Structure and Activation of the Visual Pigment Rhodopsin

ANRV411-BB39-16 ARI 2 April 2010 11:11

Structure and Activationof the VisualPigment RhodopsinSteven O. SmithDepartment of Biochemistry and Cell Biology, Stony Brook University, Stony Brook,New York 11794-5215; email: [email protected]

Annu. Rev. Biophys. 2010. 39:309–28

First published online as a Review in Advance onFebruary 10, 2010

The Annual Review of Biophysics is online atbiophys.annualreviews.org

This article’s doi:10.1146/annurev-biophys-101209-104901

Copyright c© 2010 by Annual Reviews.All rights reserved

1936-122X/10/0609-0309$20.00

Key Words

G protein–coupled receptor, retinal chromophore, light activation

AbstractRhodopsin is a specialized G protein–coupled receptor (GPCR) foundin vertebrate rod cells. Absorption of light by its 11-cis retinal chro-mophore leads to rapid photochemical isomerization and receptor acti-vation. Recent results from protein crystallography and NMR spec-troscopy show how structural changes on the extracellular side ofrhodopsin induced by retinal isomerization are coupled to the motionof membrane-spanning helices to create a G protein binding pocket onthe intracellular side of the receptor. The signaling pathway provides acomprehensive explanation for the conservation of specific amino acidsand structural motifs across the class A family of GPCRs, as well asfor the conservation of selected residues within the visual receptor sub-family. The emerging model of activation indicates that, rather thanbeing unique, the visual receptors provide a basis for understanding thecommon structural and dynamic elements in the class A GPCRs.

309

Ann

u. R

ev. B

ioph

ys. 2

010.

39:3

09-3

28. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by W

IB60

13 -

Fre

ie U

nive

rsita

et B

erlin

- F

U B

erlin

on

11/2

2/11

. For

per

sona

l use

onl

y.

ANRV411-BB39-16 ARI 2 April 2010 11:11

GPCR: Gprotein–coupledreceptor

PSB: protonatedSchiff’s base

Contents

RHODOPSIN: ALIGHT-ACTIVATED GPROTEIN–COUPLEDRECEPTOR . . . . . . . . . . . . . . . . . . . . . . 310Rod Cells Are Efficient

Photodetectors . . . . . . . . . . . . . . . . . 310Seven-Transmembrane Helix

Architecture of Rhodopsin . . . . . . 311Retinal: The Light-Sensitive Trigger

for Activation . . . . . . . . . . . . . . . . . . . 314Conservation and Constitutive

Activity . . . . . . . . . . . . . . . . . . . . . . . . . 315STRUCTURAL CHANGES UPON

RECEPTOR ACTIVATION . . . . . . 318Retinal Isomerization in a Tight

Protein Binding Pocket . . . . . . . . . 318Coupling of Retinal Isomerization

to Helix Motion . . . . . . . . . . . . . . . . 318Disruption of the Ionic Lock

and G Protein Binding . . . . . . . . . . 321RHODOPSIN AS A MODEL

FOR LIGAND-ACTIVATEDG PROTEIN–COUPLEDRECEPTORS . . . . . . . . . . . . . . . . . . . . . 321Parallels Between Rhodopsin

and Ligand-Activated GPCRs . . . 321Converting Rhodopsin into a

Ligand-Activated GPCR . . . . . . . . 323Subfamily-Specific Residues:

Evolution of the GPCRs . . . . . . . . 324

RHODOPSIN: ALIGHT-ACTIVATED GPROTEIN–COUPLED RECEPTOR

Since the discovery that the visual pig-ment rhodopsin and the ligand-activated β2-adrenoreceptor had a common structure (13),the family of G protein–coupled receptors(GPCRs) has grown to include receptors thatselectively bind a broad range of ligands, in-cluding small organic molecules, hormones,peptides, and proteins. The class A recep-tors are the largest of five distinct classes of

GPCRs and have emerged as a major drug tar-get (29). These receptors have a common seven-transmembrane helix architecture and catalyzethe exchange of GTP for GDP in intracellu-lar heterotrimeric G proteins. One of the in-triguing questions surrounding GPCRs is howa relatively simple seven-transmembrane helixstructure has evolved to be capable of respond-ing to such a diversity of signals. Rhodopsin,the low-light receptor in vertebrates, has oftenbeen considered an exception within the classA GPCRs but is now providing the basis for acommon mechanism for activation.

Rod Cells Are EfficientPhotodetectors

The capture and conversion of light into achemical signal is the first step of the visual pro-cess. Vertebrate rod cells are exquisitely evolvedphotodetectors (56). They can be triggered bythe absorption of a single photon, yet adapt toover a 100-fold range of background illumina-tion. The inner segment of the rod cell containsthe machinery for normal cellular function,while the rod outer segment is specialized forphoton capture and amplification. Human rodouter segments are ∼40 μm in length, andthe outer plasma membrane encloses a stackof roughly 1000–1500 flattened membranediscs that are separated from one anotherby ∼15 nm. Each disc contains ∼100,000receptor molecules, although activation ofjust a single receptor can be amplified suchthat within milliseconds there is the transientclosure of hundreds of sodium channels andhyperpolarization of the plasma membrane (8).

This review focuses on the initial event inthis process, the activation mechanism of thephotoreceptor rhodopsin. At the heart of thevisual receptor is the 11-cis isomer of reti-nal (vitamin A), which is attached to the pro-tein through a protonated Schiff’s base (PSB)linkage. The 11-cis retinal within its proteinbinding site is primed to convert selectively tothe all-trans isomer upon absorption of light.The quantum yield for the photoreaction is0.67, which corresponds to the probability that

310 Smith

Ann

u. R

ev. B

ioph

ys. 2

010.

39:3

09-3

28. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by W

IB60

13 -

Fre

ie U

nive

rsita

et B

erlin

- F

U B

erlin

on

11/2

2/11

. For

per

sona

l use

onl

y.

ANRV411-BB39-16 ARI 2 April 2010 11:11

photon absorption will result in isomerization.For comparison, the quantum yield for the 11-cis retinal PSB in solution is ∼0.3 (6), and ratherthan being selective, the reaction in solutiongenerates a mixture of different retinal isomers.The photoisomerization is also ultrafast, occur-ring within 200 fs (52). In contrast, the ther-mal barrier for isomerization is extremely high(∼45 kcal mol−1), such that statistically only asingle receptor is thermally activated every 470years (5).

Seven-Transmembrane HelixArchitecture of Rhodopsin

The structure of rhodopsin in its dark, in-active state has been described in detail inthe original literature (31, 40, 42) and reviews(41, 50). However, I summarize below the im-portant features of the structure to help thereader follow the signaling pathway from theextracellular surface through the transmem-brane helices to the G protein binding site onthe intracellular surface.

The extracellular, or intradiscal, side ofrhodopsin—comprising the N terminus (NT)and three extracellular loops (EL1, EL2, andEL3)—is folded into a well-organized structure(Figure 1). The N terminus is composed of twoshort strands and is glycosylated at Asn2NT andAsn15NT. The two strands (β1 and β2) stretchfrom Gly3NT to Pro12NT and adopt a typicalantiparallel β-sheet fold oriented roughlyparallel to the plane of the membrane. Nong-lycosylated rhodopsin appears to fold correctlybut shows low-light-dependent activation ofthe G protein transducin (26). Mutations atThr4NT, Asn15NT, Thr17NT, Pro23NT, andGln28NT on the N terminus can lead to auto-somal dominant retinitis pigmentosa (ADRP),an inherited human disease that causes pro-gressive retina degeneration. ADRP mutationsare often associated with misfolded receptors.

