Protein conformational gating of
electron and proton transfer in
photosynthetic reaction centres
Martina Matěnová
Supervizor: David Kaftan, PhD
Electron transport in thylakoid
membrane
4000
QA --QB electron transfer rate [s-1]
3500
3000
k  Ae

E
RT
2500
2000
1500
1000
500
0
0
10
20
30
40
Temperature [oC]
50
60
Rate constant measurement
4000
QA--QB electron transfer rate [s -1]
3500
H2O
3000
2500
2000
D2 O
1500
1000
500
0
0
10
20
30
40
Temperature [oC]
50
60
Oxygen evolution
4000
H2O
800
]
600
D2 O
400
3500
QA --QB electron transfer rate [s-1]
1000
1
Oxygen evolution rate [mmol (O2) mg ChlA-1 hour-
1200
3000
2500
2000
1500
1000
200
500
0
0
1
10
100
Light intensity [mmol (photons) m-2 s-1]
1000
0
10
20
30
40
50
60
Temperature [oC]
Major part of oxygen evolution reduction is due to decrease in electron
transport from QA to QB
Protein flexibility
4000
QA --QB electron transfer rate [s-1]
3500
3000
2500
2000
1500
1000
500
0
0
10
20
30
40
50
60
o
Temperature [ C]
Mutation in three aminoacids providing bond between D1 and D2
More termostable phenotype
Molecular dynamics simulation and Force Spectroscopy
Shlyk et al. (2006) Nature 442 (7104), 827-830
Molecular dynamic simulation
H bond dynamics between
peptides D1 and D2 through the
residues that control the
activation barrier for QA QB
electron transfer
D1-S212…D2-G207
D1-S209…D2-V204
D1-G208…D2-C211
Force spectroscopy
MDS verification
100x diluted peptide
6
4
θ (mdeg)
2
0
190
-2
200
210
220
230
-4
-6
-8
-10
λ (nm)
240
250
260
Reconstitution of peptide for FS
Nonionic
detergent
Lipids
MGDG
DDM
SQDG
DGDG
PG
Anionic
detergent
SDS
Phosphatidilglycerol
0,02
0,018
A bsorbance
MGDG
0,016
30 nM
0,014
100 nM
300 nM
0,012
H2O
0,01
0,008
0,006
PG
DGDG
SQDG
0,004
0,002
0
200
300
400
500
λ(nm)
Chloroform:Met-OH:Acetic acid:H2O
90:9:12:2
600
700
800
900
Circular dichroism of reconstituted
peptide
25
6
0.5% DDM
100x diluted
4
20
100nM PG
1% SDS
2
0.1% SDS
15
190
200
210
220
-2
230
240
250
260
θ (mdeg)
θ (mdeg)
0
80°C
10
80°C sono
80°C 0.1%
sds
5
-4
0
190
-6
200
210
220
230
-5
-8
-10
-10
λ (nm)
λ (nm)
240
250
260
CD results
Sample
Fraction of α-helix
100x diluted
3,8
0,05%SDS
94,4
0,1% SDS
96,6
1% SDS
94,8
0,25% DDM
37,7
0,5% DDM
37,9
100nM PG
14,7
80°C
4,3
80°C SDS
95,7
80°C Sono
5,4
1,756g/ml, pathlenght 0,1cm, 30AA, MW=3120
Conclusion
We have studied processes that optimize electron transfer in PS II on
molecular level
Using experimental methods of time resolved chlorophyll fluorescence
and polarimetric measurements of oxygen evolution rates we discovered
that the rate limiting step in PS II electron transfer is the proton
transfer effective already in the S1 Q -A Q B /S 2 Q A Q -B transition.
Using theoretical approaches of MDS we confirmed the existence of H
bond network connecting D helices of D1 and D2 proteins
Direct measurements of the interaction forces between the helices that
would confirm the predictions made by MDS were not conclusive due to
the misfolding of the peptides
We have successfully reconstituted peptides yielding close to 100%
a-helical structure making the force spectroscopy measurements of
peptide interactions on single molecular level possible
Acknowledgement
Alex Dulebo
Martin Lukeš
Jorge Dinamarca Cerda
Milan Durchan
David Kaftan
Vendy Lískovcová a Martin Kozák
Thanks