Transcript Objectives - Theoretical and Computational Biophysics Group

Structure-Function Relationship
of Retinal Proteins
Structure of Retinal Proteins
C
B
A
D E F G
GPCRs
Retinal proteins or rhodopsins belong to the superfamily of seventransmembrane helical (7TM) proteins. Seven helices, with N-terminus
on the extracellular side and C-terminus on the cytoplasmic side of the
membrane (not necessarily G-protein coupled)
Retinal Proteins -- Rhodopsins
Me
Me
Me
N
H
Me
Me
Me
Me
Me
N
Me
Me
Chromophore
H
• Covalently linked to a lysine
• Usually protonated Schiff base
• all-trans and 11-cis isomers
Bacteriorhodopsin -- bR

The simplest ion pump in biology

The simplest photosynthetic center

The best characterized membrane
protein

Technological applications in molecular
electronics

The first membrane protein with a
known atomic-detail 3D structures
bR role in Bioenergetics
Halobacterium
Salinarum
Light
The Purple Membrane
hn
H+
Cytoplasmic side
[H+]
ADP
ATP
ATP Synthase
Extracellular side
Proton Gradient
[H+]
Schematic proton path in bacteriorhodopsin
Cytoplasmic side H+
Asp 96
Transmambrane
helices
O
O
hv
H
H
O
H
Lys 216
N
H
Water
H
O
H
Transmambrane
helices
Asp 212
O
O
Asp 85
O
O
H+
Extracellular side
H
H
N
N
H
H
N
H
Arg 82
Active Channels Need a
‘Switch’ Mechanism
H+
H+
hn
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
What is the switch in bR? How does it work?
Photocycle of bR
Photo-induced
5 ms
3 ps
1 ms
5 ms
5 ms
40 ms
All intermediates are trapped in low temperature and have been
characterized by vibrational and absorption spectroscopy.
No membrane protein has been studied as
extensively as bR
Photo-induced
5 ms
3 ps
1 ms
5 ms
5 ms
40 ms
All intermediates have also been characterized by X-ray crystallography!
Schematic proton path in bacteriorhodopsin
Cytoplasmic side
Asp 96
Transmambrane
helices
O
O
hv
H
H
O
H
Lys 216
N
H
Water
H
O
Transmambrane
helices
H
Asp 212
O
O
Asp 85
O
O
H
H
N
N
H
H
N
Extracellular side
H
Arg 82
HOOC-D96
BR’s Photocycle
bR568
+ K216
N
H
D85-COO
cytoplasmic
H+
5ms
HOOC-E204
3ps
O645
K603
HOOC-D96
HOOC-D96
+ K216
N
N
H
+
H
K216
D85-COOH
D85-COO
OOC-E204
HOOC-E204
1ms
5ms
OOC-D96
HOOC-D96
H
+
+ K216
N
N
K216
H
D85-COOH
D85-COO
OOC-E204
HOOC-E204
N550
extracellular
L543
5ms
HOOC-D96
40ms
N
light driven proton pump
K216
D85-COOH
OOC-E204
M410
HOOC-D96
BR’s Photocycle
bR568
+ K216
N
H
D85-COO
5ms
HOOC-E204
3ps
O645
K603
HOOC-D96
HOOC-D96
+ K216
N
N
H
+
H
K216
D85-COOH
D85-COO
OOC-E204
HOOC-E204
1ms
5ms
OOC-D96
HOOC-D96
H
+
+ K216
N
N
K216
H
D85-COOH
D85-COO
OOC-E204
HOOC-E204
N550
L543
5ms
HOOC-D96
Conformational Change of Helices
Kuhlbarandt, Nature, 406,569 (2000)
40ms
N
K216
D85-COOH
OOC-E204
M410
Study of bR at three levels
Chromophore
• Analysis of the structure
• Calculation of excited state dynamics
Protein
• Chromophore-protein interaction
• QM-MM calculations
• MD simulation of the photocycle
bR in the purple membrane
Modeling of the protein in lipid
bilayers
Retinoids
Me
Me
Me
N
Me
Me
H
Retinal Schiff base
Membrane, covalently bound, chromophore
H
Me
Me
Me
O
C
O
Retinal
Me
Me
H
Me
Me
Me
O
C
O
Me
Me
Retinoic Acid
Nucleus, receptor site, ligand (no photoactivity)
Unconventioanl chemistry
Me
Me



Me




N
Me
Me
Me
7
H
Me
9
Me
11
13
Me
Me
15
N
H
The necessity of quantum mechanical treatment of the chromophore:
Conjugated p-electronic system, delocalization
The effect of protein matrix on the ligand
QM is expensive – Most of the time, one needs to use models
Effect of Conjugation on pKa (Gas Phase Proton Affinity)
Me
Me
Me
N
Me
Me
270
H
H
260
PA (kcal/mol)
H
250
syn
240
H
H
+
N
PA*
PA*(zpe)
PA**
PA**(zpe)
230
220
210
200
0
2
4
6
8
No. of conjugated double bonds
10
anti
H
n-1
H
Proton Affinity:
PA= EAH-(EA+EH)
Effect of the methyl groups on pKa
Species
6-31G*
6-31G* (zpe)
6-31G**
6-31G** (zpe)
PSB6
260.61
251.64
262.66
253.69
4-met
261.44
252.60
263.54
254.70
5-met
261.03
252.11
263.10
254.18
7-met
261.32
252.40
263.39
254.47
8-met
261.39
252.51
263.49
254.61
12cis-met
262.91
254.04
265.00
256.14
12trans-met
263.41
254.52
265.51
256.63
N-met
260.46
251.03
262.52
253.10
4,8-dimet
262.13
253.38
264.29
255.53 Lys216
Me
12,12-dimet
264.92
256.08
267.09
258.24
N,4,8-trimet
261.76
252.50
263.92
254.66
4,8,12,12-tetramet
266.08
257.46
268.33
259.71
N,4,8,12,12-pentamet
265.31
256.14
267.54
258.38
N-met-retinal Schiff
base
266.45
257.26
268.72
259.52
Me
Me
N
H
Me
Me
B1 C2 B3
B2 C3
N1
+
N16
H
C4 B5
B4 C5
C15
C6
C9
C11
C9
C11
C12
C12
C10
B6 C7
C13
C14
C8
C10
C5
C7
C8
Proton Affinity:
PA= EAH-(EA+EH)
C6
C1
No more room for additional methyl groups on the backbone
C2
What is the effect of isomerization?
hn
Isomerization State and Proton Affinity
255
cc
ct
tc
tt
H
PA (kcal/mol)
H
245
syn
H
H
+
N
anti
235
H
n-1
H
225
3
4
5
No. of conjugated double bonds
cc: B2,B3-di-cis isomer
tc: B2-strans, B3-cis isomer
6
ct: B2-s-cis, B3-trans isomer
tt: all-trans isomer.
Proton Affinity:
PA= EAH-(EA+EH)
Isomerization does not have a strong impact on PA!
What is the effect of isomerization?
hn
Retinal binding pocket in bR
lmax: ~400 570
Opsin shift
Water
Counterion:
Asp85 & Asp212
pKa: ~8.0
13.0
Asp212
Asp85
Water
WATER
Effect of the environment on PA

1.0
2.0
4.0
6.0
78.4
H
acetate 350.81
309.05
288.27
SB1
256.04
278.02
208.76
SB2
223.85
SB3
234.00 254.64 268.44 273.78 284.78
SB4
241.15 259.63 271.98 276.77 286.68
SB5
246.39
274.54
288.01
SB6
250.58
276.43
288.93
PMET 258.57
281.23
292.07
RSB
282.07
292.56
260.12
263.36
H
syn
H
H
+
N
281.86
anti
H
n-1
H
Proton Affinity:
PA= EAH-(EA+EH)
In situ isomerization and pKa
290
PA (kcal/mol)
280
PSB3
PSB4
270
260
250
240
230
1.0
2.0
4.0
6.0
78.4
Dielectric Constant
=6.0
C H3
C H3
C H3
N
C H3
H
N
O
H
C H3
H
=2.0
O
O
Asp85
Coupling of electronic excitation and
conformational change in bR
S1
S0
K
BR
C13=C14-trans
C13=C14-cis
Me
Me
Me
13
7
Me
Me
9
11
15 N
H
Ground and Excited State Potential
Energy Surfaces of Retinal
hn
trans
cis
Ab Initio QM/MM Excited State MD Simulation
QM
Quantum mechanical (QM)
treatment of the chromophore,
and force field (MM) treatment
of the embedding protein
Isomerization Barriers in retinal
B3
B1 C2
N1
Lys216
+
C4
B5
C6
C8
B2 C3
B4 C5
B6 C7
C15
C13
C11
N16
C14
C12
C12
C10
C9
Proton Affinity:
PA= EAH-(EA+EH)
C11
C9
C5
C7
C10
C8
H
C6
C4
C1
C3
C2
270
PA (kcal/mole)
265
260
255
250
Ground state
isomerization
245
B10-90
B9-90
B8-90
B7-90
B6-90
B5-90
B4-90
B3-90
B2-90
B1-90
235
all-trans
240
Rotated bond
Low barriers against double bond isomerization
A twisted chromophore in bR?
168° 165°
178°
Me
Me
Me
N
Me
Me
H
177°
176°
177°
A twisted chromophore is also experimentally reported.
X-ray structures of bR report the twisted form of chromophore
The twist is found around the terminal double bonds
It may influence pKa of the chromophore