The first crystal structure of rhodopsin re-vealed several unusual features (42). Perhapsthe most striking observation was that EL2folds into a highly ordered lid over the reti-nal binding site (40, 42). The EL2 sequence

EL1

NT

EL3

EL2

H8CT

CL1

CL2

CL3

Figure 1Crystal structure of rhodopsin (42). The helices arecolored according to their packing density (32)within the rhodopsin crystal structure.Transmembrane helices H2 (orange) and H3 (red )are the most tightly packed and contribute to astable core that does not change appreciably uponactivation. Helices H5 (dark blue) and H6 (cyan) arethe most loosely packed and undergo the largestdisplacements upon activation (17, 43). The11-cis-retinal chromophore is covalently attachedwithin the transmembrane helix bundle on theextracellular side of the receptor. CL, cytoplasmicloop; CT, C terminus; EL, extracellular loop; NT,N terminus.

EL1–3: extracellularloops 1–3

ADRP: autosomaldominant retinitispigmentosa

extends from Trp1754.64 on H4 to Thr1985.25

on H5 and consists of two short β-strands (β3and β4) constrained by a conserved disulfidebond between Cys1103.25 and Cys187EL2 and asalt bridge between Arg177EL2 and Asp190EL2.(The Ballesteros-Weinstein generic numberingsystem is used throughout the review, see side-bar below.) The disulfide bond and Asp190EL2

are critical for the correct folding of rhodopsininto a functional conformation (25). Muta-tion of Asp190EL2 leads to ADRP (23). EL2 isstabilized by a hydrogen-bonded network cen-tered on Glu181EL2. Glu181EL2 is hydrogen

www.annualreviews.org • Mechanism of Rhodopsin Activation 311

Ann

u. R

ev. B

ioph

ys. 2

010.

39:3

09-3

28. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by W

IB60

13 -

Fre

ie U

nive

rsita

et B

erlin

- F

U B

erlin

on

11/2

2/11

. For

per

sona

l use

onl

y.

ANRV411-BB39-16 ARI 2 April 2010 11:11

BALLESTEROS-WEINSTEIN GENERICNUMBERING

The amino acid numbering used in this review incorporates theresidue number from the amino acid sequence of the specificreceptor being discussed (e.g., Trp265) and a residue numberfrom a generic numbering system developed by Ballesteros andWeinstein (e.g., Trp2656.48) that gives the position of an aminoacid relative to the most conserved amino acid on a specific helix.In this example, the superscript 6.48 indicates that Trp265 is tworesidues toward the N terminus of the most conserved residue(designated 50) on H6 (i.e., Pro2676.50). Because sequence align-ments are poor for the extracellular and cytoplasmic loops, as wellas for the N and C termini, a generic superscript (e.g., EL2) isused to designate the position of a nontransmembrane residue.

H1–H7:transmembrane helices1–7

bonded to Tyr192EL2 and Tyr2686.51 and isconnected through water-mediated hydrogenbonds to Ser186EL2 and Glu1133.28, the coun-terion to the retinal PSB (40). Glu1133.28 is hy-drogen bonded to the backbone carbonyl ofCys187EL2 through a water molecule and iswithin hydrogen-bonding distance of the hy-droxyl group of Thr942.65 (40). This stablehydrogen-bonded network is thought to be im-portant for maintaining a high pKa for the PSBin the dark state of rhodopsin.

The seven transmembrane helices (H1–H7)vary in length, from 20 to 33 amino acids,and surround the retinal on the extracellularside of the receptor. Pioneering electron spinresonance studies showed that activation ofrhodopsin involves the outward rotation of H6(17). Recently, the structure of opsin was solvedand revealed many features of the activated re-ceptor (43, 51). Opsin forms after the loss of theretinal chromophore during the photoreaction.Importantly, at low pH the opsin structure cap-tures the outward rotation of H6, which is ahallmark of an active conformation.

Most of the conserved residues in class AGPCRs are located in transmembrane helices,suggesting that this region adopts a commonstructure. The conserved residues are high-lighted in Figure 2 and Table 1. In addi-tion to the retinal binding site, four regions

Table 1 Signature-conserved residues

Signature-conservedresidues

Sequence identity inclass A GPCRs

Asn551.50 99.5%Leu792.50 94.0%Asp832.54 89.4%Cys1103.25 89.2%Leu1283.43 77.6%Glu1343.49 71.7%Arg1353.50 98.1%Tyr1363.51 71.6%Trp1614.50 88.4%Cys187EL2 87.4%Pro2155.42 72.8%Tyr2235.50 93.2%Trp2656.48 69.2%Pro2676.50 80.1%Asn3023.49 85.7%Pro3037.50 98.1%Tyr3067.53 93.2%

Abbreviation: GPCR, G protein–coupled receptor.

in rhodopsin are functional microdomains thatcan operate as molecular switches to trig-ger conformational changes within the re-ceptor (3, 4, 59). On the extracellular sideare two well-characterized microdomains. Onemicrodomain involves a cluster of aromaticresidues surrounding the retinal. In the aminereceptors, a phenylalanine at position 6.52 isthought to be the ligand sensor (59). Ligandinteractions with Phe6.52 induce changes in theside chain conformations of Cys6.47, Trp6.48, andPro6.50. Rather than phenylalanine, an alanineoccurs at position 6.52 in rhodopsin. The β-ionone ring of the retinal chromophore is lo-cated in the region where the aromatic ring ofphenylalanine occurs in the amine receptors,and appears to play the same role as the ligandsensor. That is, motion of the β-ionone ring iscoupled to rotation of Trp2656.48 (12). The sec-ond microdomain on the extracellular side ofrhodopsin consists of a network of hydrogen-bonded residues centered on the His2115.38-Glu1223.37 pair. The hydrogen bonds in thisnetwork rearrange upon retinal isomerizationand receptor activation.

312 Smith

Ann

u. R

ev. B

ioph

ys. 2

010.

39:3

09-3

28. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by W

IB60

13 -

Fre

ie U

nive

rsita

et B

erlin

- F

U B

erlin

on

11/2

2/11

. For

per

sona

l use

onl

y.

ANRV411-BB39-16 ARI 2 April 2010 11:11

Q F

AAS

LM

F

M

Y

LLI

M

FGL

FNIP

LLT

Y VVT

PLN Y

L

V

L

FF

I

L

L

A L N

D AFMV

G GTTT

YTLH G C NL E G FF A TL

GGEI A LW S

S

L V

IV L A

E R YV VN

H

G M I A

F AV

M V W

T

A L A

P A A C

V L P

E S

YIVF

VFMV H F III

ILP

VF

YCFG Q L V

FK E A

R T V E

I V M

I V M I

L F A

L W C I

A Y P

F A V G

F I Y

MI YIVPV Y N

K T S A

F F A

T I P A

I F M

WP

EA L Y

Y QP

AE F

S R VVGTKNSFP

VYFN

PG

E TG N

M

Q

K K L RT

GY

F V FG

P T

VC

KP

M S NF

RF

GE

GW

RS

YI

PG

MQ

CS

CG

ID

YY P H

EETNN

TTV

KE

AA

A QQ

QE

SAT Q

K

TH

QG S D

FGP

MN K

QF M

TT

VRN

CL

CC G KNP

LGDDEASTTV

S K T E T S Q V

AA P

NT

CT

H1H1 H2H2 H3H3

H4H4H5H5 H6H6 H7H7

H8H8

H1 H2 H3

H4H5 H6 H7

H8

10

20

30

39

50

60

70

80

90

100

130

140

150

170

180

190

200

210

230240

260

270

280

300

310320

330

340

109

119

159

221

249

289

CL1

CL2

CL3

EL1

EL2

EL3

H

T

P

E

SSSS

Figure 2Sequence and conservation of rhodopsin. Two-dimensional representation of the rhodopsin sequence. The receptor has seventransmembrane helices (H1–H7) and a short amphipathic helix (H8) that lies on the cytoplasmic surface of the membrane. Amino acidsare shown in single-letter codes. Red circles denote signature-conserved residues, blue circles small and weakly polar group–conservedresidues, and green circles subfamily-specific residues. Red circles with a yellow border correspond to the residues with the highestsequence identity on each helix. Trp2656.48 (69.2%) may also be considered a signature residue. The residues in gray are between 70%and 90% conserved in the visual receptor subfamily. CL, cytoplasmic loop; CT, C terminus; EL, extracellular loop; NT, N terminus.

