal
transport in vivo. Currently these mice are used to elucidate the function of UBPY specifically in the brain and
diverse subsets of immune relevant cell types.
Editing functions of deubiquitinating enzymes. Deubiquitinating
enzymes may negatively regulate proteolysis or other signaling functions of ubiquitination such as internalization or altered protein function by removing the ubiquitin chain from the target proteins.
.
Kisser, Agnes (student) **
Hannß, Ronny (student) **/*
Fischer A, Steidl C, Wagner TU, Lang E, Jakob PM, Friedl P,
Knobeloch KP, Gessler M (2007) combined Loss of Hey1 and
HeyL causes congenital heart defects because of impaired
epithelial to mesenchymal transition Circ Res 100: 856-863.
Lenschow DJ, Lai C, Frias-Staheli N, Giannakopoulos NV, Lutz A,
Wolff T, Osiak A, Levine B, Schmidt RE, Garcia-Sastre A, Leib DA,
Pekosz A, Knobeloch KP, Horak I, Whiting Virgin IV H (2007)
IFN-stimulated gene 15 functions as a critical antiviral molecule
against influenza, herpes, and Sindbis viruses PNAS 104: 13711376.
Niendorf S, Oksche A, Kisser A, Löhler J, Prinz M, Schorle H,
Feller S, Lewitzky M, Horak I, Knobeloch KP (2007) Essential
role of ubiquitin-specific protease 8 for receptor tyrosine kinase
stability and endocytic trafficking in vivo Mol Cell Biol 27:
5029-5039.
Prinz M, Schmidt H, Mildner A, Mildner A, Knobeloch KP,
Hanisch U, Raasch J, Merkler D, Detje C, Gutcher I, Mages J,
Lang R, Martin R, Gold R, Becher B; Brück W, Kalinke U (2008)
Distinct and nonredundant in vivo functions of IFNAR on
myeloid cells limit autoimmunity in the central nervous system
Immunity 27: 675-686.
Guerra S, Caceres A, Knobeloch KP, Horak I, Esteban M
(2008) Vaccinia virus E3 protein prevents the antiviral action of
ISG15 PLoS Pathog.: e 1000096
*part of period reported **part time
Molecular Myelopoiesis
Group Leader:
Dirk Carstanjen
A
fter his medical training in internal medicine, the completion of his doctoral thesis at the Charité, and board certification in transfusion medicine and immune-hematology,
Dirk Carstanjen worked as a post-doc in Indianapolis and
Cincinnati, USA, at the laboratory of Dr. David Williams (20002002). The project focused on the function of the small RhoGTPase Rac2 in cells of the innate immune system. At the end of
2002, Dirk Carstanjen joined the group of Ivan Horak at the
department of Molecular Genetics at the FMP, first as a post-doc
and later as leader of the research group Molecular Myelopoiesis.
The research group Molecular Myelopoiesis focuses on
the genetic regulation of bone marrow derived blood cell
development. The biology of myeloid blood cell formation
is a highly complex process governed by a tightly regulated interplay of a network of transcription factors. This
genetically controlled program starts at the most primitive
hematopoietic stem cell, proceeds over several progenitor
stages, and branches into different lineages. It terminates
in mature neutrophilic, eosinophilic or basophilic granulocytes, monocytes, macrophages, mast cells, and other
bone marrow-derived nucleated cells as well as erythrocytes and platelets. A failure of this program due to an
aberrant expression of transcription factors leads to
diverse myeloid diseases as myeloproliferation, myelodysplasia or frank myeloid leukemia, or different immune
defects. The group seeks to advance the understanding of
the program that underlies myeloid development and the
function of terminally differentiated myeloid cells by
analysing the function of several key transcription factors.
Genetic tools for the manipulation of gene expression in
mice are pivotal in our approach. Utilizing gene ablation
techniques, conventional or conditional knock out-mice,
as well as transgenic over-expression with the help of
retroviral gene transfer technology allows us to investigate
the consequences of aberrant gene expression in a physiological environment.
Irf8 – a transcription factor fine tuning the myeloid
development program
Deficiency of Irf8 (Interferon regulatory factor 8 or
Interferon Consensus Sequence Binding Protein) not only
leads to an immunodeficiency due to loss of important
interferon functions but, rather surprisingly, to a myelo90 Signal Transduction/Molecular Genetics
proliferative disorder resembling human chronic myeloid
leukemia. The reason for this phenomenon is yet
unknown. To elucidate this pro-leukemic phenotype we
crossed mice lacking Irf8 with mice haploinsufficient for
Nf1 coding for neurofibromin. A lack of Irf8 and reduced
expression of neurofibromin induces forced myeloproliferation and transplantable myeloid leukemias. This cooper-
A
B
C
Figure 1. Crystal structure of the zinc finger domain of Klf4 in complex with DNA.
The zinc finger motifs are highlighted in blue (motif 1), yellow (motif
2) and green (motif 3), respectively. (A) The overall fold of the Klf4
monomer in complex with a decameric double stranded DNA molecule is presented as a cartoon model. The right model corresponds to
a 90° rotation of the monomer around the x-axis. The N- and C-terminus are marked with N or C. Zinc ions are shown as gray spheres.
(B) Consensus sequences for the zinc finger domains of Sp and Klf
factors and the entire Sp/Klf family (modified after (2)). Bold capital
letters indicate residues that are 100% conserved between all family
members (black), between all Klf proteins (green), or between all Sp
factors (red). Capital residues indicate >90% conservation, lowercase letters >75% conservation. The cysteine and histidines residues
involved in zinc coordination are highlighted in gray. The secondary
structure elements shown above the alignment correspond to murine
Klf4 (this work). Residues involved in specific base contacts are
marked with ▼, additional base interactions with Ñ, and unspecific
DNA phosphate backbone interactions with ○. (C) Schematic representation of protein-DNA interactions. DNA bases that are specifically
recognized by hydrogen bonding to Klf4 side chains are highlighted
in red, other base interactions in gray. Residues marked with * originate from a symmetry related molecule.
Dr. Rosel Blasig
Didier Nana Kouego*/**
Dr. Martina Alken*
Jessica Königsmann (doctoral student)*/**
Maja Milanovic (doctoral student)*
Anita Babic (student)**
Anne Schulze (student)*
Kyungshin, Shin (student)*/**
A
ment of eosinophil granulocytes. Mice lacking Irf8 are
unable to mount eosinophila, the normal immune
response during helminth infection. This is due to an aberrant developmental program in the eosinophil progenitor
and abnormal development of eosinophils from the
eosinophil progenitor in the absence of Irf8.
Klf4
B
(Krueppel like factor 4) is a protein of the Krueppel-like
family and Sp1 family of zinc-finger DNA binding proteins.
No specific role of Klf4 in myelopoiesis has been described
to date. We observed two novel functions of Klf4. First,
lack of Klf4 in mice leads to an absence of Th17 driven
auto-immune diseases. This is due to a lack of instructive
cytokine production in the absence of Klf4. Furthermore,
we discovered that Klf4 is essential for terminal differentiation of a distinct monocyte population. In cooperation
with Anja Schütz (PSPF, MDC) we identified the structural
and functional characteristics of DNA binding of Klf4. We
discovered that Klf4 utilizes only its two C-terminal zinc
fingers for high affinity DNA binding and activation of
transcriptional regulation. This is a novel prototypical
structural example how members of the Sp1/Klf family
regulate transactivation. Nevertheless, the first zinc finger
binds DNA with low affinity and the lack of the entire
zinc-finger domain induces an otherwise cryptic self
renewal activity of Klf4 in hematopoietic cells which is
unravelled upon deletion of the entire zinc-finger domain
STAT5
Figure 2. Lack of Klf4 in the hematopoietic system in mice inhibits
the development of experimental autoimmune encephalitis (EAE).
Mice from each group were immunized MOG35–55 peptide to
induce EAE. Mice were scored daily for the development of neurological symptoms. In some mice, histology was performed after
spinal cords were removed and fixed in 4% buffered formalin. Then,
spinal cords were dissected and embedded in paraffin before staining
with hematoxylin eosin (H&E).
Figure 2A shows the development of the disease. Mice deficient for
Klf4 showed significantly less symptoms of neurological disease.
Figure 2B shows the histology and quantification of infiltration of different leukocytes in inflamed areas in spinal cord sections of mice.
Again, mice deficient for Klf4 showed significantly reduced inflammatory spinal cord lesions as well as reduced infiltration of different
leukocyte subsets.
ation led to the induction of several genetically and phenotypic ally different tumor identities. Interestingly, loss of
Nf1heterozygosity phenotypically was not a prerequisite
for tumor progression but correlated with severe phenotypic and genotypic abnormalities. We went on to show
that Irf8, surprisingly, is required for the normal develop-
STAT5a and b are two crucial transcription factors relaying
signals from cytokine receptors towards the nucleus. In
cooperation with Jörg Rademann we are testing potential
substances that inhibit activation and homodimerization
of STAT5 proteins. These substances might be prototypes
for further pharmaceutical development. We have also
obtained mice where Stat5 can be conditionally deleted
and are testing the function of rationally designed Stat5
protein mutants to affect the function in myeloid cells.
Milanovic M, Terszowski G, Struck D, Liesenfeld O, Carstanjen
D. (2008) IFN consensus sequence binding protein (Icsbp) is
critical for eosinophil development.J Immunol. 181(7): 5045-53.
Koenigsmann J, Rudoph C, Sander S, Kershaw O, Gruber AD,
Bullinger L, Schlegelberger B, Carstanjen D (2009) Nf1
haploinsufficiency and Icsbp deficiency synergize in the
development of leukemias Blood 113(19): 4690-4701.
**part time
reporting period.
Chemical Biology
Peptide Synthesis
Group Leader:
Michael Beyermann
M
ichael Beyermann received both his diploma and
doctoral degree in chemistry from Humboldt-Universität
Berlin in the period from 1971-1979. From 1978, he
worked as research associate at the Institute of Drug Research of
the Academy of Sciences of the GDR. After a period as research
associate at the Department of Chemistry of the University of
Massachusetts (1988/89), Michael Beyermann was appointed
leader of the Peptide Synthesis research group at the FMP.
Combined Recombinant, Enzymatic and Chemical
Synthesis Strategy (CRECS)
SPPS and Native
Chemical Ligation
Thiol-Maleimide Ligation
Recombinant Synthesis
Promiscuous GPCRs can couple to multiple G proteins
thereby activating different intracellular signalling events
through one receptor. The specific activation of one activation pathway only can offer an opportunity for new
drugs with potentially less side-effects. We have demonstrated such an agonist-directed signalling for the
Corticotropin-Releasing Factor (CRF) receptor, which
belongs to the biologically important class B of GPCRs.
We are now trying to elucidate the molecular basis for this
selectivity. CRF receptors are involved in mediating anxiety
and depressive disorders and other stress-associated
pathologies. These receptors are activated by polypeptides
(Urocortins, Sauvagine, Urotensin-I, CRF). We have shown
the existence of two segregated receptor binding sites of
urocortin 1 which are connected by a helical linker. The
92 Chemical Biology
Displacement of 125I-Sauvagine
at rat brain membranes
Displacement of 125I-Sauvagine
at soluble receptor mimic
Binding cpm
Binding %
G protein-coupled receptors (GPCRs) are heptahelical
integral membrane proteins, which perform vital signalling functions in organisms ranging from the transmission of external stimuli such as vision and olfactory perception to internal signal transduction processes. GPCRs
are targets of most of today’s drugs; therefore, considerable interest exists in their structural features, which are
important for the recognition of and activation by the ligands. GPCRs as biologically important proteins are functional only when embedded into a membrane. Therefore,
it is difficult to obtain direct structural information on their
interaction with ligands through spectroscopic methods.
For this reason, we pursue indirect approaches such as
structure-activity relationship studies where we modify the
ligand or receptor structure and observe subsequent
changes of biological function. Besides GPCRs, protein
synthesis is the second main topic of the group. Here, the
intention is to synthesize soluble protein mimics of GPCRs
which can be of use for obtaining structural features of
ligand-receptor interaction and for new receptor ligands
in peptide libraries.
Enzymatic Ligation
log Urocortin M
log Urocortin M
linker is responsible for the relative orientation between
the two binding sites but not for selective G protein activation. We have shown that the signalling selectivity of
the CRF1 receptor for distinct G protein pathways is controlled by an N-terminal signalling domain of urocortin-1,
in that appropriate modifications resulted in signallingselective ligands. Appropriate analogues of urocortin 1
exhibit both full Gs activation and complete inhibition of
Gi coupling. We named this behaviour ago-antagonism
and believe that by segregating functional domains, peptides offer a way for the rational design of signallingselective receptor ligands.
