Bayro, 2011, JACS „Intermolecular structure determination of

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Bayro, 2011, JACS
„Intermolecular structure determination of
amyloid fibrils with
magic-angle spinning and
dynamic nuclear polarization NMR“
Presented by: Daniel Droste
17.10.2013
Seminar Magnetische Resonanz
Outline
1. Principal result
2. Phosphatidylinositol
3. PI and SH3 domain of PI3
4. NMR methods
5. Magic Angle Spinning
6. BASE RFDR
7. Advantages of BASE RFDR
8. Dynamic Nuclear Polarization (DNP)
9. Spectra
10. Summary
11. Applications
12. Outlook
13. Further Literature
Principal result
●
strands of the Src(cellular und sarcoma)homology 3 domain of the
Phosphatidylinositide 3-kinases are parallel
and in register
Published in: Marvin J. Bayro; Galia T. Debelouchina; Matthew T. Eddy; Neil R. Birkett; Catherine E. MacPhee; Melanie Rosay; Werner E. Maas;
Christopher M. Dobson; Robert G. Griffin; J. Am. Chem. Soc. 2011, 133, 13967-13974.
DOI: 10.1021/ja203756x
Copyright © 2011 American Chemical Society
PI and SH3 domain of PI3
Römpp online [Elektronische Ressource] : der effizientere Zugriff auf das
Wissen der Chemie, Stuttgart : Thieme, 2001-
Src(cellular und sarcoma)-homology 3 domain of the Phosphatidylinositide 3-kinase
„Biological Assembly Image for 3I5S
Crystal structure of PI3K SH3
Protein chains are colored from the N-terminal to the C-terminal using a rainbow (spectral) color gradient“,
http://www.rcsb.org/pdb/images/3i5s_bio_r_500.jpg, 12.10.2013
NMR methods
1.BASE RFDR: detection of 13Cα–13Cα contacts
2.comparison of 13Cα–13Cα correlations
3.low-temperature DNP heteronuclear spectroscopy
using mixed 15N/13C
Magic Angle Spinning
●
Abbrev. MAS
●
improves signal quality
●
●
●
removes anisotropic
interactions by
averaging
magic angle θm=54,75°
to magnetic field
(cosθm)²=1/2
http://www.magnet.fsu.edu/education/tutorials/tools/probes/images/probes-mas-angle5.jpg, 15.10.2013
BASE RFDR
●
●
without selection: correlation between aliphatic
nuclei in distance too weak to be observed
band selective radio frequency dipolar
recoupling
Angewandte Chemie International Edition
Volume 48, Issue 31, pages 5708-5710, 27 JUN 2009 DOI: 10.1002/anie.200901520
http://onlinelibrary.wiley.com/doi/10.1002/anie.200901520/full#fig1
„Schematic representation of pulse phases and durations as
a function of pulse pair index for [...] decoupling sequences: (a)
TPPM“
Vinod Chandran, C., Madhu, P. K., Kurur, N. D. & Bräuniger, T.
Swept-frequency two-pulse phase modulation (SWf-TPPM)
sequences with linear sweep profile for heteronuclear decoupling in
solid-state NMR. Magn. Reson. Chem. 46, 944 (2008), Fig. 1.
Advantages of BASE RFDR
1.stable when the experimental conditons are
not perfect
2.low 13C power levels → no depolarization
processes → no heteronuclear interference
3.recoupling by finite pulses
4.fewer unwanted 13Cα(i) −13C′(i – 1)
polarization transfer
5.attenuation of dipolar truncation
Dynamic Nuclear Polarization (DNP)
●
transfer of magnetization from electron on nuclei
●
microwaves
●
use of organic radicals
●
interactions:
–
Cross effect
–
Solid effect
–
Nuclear Overhauser Effect
–
Thermal mixing effect
Thurber, K. R. & Tycko, R. Theory for cross
effect dynamic nuclear polarization under
magic-angle spinning in solid state nuclear
magnetic resonance: The importance of level
crossings. J. Chem. Phys. 137, 084508 (2012).
K=Lysine
G=Glycine
T=Threonine
F=Phenylalanine
Y=Tyrosine
D=Aspartic acid
(a) Subsection of a BASE RFDR spectrum of microcrystalline 2-GB1 showing cross-peaks between Y45Cα and neighboring
nuclei. (b) Internuclear distances in the crystal structure of GB1 (PDB ID 2QMT) corresponding to the cross-peaks observed
between Y45Cα and other 13Cα sites, i.e., within its own strand (T44, D46, and D47), to a strand within the same molecule
(T51 and F52), and to an adjacent strand in a neighboring molecule (K13* and G14*). Asterisks denote residues in an adjacent
protein molecule in the crystal lattice. The spectrum in panel a was recorded with τmix = 24 ms and a total experimental time of
7.5 h.
DOI: 10.1021/ja203756x
Internuclear distances anticipated in parallel β-strands and resolvable 13Cα–13Cα correlations for a given residue in the middle
of three different strands, h, i, k (left), and three identical in-register strands, i, i, i (right). Interstrand correlations in the parallel
