Computational molecular modelling methods applied for a

Computational molecular modelling methods applied for a

Computational molecular
modelling methods applied for a
study of photosynthetic proteins
Students: Martin Kozák & Vendula Lískovcová
Supervisor: Dr. Michal Kutý
This project has received financal support from the Europian Social
Fund and from goverment of the Czech Republic
The aim of the project
€ Purification
and isolation of
photosynthetical membrane BBY
€ Basic skills in crystalography
€ Structure analysis of photosynthetical
reaction centres (RC) from plants and
bacteria
€ Molecular dynamics on thylakoid lipid
membrane
Puriffication and Isolation of BBY
membrane
€ Introduction:
¾
BBY (Berthold, Babcock and Yocum) membrane –
membrane purificated by BBY protocol.
€ Methods:
¾
¾
¾
¾
¾
¾
Extract from green peas
All steps in dark (only green light) and in 4°C
BBY buffers in different compositions
Put the sample in small black tubes
Store the sample at -80°C
Determinate concentration of chlorophyle in
spectrofotometre
Results:
1 – Molecular-weight marker
(kD)
2, 3 – Purified BBYmembranes of grana
thylakoids from Pisum
sativum
Remarks:
• concentration c= 1. 0 mg
chla/ml
• lane 2 => 5 μl of the sample
• lane 3 => 10 μl of the
sample
Crystalography
€ Introduction:
¾
Growing crystals for protein
structure solution by X-ray
diffraction
€ Methods:
¾
¾
¾
¾
¾
Batch method
Hanging and sitting drop techniques
Dialysis method
Microbatch crystallization under oil
Counter – diffusion technique in
capillaries
Results:
€ Capillary
method
€ Microbatch
method
€ Crash
test
Structure analysis
€ Introduction:
¾ Application of modeling software on selected proteins
(plant and bacterial reaction centres) and thylakoid lipid
bilayer
€ Methods
(modeling software):
¾ Electron density - Coot (Wincoot)
¾ Sequence alignment - ClustalX2
¾ Structure validation – Procheck
¾ Structure visualisation – VMD, DS Viewer and Swiss –
PdbViewer
¾ Molecular dynamics - Gromacs and tools for MD analysis
Experiment electron density maps
€
Source: EDS
server in Uppsala
university
€
PDB code: 3bz1
€ Fitting
biomolecular
structure into the
electron density
€
Software:
WinCoot
Experimental molecular structures
€ Protein
DataBank
€ www.pdb.org
€ Worldwide source of
biomolecular
structures
Photosynthetical proteins from
plants and Bacteria
PhotosystemII’s most importatn part – RC is formed from 2 protein
subunits D1 and D2
Similarity in D1 and L like D2 and M (L and M are parts of BRC)
PSII split water into molecular oxigen and protons and electrons
The basic look of reaction centres, compared together. 1st PSIIRC
2nd BRC
Chlorophyls in RC
Photosystem II RC
Bacterial RC
€
Method: distance measurment
€
Result: distance between Chlorophyls of special pair
in PSIIRC is 0.5Ǻ longer => different optical spectrum
Similarites between PSIIRC and BRC
€
¾ Structural aligment
¾ Align these subunits
using crystal
structures 2GNU and
3BZ1 in VMD
D1 and L
€
D2 and M
Methods:
Result:
¾ Subunits are similar,
D1 RC has a more
complex structure
Sequence alignment
Results:
D1 and L: 54 total 61 mostly
38 partly of 281 (335) (total
alignmented 153)
D2 and M: 69 total 56 mostly
35 partly of 300 (340) (total
alignmented 160)
D1 on Procheck
L on Procheck
Molecular dynamics on lipid bilayers
consisting of DGDG lipids
DGDG = digalactosyldiacylglycerol
One of active lipids in PSII
(other MGDG, SQDG a PG)
Molecular Dynamics of
DGDG lipid membrane in
water solution of NaCl
MD analysis – charts - density
Density of sugar2
1200
1100
1000
Density (kg m\S-3\N)
E (kJ mol\S-1\N)
Total Energy
-130000
-140000
-150000
-160000
-170000
-180000
-190000
-200000
-210000
-220000
-230000
-240000
-250000
-260000
-270000
-280000
-290000
-300000
900
800
700
600
500
400
300
200
100
0
0
50
100
150
0
200
2,5
5
7,5
10
Box (nm)
Time (ps)
75000
70000
65000
60000
55000
50000
45000
40000
35000
30000
25000
20000
15000
10000
5000
0
Water density
1100,0000
1000,0000
Density (kg m\S-3\N)
E (kJ mol\S-1\N)
Kinetic Energy
900,0000
800,0000
700,0000
600,0000
500,0000
400,0000
300,0000
200,0000
100,0000
0
200
Time (ps)
0,0000
0
2,5
5
Box (nm)
7,5
10
Many thanks to:
€ Dr.
Michael Kutý
€ Dr. Ivana Kutá
€ MSc.Ekaterina Sviridova
€ Academic and university centre in Nové
Hrady
This project has received financal support from the Europian Social
Fund and from goverment of the Czech Republic
Thank you for your
attention!!!