Various findings indicate that the four extracellular receptor domains contribute significantly to ligand binding of
class B GPCRs. Several other research groups have prepared single extracellular receptor domains to study their
Dr. Hartmut Berger*
Dr. Nadja Heinrich
Benoit Briand (doctoral student)*/**
Irene Coin (doctoral student)*/**
Stephan Pritz (doctoral student) */**
Christian Hoppmann (doctoral student) **
Annerose Klose (technical assistant)
Dagmar Krause (technical assistant)
Bernhard Schmikale (technical assistant)
Dagmar Michl (technical assistant)
Angelika Ehrlich (technical assistant)
binding to natural peptide ligands. Like them, we found
considerable affinity of urocortin 1, the natural ligand, for
isolated CRF receptor N-termini, but other natural ligands
like sauvagine did not exhibit any binding to single receptor domains. Therefore, we developed an approach for
the synthesis of protein mimics with non-linear backbone
topology through the so-called Combined Recombinant,
Enzymatic and Chemical Synthesis (CRECS) strategy
(Figure 1). CRECS allows the synthesis of complex protein
mimics, e.g. by expression of the receptor N-terminus in E.
coli, chemical synthesis of linear receptor loop sequences,
which were transformed into the cyclic form via native
chemical ligation, and their subsequent coupling via thiolmaleimide ligation to a template. Finally, the receptor Nterminus was bound to the template-loop construct by an
enzyme-mediated ligation using sortase A. This resulted in
a receptor mimic that binds not only urocortin 1 with high
affinity (Fig. 2) but, and this in contrast to the soluble Nterminus, also sauvagine. However, it does so with
reduced affinity compared with the wild-type receptor.
This formidable synthesis effort necessitates persistent
improvement of synthesis methodology. In the past, this
was accomplished in our group among other things by
developing the depsipeptide method for the synthesis of
“difficult” peptides (strongly competing with M.
Mutter/Lausanne and Y. Kiso/ Japan), contributions to ligation methods, such as a simple method for the preparation of peptide thiol esters (in collaboration with A. BeckSickinger/Leipzig), or the application of new photolytically
cleavable protecting groups in peptide ligation (in collaboration with V. Hagen/FMP), and the application of
Sortase A for the ligation of various compounds, such as
of cell-penetrating peptides with peptide nucleic acids
(joint work with J. Oehlke, FMP). Our methodological
studies are complemented by the ongoing investigation of
applicability of photo-switch elements for light-directed
control of peptide conformation (in collaboration with K.
Rück-Braun/TU Berlin and P. Schmieder/ FMP).
Coin I, Beyermann M, Bienert M (2007) Solid-phase peptide
synthesis: from standard procedures to the synthesis of difficult
sequences. Nat Protoc. 2(12): 3247-56.
Beyermann M, Heinrich N, Fechner K, Furkert J, Zhang W,
Kraetke O, Bienert M, Berger H. (2007) Achieving signalling
selectivity of ligands for the corticotropin-releasing factor type 1
receptor by modifying the agonist’s signalling domain. Br J
Pharmacol; 151(6): 851-9.
Pritz S, Wolf Y, Kraetke O, Klose J, Bienert M, Beyermann
M (2007) Synthesis of biologically active peptide nucleic acidpeptide conjugates by sortase-mediated ligation. J Org Chem
72(10): 3909-12.
Coin I, Beerbaum M, Schmieder P, Bienert M, Beyermann
M (2008) Solid-phase synthesis of a cyclodepsipeptide:
Cotransin. Org Lett 10 (17): 3857-3860.
Pritz S, Kraetke O, Klose A, Klose J, Rothemund S, Fechner
K, Bienert M, Beyermann M (2008) Synthesis of protein mimics with nonlinear backbone topology by a combined recombinant, enzymatic, and chemical synthesis strategy. Angew Chem
Int Ed 47 (19): 3642-3645.
Coin I, Schmieder P, Bienert M, Beyermann M (2008) The
depsipeptide technique applied to peptide segment condensation: Scope and limitations. J Pep Sci 14 (3): 299-306.
Briand B, Beyermann M (2008) {7[bis(carboxymethyl)amino]coumarin-4-yl}methoxycarbonyl derivatives for photorelease of carboxylic acids, alcohols/phenols,
thioalcohols/thiophenols, and amines. Chem Eur J 14 (5): 16211627.
Briand B, Kotzur N, Hagen V, Beyermann M (2008) A new
photolabile carboxyl protecting group for native chemical ligation. Tetrahedron Lett 49 (1): 85-87.
The research group collaborates widely within the FMP, in particular with the following RGs: RG Blasig, Dathe/ Oehlke, Freund,
Hagen, Krause, E., Keller, Klussmann, Kühne, Rademann,
Schülein. Further collaborations include the MDC research
groups Leutz and Scheidereit, at the Freie Universität RG
Multhaup, at the Charité RG Ziegler, and Prof. Neumann’s lab at
the RKI Berlin.
**part time
Chemical Biology 93
Chemical Biology
Peptide-Lipid Interaction/
Peptide Transport
Group Leaders:
Margitta Dathe
Johannes Oehlke
M
argitta Dathe received her Diploma degree in physics from
the Humboldt-Universität Berlin (1974) and her PhD degree
from the Academy of Sciences of the GDR, Berlin (1978).
From 1979-1992 she worked as research associate at the Institute of
Drug Research, followed by a position as team leader of the
Conformational Analysis Group at the FMP (1992-1999). In 1999
she was appointed team leader of the Peptide-Lipid-Interaction/
Peptide Transport research group at the FMP, a position she
shares with Johannes Oehlke.
Johannes Oehlke obtained his PhD-degree in Pharmaceutical
Chemistry at the Universität Leipzig (1975). From 1975-80 he
worked as research associate on glycoside synthesis at the Institut
von Ardenne in Dresden. Since 1980 he has been working at
what was later to become the FMP in the areas of organic chemistry, radioactive labelling, cellular uptake of peptides, oligonucleotide delivery.
The modulation of protein functions, one of the main
goals of our research at the FMP, requires the intracellular
delivery of interacting molecules. Our research focuses on
elucidating the structural requirements of peptides as targeting and uptake-promoting tools for attached
molecular cargos and lipid-based carriers and the application of antimicrobial peptides for the generation of bacteriocidal surfaces and as additives for liquid conservation of
semen of animals.
Intracellular delivery and biological activity of
peptide-tagged oligonucleotides
In order to gain insights into the effects of peptides on
intracellular delivery and bioactivity of peptide nucleic
acids (PNAs), we investigated the cellular uptake and the
antisense activity of various disulfide-bridged PNA-peptide
conjugates. Quantification of the internalized fluoresceinlabelled PNA was performed by means of capillary electrophoresis with laser-induced fluorescence detection. We
studied the biological activity of the conjugates in an assay
that was based on the down regulation of the nociceptin/orphanin FQ receptor in neonatal rat cardiomyocytes and with the “Kole splice-correction-assay”. In
contrast to a commonly held belief, the bioactivity of PNApeptide conjugates was not primarily related to their
membrane penetrating ability. Surprisingly, a high aggregation propensity combined with an enhanced endocytotic uptake proved to be beneficial for the biological activity of PNA-peptide conjugates.
94 Chemical Biology
Peptide-modified liposomal and micellar carriers
Supramolecular structures equipped with cell-targeting
and uptake-mediating peptides have attracted much
attention as drug carriers and diagnostic tools. Based on
the dipalmitoylated sequence (KRKLRKRLLR)2 (P2A2) we
developed small micelles and large vesicles and compared
their uptake into endothelial cells of blood vessels. The
different physical properties of the particles provided the
basis for cell-specific activation of transmembrane transport modes. Whereas the A2 monomer and the liposomes
entered cells non-specifically via different routes, P2A2
micelles were selectively internalized into endothelial cells
of brain capillaries by clathrin-mediated, low-densitylipoprotein-receptor-involving endocytosis (Figure 1 and
2). Thus, the micelles represent promising nanostructures
for targeting the blood-brain barrier. Furthermore, as
monitored by in-vivo magnet resonance imaging (MRI),
peptide-tagged particles containing contrast-generating
material are highly efficient in targeting atherosclerotic
plaques. The nano-sized structures exhibited high ionic
relaxivities and were effectively taken up by macrophages
of early plaques thus improving the signal intensity by a
factor 2 to 3 compared to non-targeted carriers.
Surface-Immobilized Antimicrobial Peptides
Biofilms present a serious threat to human health. We
tested the suitability of antimicrobial peptides for the generation of antimicrobial surfaces. Different strategies were
used for binding of helical antimicrobial peptides (AMPs)
on synthesis resin as model surfaces to analyze the influence of immobilization parameters upon peptide activity.
The length of spacer and the amount of surface located,
target-accessible peptide were identified as critical parameters whereas the chain position of linkage was less
important. Immobilization did not influence the activity
pattern against bacteria and conserved the membrane
permeabilizing mode of peptide action. The analyzed
parameters are relevant for the establishment of a more
general approach to obtaining efficient AMP-loaded biocidal matrices.
Small antimicrobial peptides: application for semen
conservation
Conservation of semen at a high quality is essential for the
success of assisted reproduction of animals. The restricted
applicability of conventional antibiotics in in-vitro fertilization stimulated efforts to investigate AMP as additives in
sperm conservation. Studies of the structural and functional principles of small cyclic R- and W-rich peptides lead
to compounds with a broad antimicrobial activity spectrum. Compared to standard procedures, bacterial con-
Eik Leupold (doctoral student)**
Mojtaba Bagheri (doctoral student)**
Christof Junkes (doctoral student)*/**
Katrin Zimmerling (student)*/**
Jianjun Wu (student)*/**
Heike Nikolenko (technical assistant)
Gabriela Vogelreiter (technical assistant)
b.End3
BAEC
fA2
Pritz S, Pätzel M, Szeimies G, Dathe M, Bienert M (2007)
Synthesis of a chiral amino acid with bicyclo(1.1.1)pentane moiety and its incorporation into linear and cyclic antimicrobial peptides. Org Biomol Chem 5: 1789-1794.
P2fA2-LUVs
Keller S, Böthe M, Bienert M, Dathe M, Blume A (2007) A
Simple Fluorescence-Spectroscopic Membrane Translocation
Assay. Chembiochem. 8: 546-552.
Chen W, Vucic E, Leupold E, Mulder W, Cormode D, Briley-Saebo
K, Barazza A, Fisher E, Dathe M, Fayad Z (2008) Incorporation of
an apoE derived lipopeptide in high density lipoprotein MRI contrast agents for enhanced imaging of macrophages in atherosclerosis. Contr Med Mol Imag 3(6): 233-242.
P2fA2-mics
Figure 1. CLSM images of mouse brain capillary endothelial cells
(b.End3) and bovine aortic endothelial cells (BAEC) exposed for 1h at
37oC to flourecence-labeled (green) peptide monomer (fA2), liposomes (P2fA2-LUVs) and micelles (P2fA2 mics). Endocytotic and nonendocytotic routes mediated the nonspecific cellular uptake of fA2.
The uptake of P2fA2-LUVs is nonspecific and mediated by endocytosis. PfA2 micelles selectively enter brain capillary endothelial cells but
no BAEC.
Leupold E, Nikolenko H, Beyermann M, Dathe M (2008)
Insight into the role of HSPG in the cellular uptake of
apolipoprotein E-derived peptide micelles and liposomes.
Biochim Biophys Act Biomembr 1778: 2781-2789.
Appelt Ch, Wessolowski A, Dathe, M, Schmieder P (2008)
Structures of cyclic, antimicrobial peptides in a membrane-mimicking environment define requirements for activity. J Pep Sci 14:
524-527.
Junkes Ch, Wessolowski A, Farnaud S, Evans WS, Good L,
Bienert M, Dathe M (2008) The interaction of arginine- and
tryptophan-rich cyclic hexapeptides with E. coli. Membranes. J
Pep Sci 14: 535-543.
Castanho M, Dathe M (2008) Biophysics meets membrane
active peptides. J Pep Sci 14: 365-367.
1000
b.End3
BAEC
cell flouresence (a.u.)
800
600
400
200
0
37oC
4oC
Cyto.D
Chlorpr.
Nystat.
Figure 2. FACS analysis of b.End3 cells (blue) and BAEC (grey) exposed to P2fA2 micelles at 37oC, 4oC, and in the presence of endocytotic inhibitors: cytochalasin D, chlorpromacin and nystatin. The results
point to b.End3-selective clathin-mediated endocytosis.
tamination of peptide-preserved semen was found to be
low. The peptides’ synergistic action with Gentamycin
(essential to control Proteus species) allowed a drastic
reduction of the conventional antibiotic. Additionally, several peptides favourably influenced forward motility and
movement linearity of sperms. The results provide a promising basis for the development of a peptide antibioticbased conservation medium for boar sperms.