On the intracellular side of rhodopsin, thereare two well-characterized microdomains asso-ciated with conserved structural motifs. Thefirst motif is the (E/D)RY motif, correspond-ing to the Glu1343.49-Arg1353.50-Tyr1363.51 se-quence in rhodopsin. The salt bridge betweenArg1353.50 on H3 and Glu2476.30 on H6, re-ferred to as the ionic lock, must be broken uponreceptor activation. The second motif is theconserved NPxxY sequence on H7. Tyr3067.53

in this motif rotates toward Arg1353.50 in the

H8: amphipathicC-terminal helix

CL1–3: cytoplasmicloops 1–3

active receptor and contributes to breaking theionic lock (43, 51).

The cytoplasmic side of rhodopsin is formedby the C terminus (CT) and three cytoplas-mic loops (CL1, CL2, and CL3). The C ter-minus emerges from H7 as a short amphi-pathic helix (H8) stretching from Lys311H8

to Leu321H8. The region involving theC-terminal ends of H1 and H7 through H8 ishighly conserved, suggesting a role in recep-tor structure and/or function. The C-terminal


Ann

u. R

ev. B

ioph

ys. 2

010.

39:3

09-3

28. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by W

IB60

13 -

Fre

ie U

nive

rsita

et B

erlin

- F

U B

erlin

on

11/2

2/11

. For

per

sona

l use

onl

y.

ANRV411-BB39-16 ARI 2 April 2010 11:11

Meta I(λ max = 478 nm)

Meta III(λ max = 465 nm)

Opsin + all-trans retinal(λ max = 380 nm)

N+H

Lys296

19

20

12

15

146

18

1716 7

5

11

10 1398

12

3 4

11-cis

20

N

1917 16

Excited state

Photorhodopsin(λ max = 570 nm)

Bathorhodopsin(λ max = 543 nm)

BSI (blue-shifted intermediate)(λ max = 470 nm)

Lumirhodopsin(λ max = 497 nm)

hν

200 fs

ps

ns

ns

μs

ms

min

>1 h

min

Rhodopsin(λ max = 500 nm)

Meta II(λ max = 382 nm)

Figure 3Rhodopsin photoreaction. Upon absorption of light, the 11-cis retinalchromophore isomerizes to the all-trans configuration within 200 fs and thenrelaxes thermally through a series of spectrally well-defined intermediates tothe active Meta II state, which binds and activates the G protein transducin.Hydrolysis of the Schiff’s base linkage generates opsin and frees all-trans retinal.

end of H8 is anchored to the membrane bypalmitoylation of Cys322CT and Cys323CT. A30% reduction in the rate of G protein activa-tion is observed in the absence of palmitoylation(44).

In contrast to the structured extracellularsurface of rhodopsin, the C terminus and cyto-plasmic loops are more mobile. The C terminuscontains phosphorylation sites that recognizearrestin. For rhodopsin, the major phospho-rylation sites are at Ser334CT, Ser338CT, andSer343CT (39). In the crystal structure, theC-terminal tail interacts with H8 and CL1 inthe region of these serines, which may blocktheir exposure to rhodopsin kinase. Activationof rhodopsin may change the conformation ofthe tail and facilitate phosphorylation.

Retinal: The Light-Sensitive Triggerfor Activation

The photoreactive chromophore in all vi-sual receptors is the 11-cis isomer of retinal(Figure 3). Both the retinal polyene chainand its associated methyl groups contributeto the retinal’s ability to trigger activation. Inrhodopsin, the retinal is attached to Lys2967.43

through a PSB linkage. The positive chargecreated by protonation of the Schiff’s base in-creases electron delocalization along the conju-gated retinylidene chain and shifts the absorp-tion band from the short wavelength range tothe middle of the visible spectrum. The proteincounterion to the positive charge is Glu1133.28

on H3. The Glu1133.28-PSB interaction is es-sential not only for regulating wavelength, butalso for maintaining the receptor in an inactiveconformation.

Absorption of light by the retinal chro-mophore induces cis to trans isomerization ofthe C11==C12 double bond. The fast photo-chemistry and high quantum yield are due totwo features of the retinal binding site. First,the longitudinal restriction of the retinal withinthe binding site imparts a twist about theC11==C12 double bond (40, 55, 57). In addition,the C12–C13 single bond is twisted s-cis ow-ing to a steric contact between the C20 methyl

314 Smith

Ann

u. R

ev. B

ioph

ys. 2

010.

39:3

09-3

28. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by W

IB60

13 -

Fre

ie U

nive

rsita

et B

erlin

- F

U B

erlin

on

11/2

2/11

. For

per

sona

l use

onl

y.

ANRV411-BB39-16 ARI 2 April 2010 11:11

group and the proton on C10. Together, thesetwists prime the C20 methyl group to rotatein a clockwise direction (when viewed from theSchiff’s base end of the retinal) as the retinalisomerizes. Second, the carboxylate side chainof Glu181 is negatively charged and is posi-tioned close to the C12 carbon of the retinal.Both the pretwist in the C11==C12 bond andthe position of the Glu181 side chain facilitateisomerization by lowering the C11==C12 bondorder.

Photoisomerization results in a highlystrained all-trans retinal chromophore in thefirst reaction intermediates, photorhodopsinand bathorhodopsin, because the timescale ismuch too rapid for amino acid side chainsto rearrange in the tight protein binding site.Calorimetric studies show that bathorhodopsinhas stored ∼35 kcal mol−1 of the absorbedlight energy (10). The energy is released asthe protein-retinal complex decays thermallythrough a series of distinct, spectrally definedintermediates (Figure 3). Crystal structures ofthe bathorhodopsin (36, 53), lumirhodopsin(37), and metarhodopsin I intermediates (49)have been determined. There are no large-scale changes observed in these structures rel-ative to the dark state of rhodopsin. In con-trast, a large structural transition takes placein the conversion of metarhodopsin I (Meta I)to metarhodopsin II (Meta II). Meta II corre-sponds to the active state of rhodopsin and ischaracterized by a deprotonated Schiff’s basenitrogen and a 382-nm-visible absorption max-imum. An increase in enthalpy in the Meta I toMeta II transition is compensated by a large in-crease in entropy, which may be related to therelease of water (35).

Conservation and Constitutive Activity

To understand the activation mechanism ofrhodopsin (and other class A receptors), thechallenge has been to describe in molecular de-tail the function of the conserved residues. It isimportant to consider three levels of conserva-tion. The signature amino acids with sequenceidentities of >70% are the most important set

Table 2 Group-conserved residues

Group-conservedresidues

Group conservationin class A GPCRs

Gly511.46 92.5%Ile541.49 77.7%Ala802.51 97.4%Ala822.53 82.2%Ile1233.38 71.9%Ala1243.39 77.9%Ala1323.47 99.7%Ala1534.42 81.0%Ala1644.53 92.6%Ala1684.57 87.5%Cys2646.47 76.5%Ala2957.42 83.4%Ala2997.46 78.0%

Abbreviation: GPCR, G protein–coupled receptor.

of conserved residues in the class A GPCRfamily (Table 1). The conserved prolines onH5, H6, and H7, along with the (E/D)RY andNPxxY motifs discussed above, have been ex-tensively studied. Several hydrophobic residues(e.g., Leu792.50 and Trp1614.50) are highly con-served but less well studied because it has notbeen clear how they are involved in receptorstructure or function.