in-register case are degenerate with sequential correlations within the strand. Typical internuclear distances are indicated on
the left. Dashed lines of different colors (except for black) indicate the potentially resolved cross-peaks in 13C–13C correlation
spectra.
DOI: 10.1021/ja203756x
M=Methionine
A=Alanine
R=Arganine
D=Aspatic acid
F=Phenylalanine
E=Glutamatic acid
S=Serine
Y=Tyrosine
Section of a BASE RFDR spectrum of amyloid fibrils formed by 2-PI3-SH3. Gray labels indicate sequential 13Cα–13Cα crosspeaks while black labels denote cross-peaks between 13Cα nuclei separated by two residues, with an internuclear distance
corresponding to ∼6.5 Å. Backbone–backbone correlations between sites distant in space, but near in sequence, are readily
observed for several regions of the polypeptide chain. This spectrum was recorded with τmix = 24 ms and a total experimental
time of 5 days.
DOI: 10.1021/ja203756x
D=Aspartic acid
N=Asparagine
Sections of PDSD 13C–13C correlation spectra acquired with a mixing time of 20 ms optimized for one-bond correlations of (a)
U-PI3-SH3 and (b) 2-PI3-SH3, and with a mixing time of 500 ms optimized for long-range correlations in (c) 2-PI3-SH3. The
dotted boxes in panels a and b correspond to the same region as that shown in panel c, in which asterisks identify correlations
between neighboring molecules in a parallel, in-register architecture.
DOI: 10.1021/ja203756x
missing resonances
300K,
750
MHz
carbonyl
aromatic
aliphatic
100K,
400
MHz
DOI: 10.1021/ja203756x
(a) Summary of intermolecular constraints along the PI3-SH3 sequence obtained with the methods described in the text:
Indirect CC (“>”), direct CC (“*”), mixed NC at room temperature (“– “), and mixed NC at 100 K with DNP (“+”). Filled bars
indicate residues in a β-strand conformation while empty bars mark dynamic regions that have not been assigned in the
spectra. (b) Superposition of all intermolecular constraints on a hypothetical model of PI3-SH3 amyloid fibril architecture in
which two β-sheet layers (light gray and dark gray, respectively) are formed by each half of the sequence.
DOI: 10.1021/ja203756x
Summary
-verification of parallel, in-register ß-sheet structure in amyloid
fibrils (emme-loid)
-sample used: PI3-SH3, immunoglobulin protein G
1. BASE RFDR: 2-13C glycerol: 13C-13C contacts between
adjacent strands and neighboring molecules for clearing
structural degeneracy
2. comparison of short- and long-range 13C-13C correlations :
distinguish between intra- and inter residue contact, mutually
exclusive 13C-12C and 12C-13C
3. low temperature DNP: 15N-13C temperature; big advantage
of S/N, quenching of dynamics
Applications
●
●
●
amyloid fibrils may be a cause for Alzheimer's
disease and type 2 diabetes
results at low temperatures of otherwise
missing resonances
possibility for the determination of
supramolecular interactions in general
Outlook
●
position of the turn of the ß-sheets: more
verification
●
more interactions between protein
●
additional constraints:
–
DNP
●
●
higher dimensional
higher field
Further Literature
1. Vinod Chandran, C., Madhu, P. K., Kurur, N. D. & Bräuniger, T. Swept-frequency two-pulse phase
modulation (SWf-TPPM) sequences with linear sweep profile for heteronuclear decoupling in solid-state
NMR. Magn. Reson. Chem. 46, 943–947 (2008).
2. Thurber, K. R. & Tycko, R. Theory for cross effect dynamic nuclear polarization under magic-angle
spinning in solid state nuclear magnetic resonance: The importance of level crossings. J. Chem. Phys.
137, 084508 (2012).
3. Bennett, A. E. et al. Homonuclear radio frequency-driven recoupling in rotating solids. J. Chem. Phys.
108, 9463 (1998).
4. Tycko, R. Symmetry-based constant-time homonuclear dipolar recoupling in solid state NMR. J.
Chem. Phys. 126, 064506 (2007).
5. Bayro, M. J. et al. Intermolecular Structure Determination of Amyloid Fibrils with Magic-Angle Spinning
and Dynamic Nuclear Polarization NMR. J. Am. Chem. Soc. 133, 13967–13974 (2011).
6. Debelouchina, G. T., Platt, G. W., Bayro, M. J., Radford, S. E. & Griffin, R. G. Intermolecular Alignment
in β2-Microglobulin Amyloid Fibrils. J. Am. Chem. Soc. 132, 17077–17079 (2010).
7. Bayro, M. J., Maly, T., Birkett, N. R., Dobson, C. M. & Griffin, R. G. Long-Range Correlations between
Aliphatic 13C Nuclei in Protein MAS NMR Spectroscopy. Angew. Chem. Int. Ed. 48, 5708–5710 (2009).

Bayro, 2011, JACS „Intermolecular structure determination of

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