The group collaborates with the following research groups within the FMP: Peptide Synthesis (Michael Beyermann); Biophysics
of Membrane Proteins (Sandro Keller); Cellular Imaging
(Burkhard Wiesner). Externally, it collaborates with Biosyntan,
Berlin (Rudolph Dölling); the IZW, Berlin (Stephanie Speck); the
“Institut für Fortpflanzung landwirtschaftlicher Nutztiere”,
Schönow (Burkhard Stähr); Freie Universität Berlin, Institute of
Infection Medicine (Oliver Liesenfeld); Freie Universität Berlin,
Institute of Pharmacy (Monika Schäfer Korting); Martin Luther
Universität, Halle (Alfred Blume); Max Delbrück Center for
Molecular Medicine, Berlin (Gerd Wallukat); Mount Sinai School
of Medicine, NY, USA (Willem Mulder, Zahi Fayad); Westminster
University, London, UK, School of Biosciences (Sebastien
Farnaud); University of Tartu, Estonia, Institute of Molecular and
Cellular Biology (Pille Säälik).
**part time
reporting period.
Chemical Biology 95
Chemical Biology
Mass Spectrometry
Group Leader:
Eberhard Krause
E
berhard Krause obtained his diploma degree in physical chemistry from Humboldt Universität Berlin (1975) where he continued to study for his doctoral degree (1982). From 1984-86 he
led a research group “Drug Development” in the pharmaceutical
industry. In 1987, he returned to academia as research associate at
the Institute of Drug Research, Berlin (1987-1991). Since 1992, he has
been working as senior scientist and head of the Mass Spectrometry
group at the FMP.
Mass spectrometry is a key technology in proteome
research. Our research group focuses on the elucidation of
functionally important proteins and their post-translational modifications using electrospray ionisation (ESI) and
matrix-assisted laser desorption/ionisation (MALDI) mass
spectrometry in combination with miniaturized separation
techniques and stable isotope labelling. These methods
allowed us to study protein-protein interactions which
e.g. play important roles in T-cell signalling and the assembly of spliceosomes.
Identification of phosphorylation-mediated ADAP
interaction partners using quantitative mass
spectrometry
The immune adapter protein ADAP (adhesion and
degranulation promoting adapter protein) plays a role in
integrin-dependent migration and adhesion processes as
a consequence of T-cell stimulation. ADAP becomes multiple phosphorylated during T-cell receptor (TCR) or
chemokine receptor stimulation. It is still subject to debate
how individual phosphotyrosines contribute to protein
binding and regulate cellular adhesion. In order to identify phosphorylation-dependent binding of ADAP-Tyr 625
and ADAP-Tyr 595, we have used a peptide pulldown
approach in combination with stable isotope labelling of
amino acids in cell culture (SILAC) and nanoLC-LTQOrbitrap mass spectrometry (Fig. 1). In addition to previously known SH2 domain-based interactions, we identified some novel proteins such as NCK adapter protein 1 or
Ras GTPase activating protein that belong to the larger
TCR proximal signalling complex. Comparing SILAC and
the enzymatic 18O labelling method for protein interaction
studies revealed that the 18O-method can be used as a reliable substitute for the SILAC approach, in particular if
labelling in cell culture is not feasible (Integrated FMP project “A proteomic strategy for the characterization of
phosphorylation-mediated protein-protein interaction”).
96 Chemical Biology
MS/Proteomics in cancer risk assessment
Toxicogenomic and proteomic approaches are widely
studied in order to assess their usefulness for gaining
insights into the mechanisms behind the toxic response.
Our group contributed to a joint project that sought to
demonstrate the usefulness of the 2DE/MALDITOFTOF/proteomics approach for the identification of
early biomarkers of hepatocellular cancer. Results show
that transcriptional and translational profiling should be
used complementary in order to obtain a comprehensive
result of the biochemical changes in tissues induced by
chemical injury. Proteomics data are promising for the
identification of early biomarkers for the detection of carcinogenic effects. (BMBF joint project coordinated by the
Federal Institute for Risk Assessment (BfR), Berlin).
Proteomics of Entamoeba histolytica
Entamoeba histolytica is known for its extraordinary
capacity to destroy human tissues, leading to invasive diseases such as ulcerative colitis or extra-intestinal abscesses. In this study, the proteome of axenically grown
Entamoeba histolytica parasites was explored by twodimensional gel electrophoresis (2-DE)/MS approach. The
proteome analysis has identified a range of E. histolytica
proteins which had not been reported previously to be
expressed in this parasite and thus provides a foundation
for the identification of novel proteins from E. histolytica
which are crucial for the development, survival and pathogenicity of this parasite (cooperation with I. Bruchhaus,
BNI Hamburg).
PRMT1-mediated arginine methylation of PIAS1
Protein methylation is a common post-translational modification which plays a role in STAT signaling. Inactivation
of STAT signaling is carried out by several negative regulators, amongst which the PIAS protein family operates in
the nucleus. However, it is still controversial how arginine
methylation regulates STAT signaling. Using a strategy
that involves in-gel digestion with different proteases and
nanoLC tandem MS, we identified the target arginine in
the PIAS1 protein, which is dimethylated by the protein
arginine methyltransferase PRMT1 (Fig. 2). The knockdown of PRMT1 or PIAS1 enhances the antiproliferative
effect of IFNγ. Our findings identify PRMT1 as a novel and
crucial negative regulator of STAT1 activation that controls
PIAS1-mediated repression by arginine methylation (cooperation with U.M. Bauer Philipps-University, Marburg)
Dr. Michael Schümann
Dr. Karin Lemke
Dr. Tudev Gan-Erdene*
Sabine Lange (doctoral student) */**
Stephanie Lamer (technical assistant) *
Heike Stephanowitz (technical assistant)
Balamurugan T. Varadarajan (student) */**
Jan Gropengiesser (student) */**
Lisa Vögler (student) */**
Baust T, Anitei M, Czupalla C, Parshyna I, Bourel L, Thiele C,
Krause E, Hoflack B (2008) Protein networks supporting AP-3
function in targeting lysosomal membrane proteins. Mol. Biol.
Cell 19: 1942-1951.
Huston E, Lynch MJ, Mohamed A, Collins DM, Hill EV, MacLeod
R, Krause E, Baillie GS, Houslay MD (2008) EPAC and PKA allow
cAMP dual control over DNA-PK nuclear translocation. Proc. Natl.
Acad. Sci. USA 105: 12791-12796.
Figure 1. Principle of ADAP peptide-protein interaction screen
Piotukh K, Kosslick D, Zimmermann J, Krause E, Freund C
(2007) Reversible disulfide bond formation of intracellular protein
domains probed by NMR spectroscopy. Free Radic. Biol. Med. 43:
1263-1270.
Trojan P, Rausch S, Gießl A, Klemm C, Krause E, Pulvermüller A,
Wolfrum U (2008) Light-dependent CK2-mediated phosphorylation of centrins regulates complex formation with visual G-protein. Biochim. Biophys. Acta 1783: 1248-1260.
Tolstrup J, Krause E, Tannich E, Bruchhaus I (2007) Proteomic
analysis of Entamoeba histolytica. Parasitology 134: 289-298.
Welker P, Geist B, Frühauf JH, Salanova M, Groneberg DA,
Krause E, Bachmann S (2007) Role of lipid rafts in membrane
delivery of renal epithelial Na+, K+ -ATPase, thick ascending limb.
Am. J. Physiol. Regul. Integr. Comp. Physiol. 292: 1328-1337.
Internal
Dr. Christian. Freund (regulation of scaffolding proteins by tyrosine phosphorylation, proline-rich sequence recognition, and integrated FMP project “A proteomic strategy for the characterization of phosphorylation-mediated protein-protein interaction”)
Dr. Reiner F. Haseloff (association of claudins)
Prof. Thomas Jentsch (identification of proteins binding to CLC
transporters)
Dr. Wolf-Eberhard Siems (enzymatic degradation of amyloid-ß
peptides)
Dr. Dirk Schwarzer and Dr. Philipp Selenko (integrated FMP
project “Identification and characterization of modificationdependent chromatin binding proteins”)
External
Prof. Bernard Hoflack, TU Dresden (regulation of osteoclast
functions, proteomic analysis of AP-1 and AP-3 coat assemblies)
Figure 2. MS/MS identification of the target arginine in the PIAS1
protein, which is dimethylated by the protein arginine methyltransferase PRMT1. Using extracted ion chromatograms recorded for the
unmethylated peptide at m/z 526.2 and the dimethylated peptide at
m/z 540.3 the methylation degree was determined to be 3-5%.
Prof. Iris Bruchhaus, Bernhard Nocht Institute for Tropical
Medicine, Hamburg (proteomic analysis of Entamoeba histolytica)
Prof. U. Benjamin Kaupp, Center of Advanced European
Studies and Research, Bonn, (characterization of cGMP sensitive
CNGK channels)
Prof. Miles Housley, University of Glasgow, Glasgow, Scotland
(identification of PDE4D3 interacting proteins)
Prof. J. Jankowski, Charité-Universitätsmedizin Berlin (mass
spectrometric characterization of novel angiotensin peptides in
human plasma)
**part time
Prof. Uta-Maria Bauer, Philipps-University of Marburg (protein
arginine methyltranferase 1-mediated arginine methylation of
protein inhibitor of activated STAT1)
Chemical Biology 97
Chemical Biology
Synthetic Organic Biochemistry
Group Leader:
Volker Hagen
V
olker Hagen received both his diploma and PhD-degree in
chemistry from the Humboldt Universität Berlin (1961-1970).
After two years of postdoctoral work at the HU Berlin,
he was appointed leader of a research group at the Institute for
Pharmacological Research of the Pharmaceutical Industry
(1972-1982). He moved on to become team leader at the Institute
for Drug Research of the Academy of the Sciences of the GDR
(1982-1991), where he received his Habilitation in medicinal chemistry in 1990. Since 1992 he has been working as team leader of
the Synthetic Organic Biochemistry research group at the FMP.
The core activity of our group is research in organic chemistry. We aim to design and synthesize new reagents and
tools for the investigation of biological problems. At present, our main goal is the development of so-called caged
compounds. Caged compounds are photolabile inactive
derivatives of biomolecules from which the biologically
active molecules are generated rapidly using UV/Vis or IR
light. Caging and uncaging of biomolecules are very useful techniques for studying the mechanisms and kinetics
of rapid cellular processes and their precise resolution in
time and space.
Our group designs novel caging groups and synthesizes,
photochemically characterizes, and applies caged biomolecules. Over the last two years, we have developed a
series of substituted coumarinylmethyl as well as 2nitrobenzyl moieties as novel photoremovable protecting
groups for the caging of phosphates, carboxylates,
amines, alcohols, phenols, thioalcohols, aldehydes, and
ketones. Some of these novel groups have large longwavelength absorptions (up to 430 nm) and the caged
compounds show high photoefficiencies. Additionally, the
coumarinylmethyl caged derivatives are sensitive to twophoton excitation (740–770 nm) and some compounds
are highly soluble in aqueous buffer. Our newly developed
water-soluble coumarinylmethyl caging group for aldehydes and ketones is far superior to other introduced carbonyl protecting groups.
Using the coumarinylmethyl and nitrobenzyl caging
groups, we synthesized novel caged versions of the vannilloid receptor agonist capsaicin (collaboration with S.
Frings, Heidelberg), of the gene expression activator doxycycline (collaboration with S. Cambridge, Heidelberg), of
98 Chemical Biology
different nucleotides (collaboration with U. B. Kaupp,
Bonn), of L-norepinephrine (collaboration with M. Lohse,
Würzburg), L-cysteine (collaboration with M. Beyermann,
FMP), dopamine, emetine (collaboration with D.
Eisenhardt, FU Berlin), and progesterone (collaboration
with U. B. Kaupp, Bonn).
Our caged progesterone derivatives were of especial interest. We prepared the Bhc-caged progesterone shown in
Fig. 1 and very recently also a strongly improved version of
the caged compound. Caged compounds of progesterone
have not yet been described and caged carbonyl compounds have not been used in biological systems. The
caged progesterones (cyclic ketales) are stable in aqueous
buffer and display dramatically reduced bioactivity. They
can be used to perform concentration-jump experiments
with high temporal and spatial resolution which allow us
to study the mechanisms of rapid nongenomic cellular
events caused by progesterone. The Kaupp group (Bonn)
demonstrated the usefulness of the caged derivatives by
measurement of changes in swimming behavior of single
human sperm caused by progesterone-induced Ca2+ influx
in the sperm flagellum (Fig. 2). The Ca2+ responses evoked
by photolysis of caged progesterone occurred rapidly with
almost no delay. This result strongly suggest that progesterone binds on the extracellular site of the receptor.
In another project (collaboration with M. Beyermann,
FMP) we tried to apply our caging groups to peptide
chemistry. Among others we looked for photoactivatable
SH-protecting groups that allow a wavelength-selective
photocleavage. The aim was to block two SH-groups in a
peptide by different chromophores that are photosensitive
at distinct wavelengths. In principle, we were able to
realise the aim using a coumarinylmethyl caging group
that was photosensitive at 430 nm and a novel nitrobenzyl caging group that was photosensitive at 325 nm. The
wavelength selective photocleavage was also successful
with both protecting groups at the two SH-functions of
the model peptide resact. The selective liberation of thiols
would allow separation of disulfide formation and folding. It should be useful in peptide and protein folding
studies with high temporal and spatial resolution.
Nico Kotzur (doctoral student)**
Funda Kilic (doctoral student)**
Janina Schaal (doctoral student)**
Brigitte Dekowski (technical assistant)
Gilbert D, Funk K, Dekowski B, Lechler R, Keller S, Möhrlen F,
Frings, S, Hagen V (2007) Caged capsaicins – new tools for the
examination of TRPV1 channels in somatosensory neurons
ChemBioChem 8: 89-97.
Biskup C, Kusch J, Schulz E, Nache V, Schwede F, Lehmann F,
Hagen V, Benndorf K (2007) Relating ligand binding to activation gating in CNGA2 channels. Nature 446: 440-443.
Schmidt R, Geißler D, Hagen V, Bendig J (2007) The mechanism of the photocleavage of (coumarin-4-yl)methyl esters. J.
Phys. Chem. A 111: 5768-5774.
Briand B, Beyermann, M (2008) {7-[Bis(carboxymethyl)
amino]coumarin-4-yl}methoxycarbonyl derivatives for
photorelease of carboxylic acids, alcohols/phenols, thioalcohols/thiophenols, and amines. Chem. Eur. J. 14: 1621-1627.
Figure 1. Structure of Bhc-caged progesterone. The compound
serves as source of free progesterone and allows the investigation of
non-genomic progesterone-induced rapid signal processes.
Briand B, Kotzur N, Hagen V, Beyermann M (2008) A new
photolabile carboxyl protecting group for native chemical ligation. Tetrahedron Lett. 49: 85-87.
Nache V, Kusch J, Biskup C, Schulz E, Zimmer T, Hagen V,
Benndorf K (2008) Thermodynamics of activation gating in
olfactory-type cyclic nucleotide-gated (CNGA2) channels.
Biophys. J. 95, 2750-2758.
Y coordinates (µm)
125
B. Wiesner, FMP (Two-photon uncaging)
100
75
M. Beyermann, FMP (Application of photoactivatable protecting
groups to peptide chemistry)
50
P. Wessig, Institute of Chemistry, University Potsdam
(Photochemistry of caged compounds)
R. Schmidt, Institute of Physical and Theoretical Chemistry, J.W.
Goethe-University, Frankfurt/Main (Time-resolved fluorescence
spectroscopy)
25
0
0
25
50
75
100 125
125 150 175 200
X coordinates (µm)
Figure 2. Changes in swimming behavior of a single human
sperm. Trajectory of the head before (green) and after (violet)
photorelease of progesterone.
U. B. Kaupp, Research Center Jülich and caesar Bonn (Cellular
signalling using caged compounds)
K. Benndorf, F. Schiller-University, Jena (Studies of CNG channel
gating kinetics using caged cNMPs)
S. Cambridge, MPI of Neurobiology, Munich and University
Heidelberg (Controlling of gene expression by using caged
compounds)
S. Frings, University Heidelberg (Caged compounds as tools for
studies of ion channels)
D. Eisenhardt, FU Berlin (Caged compounds as tools for studies
in honeybees)
R. Johnson, University of New York, USA (Inhibitors of adenylyl
cyclase)
**part time
Chemical Biology 99
Chemical Biology
Medicinal Chemistry
Group Leader:
Jörg Rademann
J
örg Rademann studied chemistry and biochemistry (1988-1997)
at the universities of Rutgers, USA, and Konstanz, where he
received his diploma and PhD-degree. He spent the period from
1997-1999 as a postdoctoral researcher at the Carlsberg Laboratory in
Copenhagen before returning to Germany in 1999 to become leader
of an independent research group at the Universität Tübingen in the
department of organic chemistry. Here, he obtained his venia legendi
in 2003. He moved to Berlin in 2004 to take up a professorship in
medicinal chemistry at the Freie Universität as well as a position as
leader of the department of Medicinal Chemistry at the FMP. His
awards include the "Innovationspreis Medizinische Chemie" of
the Gesellschaft Deutscher Chemiker (GDCh) and the Deutsche
Pharmazeutische Gesellschaft (DPhG).
Our research group aims at the development of chemical
tools for the validation of potential new drug targets. The
group identifies and optimizes small molecules as specific
biological effectors for studying protein structure and protein function, and as potential starting points for pharmacological intervention. In doing so, we enable the translation of biological knowledge into pharmacologically relevant small molecules.
For this purpose, we develop strategies in the areas of synthetic organic chemistry, library design, bioassays, and
high-throughput screening. Most protein targets of the
group are disease-related enzymes such as proteases and
phosphatases of relevance for clinical indications including
cancer, Alzheimer, tuberculosis, and SARS. Recently, the
targeted proteins have been extended towards receptors
and protein-protein interactions.
One focus of our recent work was the establishment of a
novel assay methodology for fragment-based ligand discovery. We found that starting fragments that contain a
reactive group (e.g. aldehyde) can be used to detect
nucleophilic fragments which undergo a template-assisted ligation reaction by using a fluorescence-based highthroughput screening assay. This “Dynamic Ligation
Screening” approach has been demonstrated for the sensitive detection of inhibitory, ligating fragments which can
be conducted iteratively to turn a peptide ligand into a
heterocycles-based drug-like inhibitor (Angew. Chem.
2008 (figure 1)). DLS was first established for the develop100 Chemical Biology
ment of inhibitors of SARS corona virus main protease, a
putative target of corona virus infection. In a second project DLS has been extended to the selective detection of
cooperatively binding fragments in a fluorescence polarization assay. The thermodynamics of the ligation reaction
on the protein surface have been determined employing a
simulation model. Employing the DLS binding assay picomolar inhibitors of the validated drug target caspase-3
have been developed (Angew. Chem. 2009 (figure 2)).
Meanwhile, the DLS methodology has been extended
towards specificity screening of phosphotyrosine binding
sites including protein tyrosin phosphatases and to the ligand development for protein-protein interaction domains.
Cellular active lead compounds have been developed for
the protein tyrosine phosphatase ptp1b and for the SH2
domain STAT5.
Inhibitor development for protein tyrosine phosphatases
has been focused on the target Shp2 (Proc. Nat. Acad. Sci.
USA, 2008). Based on a virtual screening approach we
have synthesized inhibitors that were active in several cellular metastasis models including the scattering of HGFactivated cells. The first hit compounds have been optimized iteratively in a fragment-based manner in order to
provide a lead for animal testing and protein crystallization.
Results from DLS and other screening activity are extended in several projects for the development of specific, fluorescently labelled and/or photoactivated protein probes
which can be used for cellular studies, for binding assays,
or for screening (ChemBioChem 2008).
In chemical synthesis we aim at developing novel ligation
reactions for the chemical combination of peptides with
heterocylic (drug-like) components. Such reactions will
subsequently be employed for fragment-based screening
and for the synthesis and selection of tailored protein ligands. Initially, we prepared a series of peptide electrophiles (e.g. Org. Lett. 2007) which since then have
been used successfully for dynamic ligation screening
(Angew. 2008 and 2009, in press). Along these lines we
have discovered a novel access to privileged peptide heterocycles which are currently investigated as protein turn
mimetics and inhibitors of protein-protein interactions
(Angew. Chem. 2009). Moreover, the ligation reaction has
been demonstrated to run stereoselectively in a biocompatible fashion without the need of heavy metal catalysts
as required for the current state-of-the-art.
Our central aim for the nearer future is the extension of
our strategies for protein ligand development towards
proteomic applications. In this direction we have initiated
several projects for the cellular labelling of proteins by
Dr. Boo Geun Kim*
Dr. Samuel Beligny*
Dr. Isabel Fernandez-Bachiller *
Marco Schmidt (doctoral student)*/**
Adeeb El-Dahshan (doctoral student) */**
Ahsanullah (doctoral student)*
Richard Bunnag von Briesen Raz
(doctoral student)*/**
Stefanie Grosskopf (doctoral student)*/**
André Horatscheck (doctoral student)*/**
Johannes Preidl (doctoral student) */**
Viviane Uryga-Polowy (doctoral student) */**
Jörn Saupe (doctoral student)*/**
Sina Meyer (doctoral student)*/**
Katharina Koscheck (doctoral student)*/**
Martin Richter (doctoral student)*/**
photoactivated probes which will be employed to identify
cellular interaction partners of our chemical probes.
Samina Nazir (doctoral student)*
Dr. Ludmilla Perepelittchenko
(technical assistant)
Kevin Mallow (technical assistant) *
Stefan Wagner (student) */**
Swantje Behnken (student) */**
Franziska Gottschalk (student) */**
Roland Kersten (student) */*
Kuehne R, Ouellet H, Warrier T, Alteköster M, Lee J,
Rademann J, Oschkinat H, Kaufmann SHE, Waterman MR
(2007) Small-Molecule Scaffolds for CYP51 Inhibitors Identified
by High-Throughput Screening and Defined by X-Ray
Crystallography. Antimicrob Agents Chemother 51: 3915-3923.
El-Dahshan A, Weik S, Rademann J (2007) C-acylations of
polymeric phosphoranylidene acetates for C-terminal variation of
peptide carboxylic acids” Org Lett 9: 949-952
Schmidt M, Isidro-Llobet A, El-Dahshan A, Lisurek M, Tan J,
Hilgenfeld R, Rademann J (2008) Sensitized detection of
inhibitory fragments and iterative development of non-peptidic
protease inhibitors by Dynamic Ligation Screening Angew Chem
120, 3319-3323. Angew Chem Int Ed 47: 3275-3278.
Uryga-Polowy V, Kosslick D, Freund C, Rademann J. (2008)
Resin-bound aminofluorescein for C-terminal labeling of peptides: high-affinity polarization probes binding to polyprolinespecific GYF domains. ChemBioChem 9 (15): 2452-2462.
Hellmuth K, Grosskopf S, Lum CT, Würtele M, Röder N, von
Kries JP, Rosario M, Rademann J, Birchmeier W (2008) Specific
Inhibitors of the Protein Tyrosine Phosphatase Shp2 Identified by
High-throughput Docking Proc Nat Acad Sci, 105: 7275-7280.
Ruttekolk R, Duchardt F, Fischer R, Wiesmüller KH, Rademann J,
Brock R (2008) HPMA as a scaffold for the modular assembly of
functional peptide polymers by native chemical ligation Bioconj
Chem 19: 2081-2087.
Ahsanullah, Schmieder P, Kühne R, Rademann, J (2009)
Metal-free, regioselective triazole ligations deliver locked cis-peptide mimetics, Angew Chem, 121: 5143-5147; Angew Chem Int
Ed 48: 5042-5045.
Figure 1. Dynamic Ligation Screening enables very sensitive
detection of molecular fragments binding to specific protein
sites. Ligation products are compete e.g. with substrate conversion, potent fragments display
Schmidt M, El-Dahshan A, Keller S, Rademann J (2009)
Selective identification of cooperatively binding fragments in a
high-throughput ligation assay enables the evolution of a picomolar caspase-3 inhibitor”, Angew Chem in press.
Schmidt M, Rademann J (2009) Dynamic, template-assisted
strategies in fragment-based drug discovery, Trends Biotechn.,
121: doi 10.1002/ange.200901647121, Angew Chem Int Ed 48:
doi 10.1002/anie.200901647.
Figure 2. Peptide conformations can be controlled via a novel,
metal-free and regioselective triazole ligation.
Internal: R Kühne, P Schmieder, H Oschkinat, C. Freund, E.
Klussmann, G. Krause, E. Krause;
Campus: W. Birchmeier, F. Luft (Schreiber, Kettritz), O. Daumke.
National: Robert-Koch Institut, Berlin; MPI Infektionsbiologie,
Berlin; R. Hilgenfeld, Universität Lübeck
**part time
International : F. Albericio (Barcelona, Spanien), K. Stroemgaard
(Kopenhagen, Dänemark), University of Jordan (Jordanien), F.
Delannoy (Lille, Frankreich).
Chemical Biology 101
Chemical Biology
Screening Unit
Group Leader:
Jens Peter von Kries
J
ens Peter von Kries received his diploma in biology in 1987 and
his PhD in 1991 from the University Hospital HamburgEppendorf. After 14 years as a research scientist in the Strätling
Lab in Hamburg, the Birchmeier Lab at the MDC in Berlin-Buch and
as Project Manager of the SFB 366 at the Freie Universität Berlin, in
2001 he moved into industry where he managed and established
the Screening Unit at Semaia Pharmaceuticals Berlin. In 2003 he
established the FMP-Screening Unit, which he has been managing
since.
The Screening Unit has been set up at the FMP as an open
platform for screening projects, particularly for academic
users. The Unit works to establish and improve new high
throughput screening technologies using automated
microscopes, capillary electrophoresis with LabChip mobility shift, and AlphaScreen technology. The group manages
the central compound collection of ChemBioNet by automated Remp storage and is involved in library design projects with the drug design and modelling group of the
FMP. It serves both the FMP and the ChemBioNet collection of about 50.000 compounds. Furthermore the Unit
established automated data acquisition and analysis on
the fly, which enables to process a complete screen within a few minutes generating reports about assay quality,
heat maps and hit lists for effective service. Since its establishment in 2004 the Screening Unit supported over 60
academic projects from Yale (USA), Oxford and
Cambridge (UK), Oslo and Gothenborg as well as from
Max-Planck or other research institutions in Germany.
Inhibitors of Met-receptor induced cell scattering
(cellular metastasis model)
Growth of tumours at distant sites (metastasis) is the
process responsible for over 90% of cancer deaths. The
Screening Unit focused on the inhibition of Met-receptor
induced scattering of tumor cells in vitro as a cellular
model for metastasis. A cellular assay was optimized for
HTS in 384well format using fluorescent staining of DNA,
of actin filaments and cytoplasm. Automated identification of colonies (unscattered, – HGF) and scattered cells (+
HGF) was optimized for High Content Screening
(MolDiaPacra, EU funded, HGFSF, EU funded). Inhibitors
of the phosphatase Shp2, which is a central part of Met
signalling, were profiled for specificity against several
other human phosphatases. The results may help to devel102 Chemical Biology
op novel diagnostic tools for the early detection of pancreatic tumors since Met receptors are specifically overexpressed in pancreatic tumors.
Inhibitors of protein dephosphorylation
Cellular signalling is in part regulated by phosphorylation
(kinases) and dephosphorylation (phosphatases) of proteins. Modification of specific amino acid residues in target proteins by phosphate groups creates or prevents
binding of other proteins or induces structural changes,
which switch between functional modes of proteins.
Beside the kinases, which are established drug targets,
phosphatases are more difficult to inhibit specifically,
because their substrate specificity is often mediated by
association in multiprotein complexes. In a collaboration
with Novartis (NDDI, Boston) and Oxford University we
screened a Novartis library of 44.000 compounds with
two different techniques (DiFMUP in a plate reader plus
capillary electrophoresis in LabChip-3000 with phosphorpeptides) against 5 human phosphatases. The outcome of
the screening has been analyzed and co-crystallization of
proteins with inhibitors has been started.
Mycobacterium tuberculosis enzyme inhibitor
screens
Proteins from Mycobacterium tuberculosis are selected on
the basis of gene ablation experiments (in cooperation
with MPI für Infektionsbiologie, Berlin) and reduced infectiveness of deficient bacterial strains. The structures of
selected proteins are elucidated
at the European Molecular Biology Laboratory (EMBL)
Hamburg and screened for small molecule inhibitors at
the Screening Unit in Berlin and by Combinature
(NMRscreening, Berlin).
Since autumn 2004, the Screening Unit has performed 60
primary screens for small molecule inhibitors of protein
interactions or enzyme functions. The projects included
assay set up for high-throughput screening (HTS) of the
20,000 compounds of the FMP fragment library and
about 21,000 compounds of the ChemBioNet collection,
hit analysis by structural similarity clustering, and validation of concentration-dependent mode of action. For
selected projects, virtual screens and models for binding
of inhibitors were developed by the computational
chemist of the Unit. Target proteins from M. tuberculosis,
Trypanosoma bruzei and cruzi, and human enzymes for
specificity profiling of bioactive compounds in collaboration with Oxford University, University of California (UCSF,
San Francisco) and Vanderbilt University (Nashville) have
Dr. Simone Gräber**
Dr. Michael Lisurek*
Dr. Martin Neuenschwander*
Carola Seyffarth (technical assistant)*
Franziska Hinterleitner (technical assistant)*
Christoph Erdmann (technical assistant)
Angelika Ehrlich (technical assistant)
SF + Inhibitor
Andreas Oder (technical assistant)*
Chris Eckert (technical assistant)*
Katta Kirankumar (student)*/**
SF – Inhibitor
Konkle ME, Hargrove TY, Kleshchenko YY, von Kries JP,
Ridenour W, Uddin MdJ, Caprioli RM, Marnett LJ, Nes WD,
Villalta F, Waterman MR and Lepesheva GI (2009) Indomethacin
Amides as a Novel Molecular Scaffold for Targeting Trypanosoma
cruzi Sterol 14 -Demethylase. J Med Chem, accepted.
Cellular metastasis model with MDCK cells
Cells are stained for actin filaments (red), for nuclei (green) and
for cytoplasm (blue). Cellular colonies in 384well plates (SF=
scatter factor) are presented in presence of inhibitor and scatter
factor, while in absence of inhibitor the cells get motile and
detach from colonies.
Automated microscope in laminar
flow served by an
automated pipetting
system for plate
preparation.
Nasser Eddine A, von Kries JP, Podust MV, Warrier T, Kaufmann
SH, Podust LM (2008) X-ray structure of 4,4’-dihydroxybenzophenone mimicking sterol substrate in the active site of sterol
14alpha -demethylase (CYP51). J Biol Chem. 2008 Mar 26,
doi:10.1074/jbc.M801145200
Hellmuth K, Grosskopf S, Lum CT, Würtele M, Röder N, von
Kries JP, Rosario M, Rademann J and Birchmeier W (2008)
Specific Inhibitors of the Protein Tyrosine Phosphatase Shp2
Identified by High-throughput Docking PNAS 2008
Kuehne R, Ouellet H, Warrier T, Alteköster M, Lee JS,
Rademann J, Oschkinat H, Kaufmann SH, Waterman MR
(2007) Small-molecule scaffolds for CYP51 inhibitors identified
by high-throughput screening and defined by X-ray crystallography. Antimicrob Agents Chemother 51(11): 3915-23.
been used for identification of small molecule modulators
of function.
A universal step in the biosynthesis of membrane sterols
and steroid hormones is the oxidative removal of the 14
alpha-methyl group from sterol precursors by sterol 14
alpha – demethylase (CYP51). This enzyme is a primary
target in the treatment of fungal infections in humans to
plants. The development of more potent and selective
CYP51 inhibitors is an important biological objective. The
complex structure of CYP51 from Mycobacterium tuberculosis (CYP51Mt) was determined with 4,4’-dihydroxybenzophenone (DHBP), a small organic molecule identified among top type I binding hits in a FMP library. The
newly determined CYP51Mt-DHBP structure is the most
complete to date and is an improved template for 3-D
modeling of CYP51 enzymes from fungal and prokaryotic
pathogens. The structure demonstrates the induction of
conformational fit of the flexible protein regions, and the
interactions of conserved amino acids essential for both
fungal drug resistance and catalytic function, which were
obscure in the previously characterized CYP51Mt-estriol
complex. Furthermore the inhibition of M. tuberculosis
growth by DHBP in a mouse macrophage model was
demonstrated.
The Screening Unit is the only open access screening platform of
the German Chemical Biology Network (ChemBioNet). Research
teams from all over Europa and Germany were provided assistance with assay development and HTS. Cooperations funded by
EU (MolDiaPacra, HGFSF), non funded cooperations with
University of Oslo, Umea University (Sweden) or national BMBF
funded consortia (NGFNplus; MHC-MLE; ECRC) are representative of the wide ranging service. The Unit provides screens for
companies like Jerini AG (Berlin) and DNAacos (Oslo). It also
screened for several HTS projects in the BMBF funded GoBioprogram.
**part time
Chemical Biology
Biophysics of Membrane Proteins
Group Leader:
Sandro Keller
S
andro Keller received his diploma in biophysics, biochemistry,
molecular biology, and cell biology from the University of Basel
in 2002 and his PhD in physical chemistry from the Martin
Luther University Halle-Wittenberg in 2006. Following his doctoral
studies, he was appointed leader of an independent junior research
group Biophysics of Membrane Proteins at the FMP. His awards
include the Friedrich Weygand Prize by the Max Bergmann Circle and
the Luther Certificate by the Martin Luther University HalleWittenberg. His main research interests are the physical-chemical principles underlying folding, protein-protein interactions, and ligand
binding of membrane proteins, with an emphasis on calorimetric and
spectroscopic methods.
Membrane proteins constitute about 30% of all proteins
encoded in the human genome and more than 50% of all
drug targets. Bundles of α-helical transmembrane
domains form the structural basis of the vast majority of
membrane proteins, including membrane channels and
receptors as well as G-protein-coupled receptors, which
are of particular pharmacological relevance. In spite of the
resulting enormous interest in α-helical membrane proteins, only little insight has been gained into their structural and biophysical properties. On the one hand, membrane proteins are notoriously difficult to produce in sufficient quantity and to reconstitute in a functional way. On
the other hand, their pronounced hydrophobicity makes
them hard to handle and precludes application of many
techniques developed for water-soluble proteins.
We therefore aim:
1. To understand the thermodynamic forces that govern
protein folding and protein–protein interactions in
lipid membranes,
2. To use this knowledge to optimize the recombinant
production of membrane proteins,
3. To reconstitute recombinant membrane proteins in
lipid membranes and to scrutinize the influence of
detergents and lipids on protein function,
4. To characterize reconstituted membrane proteins
biophysically and structurally, in particular with
respect to transmembrane transport and ligand
binding,
5. To explore how signals can be transmitted between
two proteins across a lipid membrane without direct
protein–protein interactions.
104 Chemical Biology
The question of how an unordered polypeptide chain
assumes its native conformation continues to be one of
the greatest challenges in molecular biophysics and cell
biology. This so-called protein folding problem not only is
of outstanding scientific interest but is also of immediate
practical relevance for the production and reconstitution
of proteins as well as for tackling numerous diseases that
are the result of protein misfolding. In vitro protein folding studies which use chemical denaturants have contributed tremendously to our understanding of water-soluble proteins. However, fully reversible denaturantinduced unfolding remains limited to a few β-barrel
porins, whereas α-helical membrane proteins have thus
far evaded this approach. We have recently discovered the
first α-helical membrane protein that can be unfolded
completely and reversibly by a chemical denaturant:
Mistic, a 110-residue protein from Bacillus subtilis, dissociates from detergent micelles or lipid vesicles and
assumes an unfolded monomeric state on titration with
urea. Using spectroscopic and microcalorimetric techniques, we exploited this unique property to provide (i) a
quantitative comparison of membrane protein stability in
different membrane-mimetic systems; (ii) an experimental
test of controversial predictions as regards the folding core
of Mistic; and (iii) a convenient setup to study the spontaneous, translocon-independent membrane insertion of
this unusual membrane protein.
A second line of research focuses on the third transmembrane helix of bacteriorhodopsin. This peptide, also
known as pHLIP, is a unique model system for studying the
interactions of a natural transmembrane domain with
lipid membranes: depending on pH, the water-soluble
peptide either adsorbs superficially or inserts as a transmembrane helix when lipid vesicles are added. Published
values for the free energies of these processes are based
on a stoichiometric model invoking two distinct sets of
binding sites. However, discrepancies between data
obtained from different experimental techniques and
inconsistencies between experimental and expected temperature dependencies cast doubt on these values. We
therefore reassessed membrane interactions of pHLIP
using titration calorimetry and fluorescence spectroscopy.
If electrostatic effects at the membrane surface are taken
into account, the data can be described quantitatively by
a partition equilibrium, but not by a stoichiometric binding model. The thermodynamics of membrane partitioning differ substantially from published values and draw a
different picture of peptide-lipid interactions. While deepening our insights into the first step of the two-stage
Dr. Oxana Krylova */**
Natalie Bordag (doctoral student)**
Jana Bröcker (doctoral student) **
Nadin Jahnke (doctoral student) */**
Sebastian Fiedler (doctoral student) */**
Monika Georgi (technical assistant)
Gerdi Hölzl (student)**
Anja Sieber (student)**
Elisabeth Fischermeier (student) */**
Georg Krainer (student) */**
model of membrane protein folding, this also sheds light
on the ability of pHLIP to drag cargo molecules across lipid
membranes.
Tsamaloukas A, Keller S, Heerklotz H (2007) Uptake and release
protocol for assessing membrane binding and permeation by
way of isothermal titration calorimetry. Nat Protoc 2: 695–704.
Keller S, Böthe M, Bienert M, Dathe M, Blume A (2007) A
simple fluorescence-spectroscopic membrane translocation assay.
ChemBioChem 8: 546–552.
Interfacial α-helix
Portwich M, Keller S, Strauss HM, Mahrenholz CC, Kramer A,
Kretzschmar I, Volkmer R (2007) A network of coiled-coil associations derived from synthetic GCN4 leucine zipper arrays.
Angew Chem Int Ed 46: 1654–1657.
Figure 1. Spontaneous membrane insertion of a transmembrane
domain.
Gilbert D, Funk K, Lechler R, Keller S, Möhrlen F, Frings S,
Hagen V (2007) Caged capsaicins: new tools for the examination of TRPV1 channels in somatosensory neurons.
ChemBioChem 8: 89–97.
Heerklotz H, Tsamaloukas A, Keller S (2009) Monitoring detergent-mediated solubilization and reconstitution of lipid membranes by isothermal titration calorimetry. Nat Protoc 4:
686–697.
A
150
Jacso T, Grote M, Daus ML, Schmieder P, Keller S, Schneider
E, Reif B (2009) The periplasmic loop P2 of the MalF subunit of
the maltose ATP binding cassette transporter is sufficient to bind
the maltose binding protein MalE. Biochemistry 48: 2216–2225.
100
∆p(µJ mol-1)
Ein mithilfe synthetischer GCN4-Leucinzipperarrays aufgedecktes
Coiled-Coil-Assoziationsnetzwerk. Angew Chem 119: 1682–
1686.
50
0
-50
-100
0
4
8
12
16
20
internal
B
Michael Beyermann, Michael Bienert, Margitta Dathe, Anne
Diehl, Christian Freund, Volker Hagen, Enno Klussmann,
Eberhard Krause, Ronald Kühne, Hartmut Oschkinat, Jörg
Rademann, Bernd Reif, Peter Schmieder
Q(kj mol-1)
10
5
external
0
-5
0
10
20
30
40
Figure 2. Monitoring membrane solubilization by way of isothermal
titration calorimetry.
Udo Heinemann (MDC), Ingo Morano (MDC), Volker Haucke
(FU), Rudolf Volkmer (Charité), Hendrik Fuchs (Charité), Andreas
Herrmann (HU), Erwin Schneider (HU), Jörg Fanghänel (Bayer
Schering Pharma AG, Berlin), Holger Strauss (Nanolytics GmbH,
Potsdam), Dirk Labudde (FH Mittweida), Anne Ulrich (Karlsruhe
Institute of Technology), Kai Hilpert (Karlsruhe Institute of
Technology), Peter Pohl (Johannes Kepler Uni, Linz, Austria), Elsa
Bárány-Wallje (Stockholm Uni, Sweden), Heiko Heerklotz (Uni
Toronto, Canada), Alekos Tsamaloukas (Rensselaer Polytechnic
Institute, Troy, USA)
**part time
Chemical Biology
Protein Chemistry
Group Leader:
Dirk Schwarzer
D
irk Schwarzer studied chemistry at the Philipps-Universität in
Marburg and completed his PhD in 2002 with Mohamed A.
Marahiel. After postdoctoral research with Philip A. Cole at
the Johns-Hopkins University, he moved to the group of Henning D.
Mootz at the Universität Dortmund in 2006. Since 2007 he is the
head of am Emmy-Noether Group at the Leibniz-Insititut für
Molekulare Pharmakologie (FMP) in Berlin.
Our group is interested in developing and utilizing chemical tools to study the physiological function of posttranslational protein modifications. A central goal is the development of probes which can be used to monitor the
dynamics of protein modifications or serves as baits to
trap, isolate and identify binding or modifying proteins.
We are also engaged in developing and applying methods
for the site-specific installation of protein modifications.
Protein phosphorylation
Phosphorylation is the most frequently found modification
of proteins. It is a common means for regulating the activity of enzymes, to stabilize or destabilize the affected proteins in the cellular context or to recruit proteins to specific locations within the cell. In the latter case dedicated
binding proteins recognize the phosphorylation site and
form a complex with the phosphoryted protein. This complex formation can be used to isolate the phosphorylbinding proteins from cellular lysates with suitable phosphorylated baits. However, protein phosphorylation is
intrinsically unstable in cellular lysates due to the presence
of protein phosphatases. To overcome these shortcomings
we use phosphonate homologues of phosphorylated
amino acids. Here, the linking oxygen is replaced by a
methylene group. These non-hydrolyzable phosphoamino acids are incorporated into peptide-based proteomic probes at known phosphorylation sites. We use a
combination of pull-down assays, Western Blotting and
mass spectrometry (in collaboration with Eberhard Krause)
to identify the dedicated binding proteins. In the following biochemical studies are conducted in order to confirm
and study the biological function of the identified phosphoryl-binding proteins.
106 Chemical Biology
Probes to detect the dynamics of protein
modifications.
In collaboration with the group of Philipp Selenko (In-cell
NMR) we develop and test methods to detect the dynamics of protein modifications by NMR-spectroscopy. The initial focus lies on lysine acetylation which represents another common modification of proteins. Lysine acetylation is
introduced by acetyl transferases and can be found in several important mammalian proteins including the regulatory p53 protein, alpha-tubulin, or histones. We use
chemical synthesis to incorporate stabile isotopic labels
into acetylated or non-acetylated lysine residues and
employ NMR-spectroscopy to monitor the presence or
absence of this modification.
Site-specific incorporation of lysine modifications in
histone proteins
Homogenously modified proteins are an important prerequisite in order to study the physiological function of
the modification. In theory, such modified proteins are
accessible through in vitro modifications with highly specific modifying enzymes. However, this course of action is
often not feasible because the corresponding enzymes are
unknown, inactive, or unspecific in in vitro assays. We use
an alternative approach to selectively install mimics of
modified lysine residues by chemical alkylation of cysteines. In this regard the unique chemical properties of the
thiol groups can be exploited. The process begins with
mutating the lysine residue of interest to cysteine. In the
following the thiol is treated with a chemically synthesized
alkylation reagent which converts the cysteine to modified
thialysine, a structural mimic of lysines. We plan to use
this approach to incorporate modified or labelled lysine
mimics into recombinant proteins and use them for structural and functional investigations.
Oliver Krätke*
Rebecca Klingberg (doctoral student)*/**
Jan-Oliver Jost (doctoral student)*/**
Alexander Dose (doctoral student)*/**
Bernhard Geltinger (technical assistant)*
Hackenberger CPR, Schwarzer D (2008) Chemoselective
Ligation and Modification Strategies of Peptides and Proteins.
Angew. Chem 120, 10182-10228; Angew. Chem. Int. Ed 47:
10030-10074.
Ludwig C, Schwarzer D, Mootz HD (2008) Interaction studies
and alanine scanning analysis of a semi-synthetic split intein
reveal thiazoline ring formation from an intermediate of the
protein splicing reaction, J Biol Chem accepted.
Schwarzer D (2008) Histone Acetylation, Encyclopedia of
Molecular Pharmacology, 2nd Ed., Springer Berlin Heidelberg
592-595.
Schwarzer D, Mootz HD (2007) Semisynthese von Proteinen,
Trendberichte Biochemie und Molekularbiologie 2006,
Nachrichten aus der Chemie 55: 276-279.
We collaborate with the groups In-Cell NMR headed by Philipp
Selenko and Mass Spectrometry lead by Eberhard Krause in the
above mentioned projects. In addition we collaborate with
Michael Beyermann (Peptide Synthesis) and Christian Freund
(Protein Design).
**part time
Scientific Service
Microdialysis
Group Leader:
Regina Richter
R
egina M. Richter received a diploma in biology and biophysics
at the Humboldt-University, Berlin and a PhD in neuropharmacology/electrophysiology at the Academy of Sciences of
the GDR (1977). In 1988 she received the Specialist Certificate in
Physiology at the Academy of Advanced Medical Training, Berlin.
From 1977-1993 she worked as head of the research group
“Electrophysiology” at the Institute for Drug Research. In 1990 she
joined the Department of Pharmacology, University of Heidelberg
and from 1993-1998 the Division of Neuropharmacology at The
Scripps Research Institute, La Jolla (USA) as research associate. At the
same time until present she held the position of head of group of
Microdialysis at the FMP.
Our group is interested in the mechanism of in vivo processing of brain neuropeptides related to cardiovascular
dysfunction and neurodegenerative processes such as
Alzheimer’s disease (AD). Our interests focus on strategies
leading to reduced levels of cerebral amyloid-β peptides
(Aβ), the major component of AD-related plaques. In
addition, studies are under way that explore the biological
activity of selected Aβ fragments. We use reverse microdialysis in combination with advanced mass spectrometric
techniques to study the clearance of these neuropeptides
in the brain of conscious rats and mice close to real-time.
To address specific questions of peptide processing, experiments with transgenic and gene-targeted mouse models
have been performed.
In vivo processing of Aβ- role of the major proteases
NEP and IDE
Previous studies revealed that proteolytic degradation of
Aβ is a key regulator of cerebral Aβ levels and AD pathogenesis. There is evidence that two metalloprotease family members, neutral endopeptidase (NEP) and insulindegrading enzyme (IDE) participate substantially in the
extracellular metabolism of Aβ. We identified a primary
cleavage site at positions 33/34 (Gly-Leu) for NEP, followed by a N-terminal directed ladder-like degradation in
the hippocampus of rats and mice.
Current studies focus on long-term treatment with protease inhibitors in combination with cerebral microdialysis.
To this end, we established a novel animal model: subcutaneously implanted Alzet osmotic minipumps that permit
the infusion of protease inhibitors into the hippocampus
108 Scientific Service
of freely moving mice for 2 to 4 weeks – contralateral
inserted to the microdialysis probe. Afterwards, the generation of Aβ fragments was successfully blocked by infusion of the specific NEP inhibitor thiorphan. The monitored cleavage pattern resemble those of NEP-knockout
mice.
Impact of β2-adrenergic receptor stimulation on the
processing of A
Several studies focus on the interplay of β2-adrenergic
receptor (β2-AR) stimulation and g-secretase activity leading to accelerated Aβ deposition (Ni et al., Nat Med 2006;
12:1390-6). In collaboration with G. Wallukat we tested
the hypothesis that β2-AR stimulation by clenbuterol may
have a stimulating effect on the processing of Aβ and the
cleavage pattern in the rat brain. These experiments provided evidence for increased cleavage activity and the
generation of specific Aβ cleavage products. Previously we
have shown that Aβ(1-42) prevent the desensitization of
the adrenergic signal cascade to β2- agonists in vitro. For
that purpose the beating rate of spontaneously beating
cultivated rat cardiomyocytes was estimated after incubation with Aβ (1-40/42), distinct C- and N-terminal Aβ fragments and the internal fragments (25-35) and (10-37).
These studies revealed potential activity of some fragments to prevent the b2-AR desensitization normally seen
for b2-adrenergic agonist clenbuterol. The lack of desensitization of the b2-AR caused permanent stimulation of
the receptor mediated signal cascade and might play a
role in apoptose induction.
Moreover, we investigated the long-term effect of Aβ(142), Aβ(1-40), Aβ(1-14) and the fragment (10-37).
Interestingly, while Aβ(1-42), Aβ(1-40) and Aβ(1-14)
diminished the pulsation rate of rat cardiomyocytes after
long-term incubation, the fragment (10-37) induced positive chronotropic effects (Figure 1). Negative chronotropic effects are induced via a proteinkinase C mediated
pathway and may play a role in the induction of toxic
properties of the amyloids and their fragments. The
potential cytotoxicity of Aβ fragments compared to the
parent molecules is currently determined using the MTT
assay in cell culture studies (in collaboration with R.
Schliebs).
Metabolic pathways and quantification of
angiotensin peptides
Analysis of the metabolic pathways of angiotensin I (Ang
I) revealed the formation of numerous bioactive
angiotensin peptides. In particular, angiotensin (1-7) [Ang-
Oliver Klein (technical assistance)*/**
Nadine Scharek (technical assistance)*/**
Anna Happe-Kramer (technical assistance)*/**
Dr. Holger Berg (technical assistance)*/**
∆PR/15 sec
Aβ(10-37)
Aβ(1-14)
Aβ(1-42)
time (min)
occurred in NEP-knockout as well as in wildtype mice.
These findings suggest a critical role rather for the
enzymes ACE and ACE 2 than for NEP in the formation of
Ang-(1-7). To study both the role of proteases and the
impact of substrate availability in greater detail we used
isotope labeling technique to quantify and separate the
signals using mass spectrometry. To this end we raised the
samples with Val(D8) labeled standards of Ang (1-9), (1-8)
and (1-7) fragments. Preliminary results indicate a time
course as for the concentration of angiotensin fragments
and the impact of ACE inhibitors (Figure 2).
Internal and external collaborations:
F. Fahrenholz, Institute of Biochemistry; Johannes GutenbergUniversity, Mainz
S. Schuchardt, Fraunhofer Institute of Toxicology and Exptl.
Medicine, Hannover
Figure 1. Effect of selected Aβ fragments on the pulsation rate (PR)
of cultivated rat cardiomyocytes. The Aβ molecules evoked positive
chronotropic [Aβ(10-37)] as well as negative chronotropic effects
[Aβ(1-14), Aβ(1-42)].
G. Wallukat, Max Delbrück-Center for Molecular Medicine,
Berlin
R. Schliebs, Paul Flechsig Institute for Brain Research, University
Leipzig
Peptide cinc (pmol/sample)
Ang(1-8)
Ang(1-9)
Ang(1-7)
time (min)
Figure 2. Time course of the concentration of three major angiotensin fragments (pmol/sample; v=2 µl) generated by in vivo processing
of angiotensin I in the hippocampus of mice. The data were estimated in dialysates by labeling the fragments with Val(D8) isotopes in
combination with mass spectrometry.
(1-7)] is thought to mimic and oppose the multiple actions
of angiotensin II. Although several proteases, such as
angiotensin-converting enzyme (ACE), its homologue
ACE2 and NEP, are involved in the formation of Ang-(1-7),
the enzymatic pathways are still not fully understood.
Previously, we have shown that major cleavage products
such as the N-terminal fragments (1-9), (1-8) and (1-7)
**part time
Scientific Service 109
Administrative and Technical Services
Computer Services
Thomas Jahn (Nework administration)
Ingrid Hermann (System administration)
Hans-Werner Pisarz (Service engineer)*
Ingo Breng (Service engineer)*
Alexander Heyne (Student)**
Björn Schümann (Student)** / *
Offices
Andrea Steuer (Department of NMR-supported Structural
Biology)
Marianne Dreißigacker (Department of Peptide,
Chemistry and Biochemistry)
Dr. Dietmar Zimmer (Scientific Coordination, Department
of Physiology and Pathology of Ion Transport)*
Directorate
Prof. Walter Rosenthal (Director until 31.12. 2008)
Prof. Hartmut Oschkinat (Acting Director from 01.01.2009)
Dr. Björn Maul (Scientific coordination, public relations)*
Dr. Almut Caspary (Scientific coordination)*
Dr. Britta Horstmann (Public relations)** / *
Dr. Anne Höner (EU-liasion officer)
Dr. Ronald Frank** / *
Maxine Saborowski (PhD-programme coordinator)**
Dörte Lohrberg (Public relations)** / *
Heidemarie Petschick (Secretary)
Alexandra Kiesling (Secretary)
Coordination Net for Drug Discovery and
Development Berlin-Brandenburg (DDDNet)
Dr. Birgit Oppmann*
Administration
Frank Schilling (Head)
Thomas Ellermann (General Administration)**
Silvia Mauks (Personnel Manager)**
Birgit Ruthenberg (General Administration)**
Christel Otto (General Administration)
Claudia Messing (General Administration)**
Kerstin Brauße (General Administration)**
Mathias Schmidt (General Administration)*
Nadin Herrfurth (General Administration)** / *
Grischa Nikolenko (General Administration)
Gabriele Schumacher (Secretary)
**part time
110
DNA Sequencing Service
Dr. Eberhard Klauschenz**
Barbara Mohs (Technical assistance)** / *
Animal Housing
Dr. Regina Richter (Head)
Eva Lojek (Technical assistance)
Annika Eggert (Technical assistance)**
Julia Hagemeister (Technical assistance)** / *
Safety Officer
Dr. Jens Furkert
Technical Service
Hans-Jürgen Mevert
Roy Moritz
Marco Mussehl
Holger Panzer
Michael Uschner
Stephanie Wendt
Roy Wolschke
Library
Dr. Michael Beyermann
Marianne Dreißigacker
For a full account of all 2007/2008 publications,
grants, collaborations, patents, innovations,
teaching activitiesas well as a series of image films
please refer to the Data CD enclosed at the back of
the report.
Structure of the FMP
Forschungsverbund
Berlin e.V.
Falk Fabich
Acting Director
Hartmut Oschkinat
Staff Council
from 01.01.2009
Structural Biology
Safety Officer
Jens Furkert
Chemical Biology
Administration,
Technical and
Scientific Services
Physiology and Pathology
of Ion Transport
Thomas Jentsch
Peptide Chemistry
& Biochemistry
Michael Bienert
Administration
Frank Schilling
Peptide Synthesis
Michael Beyermann
NMR-supported
Structural Biology
Hartmut Oschkinat
Signal Transduction
Walter Rosenthal
Protein Structure
Hartmut Oschkinat
Protein Trafficking
Ralf Schülein
Molecular
Myelopoiesis
Dirk Carstanjen
Solution NMR
Peter Schmieder
Anchored Signalling
Enno Klußmann
Biochemical
Neurobiology
Wolf-Eberhard Siems
Structural
Bioinformatics
Gerd Krause
Cellular Imaging
Burkhard Wiesner
Molecular Neuroscience
and Biophysics
Andrew Plested
Drug Design
Ronald Kühne
Science Management &
Communication
Almut Caspary
Molecular Cell
Physiology
Ingolf E. Blasig
Peptide Lipid Interaction/
Peptide Transport
Margitta Dathe
Johannes Oehlke
Mass Spectrometry
Eberhard Krause
Synthetic Organic
Biochemistry
Volker Hagen
Solid-State NMR
Bernd Reif
Medicinal Chemistry
Jörg Rademann
Protein Engineering
Christian Freund
Screening Unit
In-Cell NMR
Philipp Selenko
Biophysics of
Membrane Proteins
Sandro Keller
Computer Services
Thomas Jahn
Technical Services
Hans-Jürgen Mevert
Library
Michael Beyermann
Marianne Dreißigacker
Microdialysis Service
Regina Richter
DNA Sequencing Service
Erhard Klauschenz
Protein Chemistry
Dirk Schwarzer
(round corners): sections
departments and independent groups
scientific groups associated with departments or with independent groups
junior research groups (temporary)
Structure of FMP 111
Common Facilities
A 8 Gate House with Café Max and apartments
A 9 Reception
A 13 Life Science Learning Lab; CampusInfoCenter
A 14 Cafeteria
Guesthouses of the MDC
B 54 Hans-Gummel-Guest House
B 61 Kindergarden; Salvadore-Luria-Guest House
Research
Max Delbrück Center for Molecular Medicine (MDC)
C 27 Walter-Friedrich-House
C 31 Max-Delbrück-House
C 83 Max-Delbrück-Communications Center
C 84 Hermann-von-Helmholtz-House
C 87 Timoféeff-Ressovsky-House
C 71
B 63 Research services
B 64
A 10 Library
C 81 Leibniz-Institut für Molekulare Pharmakologie (FMP)
}
Clinical Research
B46-51 Clinical Research
112
Companies
A 15
car mechanics, EZAG, Charles River, WISAG
B 55
Oskar und Cécile Vogt House
BBB Post office, patent lawyer Dr. Baumbach, FILT,
ConGen, E.R.D.E., Höppner, HUMAN, Zell GmbH,
TECAN, Dr. Scherrer, ART-CHEM, Roboklon,
Gressus, Fresenius, 8sens.biognostic, neptuntec
B 64 epo
D16/23 Eckert & Ziegler AG, NEMOD, Eurotope, Glykotope,
BEBIG, Eckert Consult, Isotope Products
D 79
Erwin Negelein House
Glycotope, Isotope Products, celares, imaGenes,
BioTeZ, Bavarian Nordic (House 31.1)
D 80
Otto Warburg House
ALRISE, Silence Therapeutics, Combinature,
PolyPhag Evotec AG
D 82
Karl-Lohmann-House:
Eckert & Ziegler, BEBIG, AJ Innuscreen
D 85
Arnold Graffi House
BBB, I.M.S.M., INvitek, aokin, Biosyntan, L.O.S.,
Clin. Research, rennesens, Prof. Wanker, MerLion,
emp, Akademie der Gesundheit, Geneo
BioTechProducts
Campus Berlin-Buch
A11
A10
A24
Prenzlau
Berliner Ring
Hamburg
A 10 Exit Weißensee
Tegel
Berlin
Tempelhof
A2
Potsdam
Hannover
A12
Schönefeld
A9
A13
Leipzig
Dresden
Frankfurt/Oder
Imprint:
Leibniz-Institut für Molekulare Pharmakologie (FMP)
Campus Berlin-Buch
Robert-Rössle-Str. 10
13125 Berlin
Germany
Phone:
Fax:
e-mail:
0049 30 94793 102
0049 30 94793 109
caspary@fmp-berlin.de
Editorial Board:
Michael Bienert, Thomas Jentsch, Hartmut Oschkinat
Feature Articles, Coordination & Editing:
Russ Hodge
Science Groups:
Coordination & Editing: Almut Caspary
Cover, Design and Layout:
Nicola Graf
Photos:
Maj Britt Hansen
Copyright MDC, von David Ausserhofer (S.10, S.14)
Print:
Colordruck Leimen
113
Index of all FMP staff
A
Agarwal, Vipin .......................................................71
Ahsanullah ...........................................................101
Albert, Gesa ...........................................................73
Alder-Behrens, Nele ................................................73
Alken, Martina .......................................................91
Asami, Sam ............................................................71
Ash, Miriam Rose ...................................................73
B
Babic, Anita ............................................................91
Bagheri, Mojtaba ....................................................95
Ball, Linda ..............................................................62
Becker, Johanna .....................................................63
Beerbaum, Monika .................................................65
Bekei, Beata ...........................................................75
Behnken, Swantje ................................................101
Beligny, Samuel ....................................................101
Bengtsson, Luiza ....................................................86
Berg, Holger .........................................................109
Berger, Hartmut ......................................................93
Bergsdorf, Eun-Yeong .............................................87
Beulshausen, Sven ..................................................79
Beyermann, Michael ................. 53, 56, 92, 110, 111
Bibow, Stefan .........................................................71
Bienert, Michael .....................................58, 111, 113
Billig, Gwendolyn ...................................................87
Blasig, Ingolf ............................................25, 82, 111
Cording, Jimmi .......................................................83
Cremer, Nils ............................................................63
D
Da Costa Gonsalves, Andrey Christian ....................79
Dasari, Muralidhar ..................................................71
Dathe, Margitta ..............................................94, 111
Davies, Victoria Ann ...............................................62
Dekowski, Brigitte ..................................................99
Diehl, Annette ........................................................63
Dietrich, Nils ...........................................................85
Dorn, Matthias .......................................................65
Dose, Alexander ...................................................107
Dowler, Elizabeth ....................................................63
Dreißigacker, Marianne .................................110, 111
E
Eckert, Chris ...................................................23, 103
Eggert, Annika .....................................................110
Ehrlich, Angelika ............................................93, 103
Eichhorst, Jenny .....................................................81
Eilemann, Barbara ............................................31, 83
Eisenmenger, Frank ................................................69
Eisermann, Beate ....................................................79
El-Dahshan, Adeeb ...............................................101
Ellermann, Thomas ...............................................110
Erdmann, Christoph .............................................103
Erdmann, Natalja ....................................................63
Ezerski, Verena .......................................................79
Blasig, Rosel ...........................................................91
Bogum, Jana ..........................................................79
F
Boltzen, Mareike ....................................................79
Fabich, Falk ..........................................................111
Bordag, Natalie ....................................................105
Faelber, Katja ..........................................................71
Borowski, Tordis ...............................................79, 85
Fast, Alexander .......................................................87
Brauße, Kerstin .....................................................110
Fernandez-Bachiller, Isabel ....................................101
Breng, Ingo ..........................................................110
Fidzinski, Pawel ......................................................86
Briand, Benoit ........................................................93
Fiedler, Sebastian ..................................................105
Brito, Rui M. ...........................................................62
Fink, Uwe ...............................................................71
Bröcker, Jana ........................................................105
Fischermeier, Elisabeth ..........................................105
Büssow, Cindy ........................................................73
Frank, Ronald .......................................................110
C
Freund, Christian ......................................43, 72, 111
Freyert, Inga ...........................................................87
Carstanjen, Dirk .............................................90, 111
Friedl, Sabine ..........................................................78
Caspary, Almut .....................................110, 111, 113
Fritzsche, Susanne ..................................................83
Castro Villela, Victor Manuel ..................................83
Führl, Stephanie .....................................................85
Chevelkov, Veniamin ..............................................71
Furkert, Jens .................................................110, 111
Christian, Frank ......................................................79
114 Index
Choudhary-Mohr, Parveen ......................................62
G
Cirauqui, Nuria .......................................................69
Gagell, Corinna ......................................................83
Coin, Irene .............................................................93
Gan-Erdene, Tudev .................................................97
Ge, Feng ................................................................63
Jahn, Thomas ...............................................110, 111
Geelhaar, Andrea ...................................................79
Jentsch, Thomas ...............................35, 86, 111, 113
Gehring, Claudia ....................................................83
Joshi, Mangesh ......................................................71
Geithner, Sabine .....................................................73
Jost, Jan-Oliver ...............................................55, 107
Geltinger, Bernhard ..............................................107
Junkes, Christof ......................................................95
Georgi, Monika ....................................................105
Jurk, Marcel ...........................................................65
Göritz, Petra ...........................................................87
Göttert, Hendrijke ..................................................79
K
Götz, Frank ............................................................78
Kahlich, Bettina ......................................................85
Gomoll, Michael .....................................................79
Kamdem, Nestor ....................................................63
Gottschalk, Franziska ............................................101
Keller, Sandro ...............................................104, 111
Grantcharova, Evelina .............................................79
Kersten, Roland ....................................................101
Gräber, Simone ..............................................15, 103
Kiesling, Alexandra ...............................................110
Gropengiesser, Jan .................................................97
Kilic, Funda ............................................................99
Grosskopf, Stefanie ........................................21, 101
Kim, Boo Geun .....................................................101
Grossmann, Solveig ................................................78
Kirankumar, Katta ................................................103
Grzesik, Paul ..........................................................67
Kisser, Agnes ..........................................................89
Klauschenz, Eberhard ...................................110, 111
H
Klein, Eva ...............................................................65
Haas, Ann-Karin .....................................................67
Klein, Oliver ..........................................................109
Hagemeister, Julia .................................................110
Kleinau, Gunnar .....................................................67
Hagen, Volker ................................................98, 111
Klingberg, Rebecca ...............................................107
Hahn, Janina ..........................................................65
Klose, Annerose ...............................................56, 93
Handel, Lieselotte ...................................................63
Klußmann, Enno ............................................78, 111
Hannß, Ronny ........................................................89
Knobeloch, Klaus-Peter ..........................................88
Happe-Kramer, Anna ............................................109
Knöbel, Vivian ........................................................83
Haseloff, Reiner ......................................................83
Köhler, Christian .....................................................63
Heidenreich, Matthias ............................................87
Körner, Jana ...........................................................62
Heinrich, Nadja .......................................................93
Königsmann, Jessica ...............................................91
Heinze, Matthias ....................................................73
Kofler, Michael .................................................45, 73
Helmbrecht, Tolga ..................................................65
Koschek, Katharina ..............................................101
Hermann, Ingrid ...................................................110
Kosslick, Daniela .....................................................73
Herrfurth, Nadin ...................................................110
Kotzur, Nico ...........................................................99
Herzig, Michaela ....................................................75
Krabben, Ludwig ....................................................62
Heuser, Markus ......................................................73
Krätke, Oliver .......................................................107
Heyne, Alexander .................................................110
Krainer, Georg ......................................................105
Hinterleitner, Franziska .........................................103
Krause, Dagmar .....................................................93
Hiller, Matthias .......................................................63
Krause, Eberhard ......................................43, 96, 111
Holtmann, Jan Hendrik ...........................................63
Krause, Gerd ............................................19, 66, 111
Hölzl, Gerdi ..........................................................105
Krönke, Nicole ........................................................87
Höner, Anne ...................................................79, 110
Krylova, Oxana .....................................................105
Hoppmann, Christian .............................................93
Kuego, Didier Nana ................................................91
Horatscheck, André ..............................................101
Kühne, Ronald .........................................20, 68, 111
Horstmann, Britta .................................................110
Kunert, Britta .........................................................63
Hübel, Stefan .........................................................69
Hundsrucker, Christian ...........................................78
L
Lamer, Stephanie ....................................................97
J
Lange, Sabine ...................................................45, 97
Jabs, Sabrina ..........................................................87
Lange, Sascha ..................................................81, 63
Jacso, Tomas ..........................................................71
Lange, Vivien ..........................................................63
Index 115
Lauterbach, Ina ......................................................87
Neumann, Anita .....................................................79
Lautz, Katja ............................................................81
Neumann, Juliane ...................................................83
Leben, Rainer .........................................................87
Nikolenko, Heike ....................................................95
Lehmann, Roland ...................................................73
Nikolenko, Grischa ...............................................110
Leibrandt, Benjamin ...............................................79
Niquet, Sylvia .........................................................79
Leidert, Martina ......................................................63
Noack, Claudia .......................................................79
Leisle, Lilja ..............................................................87
Novarino, Gaia .......................................................86
Lemke, Karin ..........................................................97
Leupold, Eik ...........................................................95
O
Liebold, Janet .........................................................87
Oder, Andreas ................................................15, 103
Linden, Arne ..........................................................63
Olal, Daniel ............................................................63
Linser, Rasmus ........................................................71
Oehlke, Johannes ...........................................94, 111
Liokatis, Stamatios ..................................................75
Opitz, Robert ..........................................................69
Lisurek, Michael .............................................69, 103
Oppmann, Birgit ...................................................110
Lohrberg, Dörte ..............................................83, 110
Otto, Christel .......................................................110
Lojek, Eva .............................................................110
Oczko, Brunhilde ....................................................81
Lopez del Amo, Juan Miguel ..................................71
Oschkinat, Hartmut ....................6, 62, 110, 111, 113
Lorenz, Dorothea ....................................................81
P
M
Pankow, Kristin ......................................................85
Mainz, Andi ...........................................................71
Panzer, Holger ......................................................110
Mallow, Kevin ......................................................101
Papsdorf, Gisela .....................................................76
Malkewitz, Jürgen ..................................................79
Pareja, Ruth ............................................................87
Markovic, Stefan ....................................................63
Pechstein, Arndt .....................................................63
Marsch, Karola .......................................................63
Perepelittchenko, Ludmilla ....................................101
Mauks, Silvia ........................................................110
Perneczky, Verena ...................................................87
Maul, Björn ..........................................................110
Petrucci, Oskar .......................................................79
Meineke, Bernhard .................................................73
Petschick, Heidemarie ...........................................110
Messing, Claudia ..................................................110
Pfeffer, Carsten ......................................................86
Metzner, Katharina .................................................83
Piehl, Christian .......................................................83
Mevert, Hans-Jürgen ....................................110, 111
Piontek, Jörg ....................................................28, 83
Meyer, Johanna ......................................................63
Piotukh, Kirill ..........................................................73
Meyer, Sina ..........................................................101
Pisarz, Hans-Werner .............................................110
Michl, Dagmar .......................................................93
Plans, Vanessa ........................................................86
Milanovic, Maja ......................................................91
Popara, Viola ..........................................................79
Mohs, Barbara ......................................................110
Preidl, Johannes ...................................................101
Morandi, Federica ..................................................69
Preston, Patricia ......................................................87
Moritz, Roy ..........................................................110
Pritz, Stephan ...................................................56, 93
Motzny, Kathrin ......................................................73
Müller, Sebastian ....................................................67
R
Münzer, Matthias ...................................................85
Rademann, Jörg .....................................15, 100, 111
Mussehl, Marco ....................................................110
Radetzki, Silke ........................................................63
Rehbein, Kristina ....................................................63
116 Index
N
Reif, Bernd .....................................................70, 111
Natho, Kristin .........................................................87
Richter, Martin ......................................................101
Nazir, Samina .......................................................101
Richter, Regina .....................................108, 110, 111
Neagoe, Ioana ........................................................86
Richter, Sandra .......................................................83
Nedvetzky, Pavel .....................................................78
Ridelis-Rivas, Ingrid .................................................76
Neuenschwander, Martin ......................................103
Riemann, Katja .......................................................63
Ringling, Martina ....................................................81
Stephanowitz, Heike ..............................................97
Röben, Marco ........................................................65
Steuer, Andrea .....................................................110
Rohleder, Cathleen .................................................87
Sticht, Jana .............................................................73
Rosenthal, Walter .....................................6, 110, 111
Sun, Xiaoou ...........................................................85
Rückert, Christine ...................................................83
Sylvester, Marc .......................................................73
Rupp, Bernd ............................................................69
Szaszak, Marta .......................................................78
Ruthenberg, Birgit ................................................110
Rutz, Claudia ..........................................................76
S
T
Tabor, Vedrana .......................................................78
Thao, Thi Bich ........................................................63
Saborowski, Maxine .............................................110
Techen, Daniel ........................................................67
Santamaria, Katja ...................................................79
Teichmann, Anke ....................................................81
Saupe, Jörn ..........................................................101
Thiemke, Katharina ................................................73
Schaal, Janina .........................................................99
Thongwichian, Tim .................................................75
Schäfer, Gesa .........................................................79
Tourel, Silvain .........................................................75
Scharek, Nadine ...................................................109
Tröger, Jessica .........................................................79
Schilling, Frank .............................................110, 111
Schillinger, Christian ...............................................67
U
Schläger, Peter ........................................................83
Uryga-Polowy, Viviane ..........................................101
Schlegel, Brigitte ....................................................65
Uschner, Michael ..................................................110
Schlundt, Andreas ..................................................73
Schmidt, Antje .......................................................76
V
Schmidt, Marco ....................................................101
Schmieder, Peter ............................................64, 111
Van Rossum, Barth-Jan ...........................................62
Schmikale, Bernhard ...............................................93
Van Rossum, Marleen .............................................75
Schröder, Leif ...........................................................8
Varadarajan, Balamurugan T. .................................. 97
Schumacher, Gabriele ...........................................110
Vargas, Carolyn ......................................................78
Schülein, Ralf .................................................76, 111
Verzini, Silvia ..........................................................75
Schümann, Björn ..................................................110
Vögler, Lisa .............................................................97
Schümann, Michael ..........................................45, 97
Vogelbein, Susanne ................................................76
Schulz, Katharina ...................................................76
Vogelreiter, Gabriela ...............................................95
Schulze, Anne ........................................................91
Von Bock, Anyess ...................................................87
Schrey, Anna ..........................................................69
Von Briesen Raz, Richard Bunnag .........................101
Schwarzer, Dirk ................................56, 58, 106, 111
Von Kries, Jens Peter ..............................15, 102, 111
Schwiebs, Anja .......................................................85
Seedorff, Sabine .....................................................65
W
Seidler, Patrick ........................................................87
Wagner, Florian ......................................................87
Seja, Patricia ...........................................................87
Wagner, Stefan ....................................................101
Selenko, Philipp ..............................................74, 111
Walter, Juliane ........................................................83
Semprich, Claudia ..................................................83
Wartenberg, Anne ..................................................63
Seyffarth, Carola ..................................................103
Wartosch, Lena ......................................................86
Shin, Kyungshin .....................................................91
Weinert, Stefanie ...................................................86
Sieber, Anja ..........................................................105
Wendt, Stephanie ................................................110
Siems, Wolf-Eberhard .....................................84, 111
Wenzel, Ariane .......................................................83
Silipo, Alba .............................................................62
Werther, Tobias ......................................................62
Skroblin, Philipp .....................................................79
Wernick, Stephanie ................................................87
Spitzmaul, Guillermo ..............................................86
Westendorf, Carolin ...............................................76
Stauber, Tobias .......................................................86
Wichard, Jörg .........................................................69
Steinhagen, Kerstin ................................................71
Wiesner, Burkhard ..........................................80, 111
Index 117
Winkler, Franziska ..................................................67
Winkler, Lars ..........................................................83
Wolkenhauer, Jan ...................................................76
Wolschke, Roy ......................................................110
Worth, Catherine ...................................................67
Wu, Jianjun ............................................................95
Z
Zapke, Janet ...........................................................63
Zhang, Jingjing .......................................................83
Ziegler, Andreas ......................................................63
Zieger, Martin .........................................................65
Zillmann, Silke ........................................................87
Zimmer, Dietmar ............................................86, 110
Zimmerling, Katrin ..................................................95
118 Index
im Forschungsverbund Berlin e.V.
Leibniz-Institut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V.

Research Report 2007 2008

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