The second level of conservation involvesthe group-conserved residues in the class AGPCR family, with conservation of >70% whenconsidered as a group of small and weaklypolar residues (Ala, Gly, Ser, Cys, and Thr)(Table 2). These amino acids facilitate closehelix packing and the formation of interheli-cal hydrogen bonds (14, 32). We have previ-ously suggested (32), on the basis of the loca-tion of the group-conserved positions (40), thatH1–H4 (and possibly H7) form a tightly packedcore.

The third level of conservation includesthose residues that have sequence identities of>90% in the visual receptor subfamily. Eachclass A GPCR subfamily contains a set ofresidues that makes it uniquely able to respondto its own ligand. For rhodopsin, these residuesare shown in green in Figure 2 and are listed


Ann

u. R

ev. B

ioph

ys. 2

010.

39:3

09-3

28. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by W

IB60

13 -

Fre

ie U

nive

rsita

et B

erlin

- F

U B

erlin

on

11/2

2/11

. For

per

sona

l use

onl

y.

ANRV411-BB39-16 ARI 2 April 2010 11:11

Table 3 Rhodopsin subfamily-specific residues

Rhodopsinsubfamily-specificresidues

Subfamilyconservation

Lys66CL1 93.4%Leu68CL1 97.8%Arg69CL1 90.9%Asn732.44 97.9%Asn782.49 97.9%Gly106EL1 95.5%Gly1213.36 99.2%Val1383.53 90.1%Pro1714.60 99.1%Asp190EL2 96.2%Ile2195.46 89.9%Glu2476.30 97.5%Trp2656.48 87.2%Tyr2686.51 99.9%Lys2967.43 100%Arg314H8 89.7%

in Table 3. For example, lysine at position7.43 in the visual receptor subfamily is 100%conserved, because this is the site of covalentattachment of the retinal. Tyr2686.51 (99.9%)and Gly1213.36 (99.2%) have the next high-est levels of subfamily conservation, indicatingkey roles in activation. Val1383.53 (90.1%) andGlu2476.30 (97.5%) are part of the intracellularionic lock: Val1383.53 helps form the hydropho-bic cage around the ionic lock, and Glu2476.30

is the counterion for Arg1353.50. Whereas the(E/D)RY motif consists of signature residues,the rest of the ionic lock tends to be subfamilyconserved, suggesting that different subfamilieshave evolved different strategies for stabilizingthe Arg3.50 side chain in the inactive receptorand breaking the ionic lock upon activation.

Figure 4 presents the core conserved trans-membrane region of rhodopsin to illustratethe interplay between the signature, group-conserved, and subfamily-specific amino acids.The most highly conserved residue in theclass A GPCRs is Asn551.50. Close analysisof the amide functional group shows thatthe amine NH2 is hydrogen bonded to the

backbone carbonyls of Gly511.46 and Ala2997.46,both of which are group-conserved aminoacids. Asp832.54 is part of the highly con-served LAXAD sequence on H2. Leu792.50

and Asp832.54 are signature residues, whereasAla802.51 and Ala822.53 are group conserved.The very high group conservation of Ala802.51

(97%) allows close H1-H2 packing and theformation of a specific Asn551.50-Asp832.54

hydrogen-bonding contact. Ala822.53 mediatesthe packing of H2 with H3 (Ile1233.38 andAla1243.39) and H4 (Trp1614.50). The intricatepacking of the signature and group-conservedresidues in the transmembrane core suggeststhat they do not appreciably change positionupon activation.

The conserved transmembrane core liesbetween the Glu1133.28-PSB salt bridge onthe extracellular side of the receptor and theArg1353.50-Glu2476.30 ionic lock on the cyto-plasmic side of the receptor. There is a grow-ing body of evidence that these two ionic pairsform protonation switches (PS1 and PS2) thatcontrol rhodopsin activation (33). Internal pro-ton transfer occurs from the retinal PSB toGlu1133.28 upon activation, which allows H6to pivot and rotate outward on the intracellularside of rhodopsin. This motion of H6 breaks theionic lock on the intracellular side of the proteinand allows the uptake of a proton from the sol-vent by Glu1343.49, thereby stabilizing the ac-tive state. The sequence of events in the activa-tion mechanism (i.e., protonation of Glu1133.28

by an internal proton transfer, an outward ro-tation of H6, and protonation of Glu1343.49) isassociated with a series of Meta II substates (27).

Constitutively active GPCRs can provide di-rect insights into the activation mechanism ofthe wild-type receptors. Constitutively activemutations in rhodopsin are particularly reveal-ing, because the receptor has evolved to havevery low thermal activity and requires a sub-stantial input of energy to convert it to the ac-tive state. The retinal chromophore serves asan inverse agonist to activation. Without theretinal there is a small, but measurable, amountof activity that is eliminated with 11-cis retinalbinding.

316 Smith

Ann

u. R

ev. B

ioph

ys. 2

010.

39:3

09-3

28. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by W

IB60

13 -

Fre

ie U

nive

rsita

et B

erlin

- F

U B

erlin

on

11/2

2/11

. For

per

sona

l use

onl

y.

ANRV411-BB39-16 ARI 2 April 2010 11:11

Met257Met257

Asn302Asn302

Trp161Trp161Ile123Ile123

Gly51Gly51

Asn55Asn55

Ala80Ala80

Ala82Ala82Ala124Ala124

Leu128Leu128

Leu79Leu79Asp83Asp83

Met257

Asn302

Trp161Ile123

Gly51

Asn55

Ala80

Ala82Ala124

Leu128

Leu79Asp83

Glu113Glu113

Arg135Arg135

Retinal Retinal Glu113

Glu247

Arg135

Retinal

a b

c

PS1

PS2

H6

Figure 4Signature and group-conserved residues in the transmembrane core of rhodopsin. (a) View of rhodopsin showing the conserved core ofsignature (red ), group-conserved (blue), and subfamily-specific ( green) amino acids as van der Waals spheres. Transmembrane helix H6is highlighted in black. (b) View of rhodopsin rotated 90◦ about the vertical relative to panel a. The conserved core is midway betweenthe two protonation switches (PS1 and PS2) that control activation. (c) View from the extracellular surface of rhodopsin showingsignature and group-conserved amino acids within the transmembrane core. Met2576.40, a subfamily-specific residue, is packedbetween Leu1283.43 and Asn3027.49. Replacement of Met2576.40 with isoleucine, a subfamily-specific residue in the amine subfamily ofclass A GPCRs, converts rhodopsin into a receptor activated by exogenous all-trans retinal, i.e., into a ligand-activated receptor.

Not surprisingly, constitutively active mu-tations are associated with two human reti-nal diseases, congenital night blindness andADRP (46). Three mutations (G90D, A292E,and T94I) cause congenital night blindness.Each of these mutations leads to constitutiveactivity by disrupting the salt bridge betweenthe PSB and Glu1133.28 (24). The G90D andA292E mutations introduce charges that re-place Glu1133.28 as the counterion, and T94Iremoves a potentially stabilizing hydrogenbond with Glu1133.28. Night blindness resultsfrom rod cells adapting to what they sense as alight signal generated by the mutant opsins anddesensitizing until the cell no longer responds.

ADRP is a more severe degenerative dis-ease of the retina that has more than one cause.Mutations in the visual receptor rhodopsin ac-count for ∼30% of ADRP cases (47). Most of

the ADRP mutations appear to cause destabi-lization of the opsin structure. For example,Gly511.46 and Ala1644.53 are group-conservedsites where the ADRP mutation results in ther-mally destabilized receptors (7). In contrast, theK296E and K296M ADRP mutations result inconstitutive activity (18, 48) because they elim-inate the Glu1133.28-Lys2967.43 salt bridge andcannot bind retinal. However, unlike the situ-ation for night blindness, receptors containingthese mutations eventually get shut down ow-ing to phosphorylation and irreversible bindingof arrestin.

The constitutively active mutations thatbreak the Glu1133.28-Lys2967.43 salt bridge sug-gest that this protonation switch holds H6 inan inactive position in the absence of reti-nal. Below, I discuss other constitutively ac-tive mutations in rhodopsin that are within the


Ann

u. R

ev. B

ioph

ys. 2

010.

39:3

09-3

28. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by W

IB60

13 -

Fre

ie U

nive

rsita

et B

erlin

- F

U B

erlin

on

11/2

2/11

. For

per

sona

l use

onl

y.

ANRV411-BB39-16 ARI 2 April 2010 11:11

cytoplasmic ionic lock (Glu1343.49) and at thecontact point between H6 and the conservedtransmembrane core (Met2576.40).

STRUCTURAL CHANGES UPONRECEPTOR ACTIVATION

Within the transmembrane domain ofrhodopsin, roughly one-third of the aminoacids are conserved at one of three levels ofconservation presented in Figure 2. Thissection discusses the signaling pathway fromthe retinal binding pocket through the trans-membrane helices to the cytoplasmic ioniclock, with an emphasis on explaining aminoacid conservation.

Retinal Isomerization in a TightProtein Binding Pocket

Retinal isomerization controls the first proto-nation switch (PS1) shown in Figure 4b (i.e.,the internal proton transfer from the retinalPSB to Glu1133.28), as well as facilitates the mo-tion of H5 and H6. In the dark state, the retinalbinding site is shaped to accommodate the bent11-cis isomer of retinal, but not the straight all-trans form (34). Within this restricted space,the rapid 11-cis to all-trans isomerization trig-gers receptor activation by forcing the proteinto adapt to the all-trans isomer.

In the initial photoreaction, the largest dis-placement of the retinal involves rotation ofthe C20 methyl group toward EL2 (36, 53).A cavity within the retinal binding pocket ex-tends toward EL2 and provides a pathway forthe motion of this methyl group. NMR exper-iments indicate that the final position of theC20 methyl group in the active Meta II inter-mediate is close to Gly1143.29, suggesting thatit has moved past EL2 during isomerization(1). The trajectory of the C20 methyl groupsuggests that the N-H proton of the retinalSchiff’s base rotates toward the protein inte-rior. Displacement of the Schiff’s base protoninto a more hydrophobic environment wouldlower the Schiff’s base pKa and induce depro-tonation. A similar mechanism for controlling

the protonation state of the Schiff’s base oc-curs in bacteriorhodopsin, a light-driven pro-ton pump. These observations indicate that theprotein binding pocket has evolved to guide theisomerization trajectory as a way to increasethe quantum yield, to lower the Schiff’s basepKa, and possibly to guide steric contacts withthe surrounding protein. When the retinal C20methyl group is removed, the photoreaction isslowed (61) and the quantum yield is reduced(28).

The retinal C19 methyl group in rhodopsinis tightly constrained in the retinal binding site,where it packs against Thr1183.33, Ile189EL2,Tyr191EL2, and Tyr2686.51. Tyr191EL2 andTyr2686.51 are part of the hydrogen-bondingnetwork connecting EL2 to the retinal PSB-Glu1133.28 protonation switch. Tyr2686.51 ishighly conserved in the rhodopsin subfamilyand C19 is essential for activation. Remov-ing the C19 methyl group (11) or mutatingTyr2686.51 to phenylalanine (38) reduces trans-ducin activation. NMR chemical shift mea-surements have shown that the hydrogen-bonding interactions involving Tyr2686.51 andTyr191EL2 are altered in Meta II (2), possiblybecause of their direct steric contact with theC19 methyl group during the rhodopsin pho-toreaction. In addition, NMR distance mea-surements have shown that the β4-strand ofEL2 is displaced from the retinal binding sitein Meta II (2) (Figure 5b). Increasing the sizeof the C19 methyl group to an ethyl or propylgroup results in a small level of receptor activity,even though the retinal is still covalently boundand in its 11-cis conformation (19). This darkactivity is consistent with a direct steric inter-action between the C19 methyl group and EL2in the activation mechanism.

Coupling of Retinal Isomerizationto Helix Motion

Retinal isomerization leads to strong steric in-teractions within the protein binding site anddeprotonation of the Schiff’s base, which to-gether serve to trigger the motion of H5 andH6. Motion of the extracellular end of H5

318 Smith

Ann

u. R

ev. B

ioph

ys. 2

010.

39:3

09-3

28. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by W

IB60

13 -

Fre

ie U

nive

rsita

et B

erlin

- F

U B

erlin

on

11/2

2/11

. For

per

sona

l use

onl

y.

ANRV411-BB39-16 ARI 2 April 2010 11:11

H5

EL2

H7

H5

H6

Tyr206 Trp126

Ser186

Asn302

a

c

H6

Met207

His211

Tyr268

H2O

Retinal

Ala295

Trp265

Ile289

Gly188Cys187

H3

H7

Gly121

Retinal Trp265

Ala295

Lys296

Phe208

His211

Glu122

b

d

H2O

Figure 5Retinal motion within the binding site triggers protein conformational changes. (a) Side view of the retinal binding site in rhodopsinshowing retinal interactions with EL2 and H5. (b) Side view of the retinal binding site in a model of Meta II derived from guidedmolecular dynamics simulations (1). NMR distance constraints used in the simulations reveal that EL2 is displaced and Trp2656.48

rotates toward the extracellular surface. Tyr2686.51 has the highest subfamily-specific conservation in the rhodopsin subfamily and is ina key position between EL2 and the retinal to form stabilizing contacts in both the inactive and active conformations of rhodopsin.(c) View of the retinal binding site in rhodopsin from the extracellular surface. Trp2656.48 packs between two highly conserved residues,Gly1213.36 and Ala2957.42. Gly1213.36 has the second highest rhodopsin subfamily-specific conservation. Mutation of Gly1213.36 toresidues with larger side chains results in dark activity of rhodopsin, consistent with the observation that Trp2656.48 is displaced uponactivation (19). (d ) View from the extracellular surface in a model of Meta II derived from guided molecular dynamics simulationsshowing that motion of the β-ionone ring toward H5 leads to a rearrangement of the hydrogen-bonding interactions involvingHis2115.38.


Ann

u. R

ev. B

ioph

ys. 2

010.

39:3

09-3

28. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by W

IB60

13 -

Fre

ie U

nive

rsita

et B

erlin

- F

U B

erlin

on

11/2

2/11

. For

per

sona

l use

onl

y.

ANRV411-BB39-16 ARI 2 April 2010 11:11

results from displacement of EL2 (2) and trans-lation of the retinal β-ionone ring in Meta II(1) (Figure 5d ). When viewed from the ex-tracellular surface of rhodopsin, the β-iononering is packed against Glu1223.37. Retinal move-ment disrupts the hydrogen bond between themain chain carbonyl of His2115.38 and the sidechain of Glu1223.37, and a new hydrogen bondforms between Glu1223.37 and the imidazoleδ-nitrogen of His2115.38. These interactions ex-plain the requirement for an intact retinal β-ionone ring for rhodopsin activation (22, 60)and the role of the His2115.38 side chain in MetaII stability and activation (30).

The switch for H6 motion has long beenassociated with a conserved aromatic clusteron the extracellular end of the helix (54).In rhodopsin, the aromatic cluster is formedby three residues with relatively high iden-tity across the class A GPCRs: Trp2656.48,Phe2616.44, and Tyr2686.51. Trp2656.48 lieswithin the arc formed by the retinal polyenechain and the Lys2967.43 side chain, and ispacked between Gly1213.36 and Ala2957.42. Theretinal appears to function as a clamp to prevent

motion of Trp2656.48 and H6. Isomerization ofthe retinal and motion of the β-ionone ringtoward H5 appear to be essential for rotationof Trp2656.48 toward the extracellular surface(12).

Figure 6a shows the signaling pathway fromTrp2656.48 through Asn3027.49 to Tyr3067.53.Asn3027.49 is not directly hydrogen bondedto polar residues within the protein interior;rather a shell of water molecules surroundsthe Asn3027.49 side chain. Trp2656.48 is con-nected to Asn3027.49 through water-mediatedhydrogen bonds. Asn3027.49 is part of theconserved transmembrane core within GPCRsthat appears to direct the outward rotation ofH6. The sequence of events following retinalisomerization is likely simple. The rotationof Trp2656.48 disrupts the hydrogen-bondingcontacts with Asn3027.49 (45). One can specu-late that the Asn3027.49 side chain then rotatestoward the conserved Asp832.54 on H2. Such arotation is consistent with an observed increasein hydrogen bonding of the Asp832.54 sidechain in the Meta I to Meta II transition (16).Motion of Asn3027.49 then sets the stage for

Gly51Ala299

Asn55

Ala80

Ala82

Trp161

Asp83

Ile123

Ala124

Leu128

Met257

Leu79

Asn302

Trp265

Ala295

Met257

Asn302

Gly51

Asn55

Phe313

Asn73

Tyr306

Ala299

Asp83

a b

H6

H7

H1

H7

H1

H4

H6

H8

Figure 6Signal transduction pathway through the conserved transmembrane core of rhodopsin. (a) View of the rhodopsin crystal structureshowing the connection of Trp2656.48 with Asn3027.49 of the NPxxY sequence. Asn3027.49 and Tyr3067.53 are signature residues on H7that form stabilizing interactions with subfamily-specific residues ( green) on H6 and H2, respectively. Tyr3067.53 rotates into the spaceoccupied by Met2576.40 upon activation (43, 51). (b) View of the conserved core of rhodopsin from the extracellular side of the receptorshowing the relative positions of the signature (red ) and group-conserved (blue) amino acids.

320 Smith

Ann

u. R

ev. B

ioph

ys. 2

010.

39:3

09-3

28. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by W

IB60

13 -

Fre

ie U

nive

rsita

et B

erlin

- F

U B

erlin

on

11/2

2/11

. For

per

sona

l use

onl

y.

ANRV411-BB39-16 ARI 2 April 2010 11:11

the outward rotation of the cytoplasmic end ofH6 (see below).

H5, H6, and H7 each contains a highlyconserved proline (Pro2155.42, Pro2676.50, andPro3037.50) in the middle of the transmembranesequence. The prolines are thought to facilitatehelix motion. There are three additional pro-lines (Pro1704.59, Pro1714.60, and Pro2917.38)with high subfamily-specific conservation. Thedefining feature of a proline in transmembranehelices is that it is not able to form a back-bone hydrogen bond to the carbonyl group onehelical turn away. In the case of Pro1714.60,Pro2155.42, and Pro3037.50, the free backbonecarbonyl makes key hydrogen-bonding con-tacts. However, the free backbone carbonylsassociated with Pro2676.50 and Pro2917.38 areoriented toward the lipids and do not formhydrogen bonds in the crystal structures ofrhodopsin and opsin, suggesting that they al-low the helical segments to easily swivel. Theseobservations raise the possibility that the ex-tracellular helix-loop-helix segment stretchingfrom Pro2676.50 to Pro2917.38 pivots upon reti-nal isomerization. Coordinated motion of theextracellular ends of H6 and H7 is part of aglobal toggle switch mechanism proposed forGPCR activation (15).

Disruption of the Ionic Lockand G Protein Binding

The motion of H5, H6, and H7 con-verge on the Arg1353.50-Glu2476.30 ioniclock on the intracellular side of rhodopsin.Three residues contribute directly to disrupt-ing the ionic lock upon rhodopsin activa-tion: Tyr2235.50, Met2576.40, and Tyr3067.53.Tyr2235.50 is oriented toward the surround-ing lipid in rhodopsin and rotates towardArg1353.50 in Meta II. The Tyr2235.50 side chainin the active conformation observed in the opsincrystal structure is packed against Ala1323.47,a group-conserved residue, suggesting that thesmall side chain at this position creates a pocketfor orienting the tyrosine side chain.

Met2576.40 on H6 shifts from its locationnext to Asn3027.49 in the transmembrane core

to a position close to Arg1353.50 as H6 rotatesoutward (Figure 7). This motion allows twoadditional salt bridges to form on the intracel-lular surface of the receptor: Glu2476.30-Lys2315.58 and Glu2496.28-Lys311CT

(Figure 7c). In rhodopsin, the position ofTyr3067.53 is constrained by hydrogen bondingwith Asn732.44 and Phe313H8, both subfamily-conserved residues. Tyr3067.53 moves into thehydrophobic pocket vacated by Met2576.40

when H6 rotates outward (Figure 7b).The ability of the receptor structure to bind

the C terminus of the α-subunit of transducinwas shown by the opsin structure in complexwith a synthetic peptide (51). The Gα peptidebinds to opsin in an α-helical conformation ina site opened by the outward rotation of H6(Figure 7d ). An important unanswered ques-tion is how receptor binding propagates a signalfrom the receptor surface to the GDP bindingsite of the G protein to facilitate GDP ⇒ GTPexchange.

RHODOPSIN AS A MODELFOR LIGAND-ACTIVATEDG PROTEIN–COUPLEDRECEPTORS

The activation mechanism that emerges fromstructural and functional studies on rhodopsinprovides an explanation for how the conservedresidues interact to create a light-activatedGPCR. Consideration of the different levels ofconservation reveals how the visual receptorsare designed to be locked in an inactive con-formation in the dark and to rapidly shift to afully active conformation in the light. In thislast section, I describe some of the similaritiesand differences between the visual and ligand-activated receptors to illustrate how differencesthat occur at all three levels of conservation giveeach receptor subfamily its distinct twist on acommon structure and activation mechanism.

Parallels Between Rhodopsinand Ligand-Activated GPCRs

Over the past two years, the crystal structuresof several GPCRs in the amine subfamily of


Ann

u. R

ev. B

ioph

ys. 2

010.

39:3

09-3

28. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by W

IB60

13 -

Fre

ie U

nive

rsita

et B

erlin

- F

U B

erlin

on

11/2

2/11

. For

per

sona

l use

onl

y.

ANRV411-BB39-16 ARI 2 April 2010 11:11

a

c

b

d

Met257

Tyr306

H8

Lys311

Phe313

H6Glu247

Thr251

Tyr223

H5

H3

Tyr136

Glu134

H7

Arg135Arg135Arg135

Lys311

Glu247

Glu249

Lys231

Tyr306Met257

Tyr223

Gα peptide

Glu134

Tyr136

Tyr223

Glu247

Met257

Tyr306

Arg135

Arg135

Tyr306 Tyr223

H6H5

H3

H7

H8

H6H5

H3

H7H5

H8

H7

Phe313

Figure 7The Arg1353.50-Glu2476.30 ionic lock in rhodopsin. (a) View of the intracellular side of rhodopsin in region of the ionic lock.Glu1347.49 is charged in the dark state and stabilizes the Arg1353.50-Glu2476.30 interaction. Mutation of Glu1347.49 to glutamineresults in constitutive activity. (b) View from the extracellular surface showing that rotation of Tyr2235.50, Met2576.40, and Tyr3067.53

contributes to breaking the ionic lock between Arg1353.50 and Glu2476.30. The figure presents a superposition of the rhodopsin ( grayhelices and gray signature residues) and opsin (orange helices and red signature residues) crystal structures. (c) View of the intracellular side ofopsin in the region of the ionic lock. (d ) View from the intracellular surface of opsin showing the position of the C-terminal Gα peptide(blue spheres).

receptors have been determined with eitherantagonists or partial inverse agonists bound(9, 21, 62). The structures of these receptorsare remarkably similar to that of the dark-stateof rhodopsin containing the 11-cis retinal PSBchromophore.

The conserved transmembrane core of theβ2-adrenoreceptor is shown in Figure 8.Importantly, the intricate packing within the

cluster of signature and group-conservedresidues shown in Figure 6b for rhodopsin isalso observed in the β2-adrenoreceptor. How-ever, there are three key differences betweenrhodopsin and the β2-adrenoreceptor. First, thegroup-conserved residue at position 1.46 is anisoleucine, which is an exception to the smalland weakly polar residue type that typically oc-curs at this position in class A GPCRs, and may

322 Smith

Ann

u. R

ev. B

ioph

ys. 2

010.

39:3

09-3

28. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by W

IB60

13 -

Fre

ie U

nive

rsita

et B

erlin

- F

U B

erlin

on

11/2

2/11

. For

per

sona

l use

onl

y.

ANRV411-BB39-16 ARI 2 April 2010 11:11

Ile47

Ser319

Asn51

Ala76

Ala78

Trp158

Asp79

Ala119

Ser120

Leu124

Ile278

Leu75

Asn322

a b

H7

H1

H3

H4

H6

Trp286

Gly315

Ile278

Asn318

Ile47

Asn51

Phe332

Asn69

Tyr326

Ser319

Asp79

H6

H7

H1

Asn322

H8

Figure 8The signaling pathway and conserved core in the β2-adrenoreceptor. (a) View of the β2-adrenoreceptor crystal structure showing theconnection of Trp2866.48 with Asn3227.49 of the NPxxY sequence. Comparison with Figure 6a reveals a common structure.(b) Conserved core of the β2-adrenoreceptor showing the relative positions of the signature-conserved (red ), group-conserved (blue),and subfamily-specific ( green) amino acids.

be responsible for a shift in the position of H1between rhodopsin and the β2-adrenoreceptor.Second, an asparagine (Asn7.45), which is con-served in the amine subfamily, is positionedbetween Trp2866.48 and Asn3227.49 in the β2-adrenoreceptor (Figure 8a) and likely medi-ates their interaction. The amino acid typeat position 7.45 tends to reflect the specificsubfamily. For example, histidine is conserved(93.4%) at position 7.45 in the chemokine re-ceptors. Third, the subfamily-specific residueat position 6.40 is a methionine in rhodopsinbut an isoleucine in the β2-adrenoreceptor.β-branched amino acids tend to be commonat this position in the class A GPCRs, possiblybecause of their restricted motion in α-helicalsecondary structure.

Converting Rhodopsin into aLigand-Activated GPCR

One of the strongest arguments that the vi-sual receptor rhodopsin can serve as a modelclass A GPCR is that a single mutationin the transmembrane core can convert the

receptor into a ligand-activated receptor. Mu-tation of Met2576.40 allows activation of opsinby the addition of all-trans retinal as a diffusibleligand (20). Most substitutions of Met2576.40

exhibit low, but measurable, activity withoutbound retinal. These receptors are locked offby covalent attachment of the inverse ago-nist 11-cis retinal but exhibit activities com-parable to the light-activated receptor uponbinding the agonist all-trans retinal. For ex-ample, the M257I mutation (where isoleucineis the wild-type residue in this position inmany class A GPCRs) exhibits 4.4% opsin ac-tivity, 1.0% activity with bound 11-cis retinal,and 71% activity with the addition of all-transretinal. These results suggest that methionineat position 257 stabilizes the inactive state ofrhodopsin, whereas isoleucine allows H6 to eas-ily rotate into an active orientation upon lig-and binding. More polar substitutions at posi-tion 257 in the M257Y, M257N, and M257Smutants appear to stabilize a direct interactionwith Arg1353.50, hold H6 in an active orien-tation, and result in high levels of constitutiveactivity.


Ann

u. R

ev. B

ioph

ys. 2

010.

39:3

09-3

28. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by W

IB60

13 -

Fre

ie U

nive

rsita

et B

erlin

- F

U B

erlin

on

11/2

2/11

. For

per

sona

l use

onl

y.

ANRV411-BB39-16 ARI 2 April 2010 11:11

Subfamily-Specific Residues:Evolution of the GPCRs

One of the future challenges in understand-ing GPCR activation is to establish how eachsubfamily has modified the class A GPCRframework to recognize and respond to itsown unique set of ligands. In this review, Ihave emphasized the importance of the PSB-Glu1133.28 salt bridge, the Trp2656.48 rotamertoggle switch, and the Arg1353.50-Glu2476.30

ionic lock in controlling rhodopsin activa-tion. Yet, the PSB-Glu1133.28 salt bridge andGlu2476.30 in the intracellular ionic lock areonly subfamily conserved, whereas Trp2656.48

with an overall conservation of 69.2% falls justshort of being a true signature residue. Thelack of high conservation at these sites suggeststhat different subfamilies have evolved varia-tions on the rhodopsin framework for both lig-and recognition and activation.

The regions on the extracellular side of thereceptor that recognize and bind ligands arelikely to be appreciably different between dif-ferent subfamilies. For example, in the crys-tal structures solved to date, EL2 adopts dif-ferent folds. In the β2-adrenoreceptor, EL2has a short helical segment (9), whereas in theA2A adenosine receptor EL2 lacks secondarystructure (21). Nevertheless, structure-functionstudies of ligand-activated GPCRs show thatEL2 mediates activation in many specific cases

(see Reference 2). For example, in the free fattyacid receptor, there appear to be two glutamicacid residues in EL2 that form ionic locks withtwo arginine residues, Arg1835.31 in H5 andArg2587.35 in H7 (58). Mutation of either ofthe glutamates to alanine results in constitutiveactivity.

In many receptor subfamilies, the classicionic lock between Arg3.50 and Glu6.30 is ab-sent. For example, in the chemokine recep-tors, the intracellular end of H6 is highly pos-itively charged and lacks a negatively chargedresidue at or near position 6.30. Instead, thereare two highly subfamily-conserved residues(Thr2.43, 97%; Asp2.44, 85%) at the intracel-lular end of H2 that are positioned close toArg3.50 in homology models of the chemokinereceptors. These residues may replace Glu6.30

to stabilize the inactive receptor conformation.Chemokine receptors also have low conserva-tion (52%) of Tyr7.53 in the NPxxY motif on H7,consistent with a different type of intracellu-lar ionic lock. Nevertheless, conservation of thetransmembrane core and the conserved Tyr5.50

on H5 suggests that the signaling pathway is notdramatically different from that described forrhodopsin. These and other examples empha-size the interplay of the signature-conserved,group-conserved, and subfamily-specific aminoacids in our understanding of the similaritiesand differences of how the class A GPCRs areactivated.

SUMMARY POINTS

1. Retinal isomerization leads to steric strain within the retinal binding site and internal pro-ton transfer from the retinal PSB to Glu1133.28. These changes trigger the displacementof EL2 and the motion of transmembrane helices H5 and H6.

2. Motion of the β-ionone ring is coupled to the rotation of Trp2656.48 toward the extracel-lular surface, which in turn changes the hydrogen-bonding environment of Asn3027.49

in the conserved transmembrane core.

3. Rotation of Tyr2235.50, Met2576.40, and Tyr3067.53 converges to disrupt the intracellularionic lock between H3 and H6.

4. The activation mechanism that emerges from structural and functional studies onrhodopsin provides an explanation for how the signature-conserved, group-conserved,and subfamily-specific residues interact to create light-activated GPCRs.

324 Smith

Ann

u. R

ev. B

ioph

ys. 2

010.

39:3

09-3

28. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by W

IB60

13 -

Fre

ie U

nive

rsita

et B

erlin

- F

U B

erlin

on

11/2

2/11

. For

per

sona

l use

onl

y.

ANRV411-BB39-16 ARI 2 April 2010 11:11

FUTURE ISSUES

1. The role of water in receptor activation is not yet understood. A large increase in enthalpyoccurs in the Meta I to Meta II transition, which is compensated by a large increase inentropy. The nature of the entropy increase has not been established but may be relatedto the release of water in the Meta I to Meta II transition.

2. The role of receptor dynamics and the membrane environment are not fully understood.Rhodopsin requires a fluid membrane environment, and specific lipids can modulatereceptor activity. Does the motion of EL2 precede or occur simultaneously with themotion of H5 and H6? Do H5 and H6 move in a concerted fashion or sequentially?

3. The nature of how the full heterotrimeric G protein binds to the intracellular surfaceand is activated by rhodopsin has yet to be determined. Similarly, it is not known howactivated rhodopsin is recognized by and interacts with rhodopsin kinase.

DISCLOSURE STATEMENT

The author is not aware of any affiliations, memberships, funding, or financial holdings that mightbe perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

I thank the many colleagues and students that contributed to the research underlying this re-view, including Shivani Ahuja, Prashen Chelikani, Evan Crocker, Markus Eilers, Sina Erfani, JoeGoncalves, Viktor Hornak, H. Gobind Khorana, Brian Kobilka, Ashish Patel, Phil Reeves, TomSakmar, Mudi Sheves, Reiner Vogel, Elsa Yan, and Martine Ziliox. This work was supported by agrant from the National Institutes of Health (RO1-GM41412).

LITERATURE CITED

1. Ahuja S, Crocker E, Eilers M, Hornak V, Hirshfeld A, et al. 2009. Location of the retinal chromophorein the activated state of rhodopsin. J. Biol. Chem. 284:10190–201

2. Ahuja S, Hornak V, Yan ECY, Syrett N, Goncalves JA, et al. 2009. Helix movement is coupled to dis-placement of the second extracellular loop in rhodopsin activation. Nat. Struct. Mol. Biol. 16:168–75

3. Ballesteros J, Kitanovic S, Guarnieri F, Davies P, Fromme BJ, et al. 1998. Functional microdomains inG-protein-coupled receptors—the conserved arginine-cage motif in the gonadotropin-releasing hormonereceptor. J. Biol. Chem. 273:10445–53

4. Ballesteros JA, Shi L, Javitch JA. 2001. Structural mimicry in G protein-coupled receptors: implicationsof the high-resolution structure of rhodopsin for structure-function analysis of rhodopsin-like receptors.Mol. Pharmacol. 60:1–19

5. Baylor DA. 1987. Photoreceptor signals and vision. Invest. Ophthalmol. Vis. Sci. 28:34–496. Becker RS, Freedman K. 1985. A comprehensive investigation of the mechanism and photophysics of

isomerization of a protonated and unprotonated Schiff-base of 11-cis-retinal. J. Am. Chem. Soc. 107:1477–85

7. Bosch L, Ramon E, del Valle LJ, Garriga P. 2003. Structural and functional role of helices I and IIin rhodopsin—a novel interplay evidenced by mutations at Gly-51 and Gly-89 in the transmembranedomain. J. Biol. Chem. 278:20203–9

8. Chabre M. 1985. Trigger and amplification mechanisms in visual phototransduction. Annu. Rev. Biophys.Biophys. Chem. 14:331–60


Ann

u. R

ev. B

ioph

ys. 2

010.

39:3

09-3

28. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by W

IB60

13 -

Fre

ie U

nive

rsita

et B

erlin

- F

U B

erlin

on

11/2

2/11

. For

per

sona

l use

onl

y.

ANRV411-BB39-16 ARI 2 April 2010 11:11

9. Cherezov V, Rosenbaum DM, Hanson MA, Rasmussen SGF, Thian FS, et al. 2007. High-resolutioncrystal structure of an engineered human β2-adrenergic G protein-coupled receptor. Science 318:1258–65

10. Cooper A. 1979. Energy uptake in the first step of visual excitation. Nature 282:531–3311. Corson DW, Cornwall MC, MacNichol EF, Tsang S, Derguini F, et al. 1994. Relief of opsin desensiti-

zation and prolonged excitation of rod photoreceptors by 9-desmethylretinal. Proc. Natl. Acad. Sci. USA91:6958–62

12. Crocker E, Eilers M, Ahuja S, Hornak V, Hirshfeld A, et al. 2006. Location of Trp265 in metarhodopsinII: implications for the activation mechanism of the visual receptor rhodopsin. J. Mol. Biol. 357:163–72

13. Dixon RAF, Kobilka BK, Strader DJ, Benovic JL, Dohlman HG, et al. 1986. Cloning of the gene andcDNA for mammalian β-adrenergic receptor and homology with rhodopsin. Nature 321:75–78

14. Eilers M, Patel AB, Liu W, Smith SO. 2002. Comparison of helix interactions in membrane and solubleα-bundle proteins. Biophys. J. 82:2720–36

15. Elling CE, Frimurer TM, Gerlach LO, Jorgensen R, Holst B, Schwartz TW. 2006. Metal ion site en-gineering indicates a global toggle switch model for seven-transmembrane receptor activation. J. Biol.Chem. 281:17337–46

16. Fahmy K, Jager F, Beck M, Zvyaga TA, Sakmar TP, Siebert F. 1993. Protonation states of membrane-embedded carboxylic acid groups in rhodopsin and metarhodopsin II: a Fourier-transform infrared spec-troscopy study of site-directed mutants. Proc. Natl. Acad. Sci. USA 90:10206–10

17. Farrens DL, Altenbach C, Yang K, Hubbell WL, Khorana HG. 1996. Requirement of rigid-body motionof transmembrane helices for light activation of rhodopsin. Science 274:768–70

18. Govardhan CP, Oprian DD. 1994. Active site-directed inactivation of constitutively active mutants ofrhodopsin. J. Biol. Chem. 269:6524–27

19. Han M, Groesbeek M, Sakmar TP, Smith SO. 1997. The C9 methyl group of retinal interacts withglycine-121 in rhodopsin. Proc. Natl. Acad. Sci. USA 94:13442–47

20. Han M, Smith SO, Sakmar TP. 1998. Constitutive activation of opsin by mutation of methionine 257 ontransmembrane helix 6. Biochemistry 37:8253–61

21. Jaakola VP, Griffith MT, Hanson MA, