Structure-Function Relationship of Retinal

Proteins

Retinal proteins or rhodopsins belong to the superfamily of seven-transmembrane 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)

AA BB CC DD EE FF GG

Structure of Retinal Proteins

GPCRs

Retinal Proteins -- Rhodopsins

N

Me Me Me

MeMe

H

N

Me Me Me

MeMe

H

• Covalently linked to a lysine• Usually protonated Schiff base• all-trans and 11-cis isomersChromophore

The simplest ion pump in biology

The best characterized membrane protein

The simplest photosynthetic center

Technological applications in molecular electronics

Bacteriorhodopsin -- bR

The first membrane protein with a known atomic-detail 3D structures

Halobacterium Salinarum

The Purple Membrane

H+h [H+]

[H+]

ADP ATPCytoplasmic side

Extracellular side

ATP Synthase

h

bR role in Bioenergetics Light

Proton Gradient

N

H

Lys216

OHH

O

O

Asp85O

O

Asp212

H H

N

H

NH

N

Arg82

H

Water

hv H

OO

OHH

Asp96

Cytoplasmic side

Extracellular side

Schematic proton path in bacteriorhodopsin

Transmambrane helices

Transmambrane helices

H+

H+

Active Channels Need a ‘Switch’ Mechanism

H+ H+H+

H+

H+

H+ H+H+

H+H+

H+H+ h

H+

H+

What is the switch in bR? How does it work?

Photocycle of bRPhoto-induced

All intermediates are trapped in low temperature and have been characterized by vibrational and absorption spectroscopy.

3 ps

s

40 s5 ms

5 ms

5 ms

No membrane protein has been studied as extensively as bR Photo-induced

3 ps

s

40 s5 ms

5 ms

5 ms

All intermediates have also been characterized by X-ray crystallography!

N

H

Lys216

OHH

O

O

Asp85O

O

Asp212

H H

N

H

NH

N

Arg82

H

Water

hv H

OO

OHH

Asp96

Cytoplasmic side

Extracellular side

Schematic proton path in bacteriorhodopsin

Transmambrane helices

Transmambrane helices

cytoplasmic

extracellular

H+

light driven proton pump

+

H

D85-COO

HOOC-E204

HOOC-D96

N

+

H

D85-COO

HOOC-E204

HOOC-D96

N

K216

K216

bR568

K603

+

HD85-COO

HOOC-E204

HOOC-D96

NK216

D85-COOH

OOC-E204

HOOC-D96

N

K216

L543

M410

+

H

D85-COOH

OOC-E204

OOC-D96

N

K216

N550

+

H

D85-COOH

OOC-E204

HOOC-D96

N K216

O645

3ps

s

40s5ms

5ms

5ms

BR’s Photocycle

+

H

D85-COO

HOOC-E204

HOOC-D96

N

+

H

D85-COO

HOOC-E204

HOOC-D96

N

K216

K216

bR568

K603

+

HD85-COO

HOOC-E204

HOOC-D96

NK216

D85-COOH

OOC-E204

HOOC-D96

N

K216

L543

M410

+

H

D85-COOH

OOC-E204

OOC-D96

N

K216

N550

+

H

D85-COOH

OOC-E204

HOOC-D96

N K216

O645

3ps

s

40s5ms

5ms

5ms

Kuhlbarandt, Nature, 406,569 (2000)

Conformational Change of Helices

BR’s Photocycle

bR in the purple membraneModeling of the protein in lipid bilayers

Chromophore•Analysis of the structure•Calculation of excited state dynamics

Protein• Chromophore-protein

interaction• QM-MM calculations• MD simulation of the

photocycle

Study of bR at three levels

Retinal Schiff baseMembrane, covalently bound, chromophore

C

O

O

MeMeMe

MeMe

H

Retinoic AcidNucleus, receptor site, ligand (no photoactivity)

N

Me Me Me

MeMe

H

Retinoids

C

O

O

MeMeMe

MeMe

H

Retinal

N

Me Me Me

MeMe

H

The necessity of quantum mechanical treatment of the chromophore:

Conjugated -electronic system, delocalization The effect of protein matrix on the ligand

N

Me Me Me

MeMe

H

7 9 11 13 15

Unconventioanl chemistry

QM is expensive – Most of the time, one needs to use models

200

210

220

230

240

250

260

270

0 2 4 6 8 10

No. of conjugated double bonds

PA

(kc

al/m

ol)

PA*

PA*(zpe)

PA**

PA**(zpe)

N

Me Me Me

MeMe

H

N

H

H H

H

H

H

syn

anti

n-1 +

Proton Affinity:PA= EAH-(EA+EH)

Effect of Conjugation on pKa (Gas Phase Proton Affinity)

N1

C2

C3

C4

C5

C6

C7

C8

C9

C10

C11

C12

C6

C1

C2

C3

C4

C5C7

C8

C9

C10

C11

C12

C13

C14

C15

N16

Lys216

H

B1

B2

B3

B4

B5

B6

+

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

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

N

Me Me Me

MeMe

H

Proton Affinity:PA= EAH-(EA+EH)

Effect of the methyl groups on pKa

No more room for additional methyl groups on the backbone

h

What is the effect of isomerization?

225

235

245

255

3 4 5 6No. of conjugated double bonds

PA

(k

cal/m

ol)

cc ct tc tt

N

H

H H

H

H

H

syn

anti

n-1 +

Proton Affinity:

PA= EAH-(EA+EH)cc: B2,B3-di-cis isomer ct: B2-s-cis, B3-trans isomertc: B2-strans, B3-cis isomer tt: all-trans isomer.

Isomerization State and Proton Affinity

Isomerization does not have a strong impact on PA!

h

What is the effect of isomerization?

Water

Asp85

Asp212

Water

Counterion:Asp85 & Asp212

WATER

pKa: ~8.0 13.0

max: ~400 570Opsin shift

Retinal binding pocket in bR

1.0 2.0 4.0 6.0 78.4

acetate 350.81 309.05 288.27

SB1 208.76 256.04 278.02

SB2 223.85 263.36 281.86

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 260.12 282.07 292.56

Proton Affinity:PA= EAH-(EA+EH)

Effect of the environment on PA

230

240

250

260

270

280

290

1.0 2.0 4.0 6.0 78.4

Dielectric Constant

PA

(k

ca

l/m

ol)

PSB3

PSB4

N

CH3CH3CH3

CH3

CH3

NH

OH

H

O

O

Asp85

=6.0

=2.0

In situ isomerization and pKa

S0

S1

KBR

C13=C14-trans C13=C14-cis

Coupling of electronic excitation and conformational change in bR

N

Me Me Me

MeMe

H

7 9 11

13

15

h

trans

cis

Ground and Excited State Potential Energy Surfaces of Retinal

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

235

240

245

250

255

260

265

270

all-

tran

s

B1-

90

B2-

90

B3-

90

B4-

90

B5-

90

B6-

90

B7-

90

B8-

90

B9-

90

B10

-90

Rotated bond

PA

(kc

al/m

ole)

N1

C2

C3

C4

C5

C6

C7

C8

C9

C10

C11

C12

C6

C1

C2

C3

C4

C5C7

C8

C9

C10

C11

C12

C13

C14

C15

N16

Lys216

H

B1

B2

B3

B4

B5

B6

+

Proton Affinity:PA= EAH-(EA+EH)

Low barriers against double bond isomerization

Ground state isomerization

Isomerization Barriers in retinal

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

N

Me Me Me

MeMe

H

165°168°178°

177°176°177°

A twisted chromophore in bR?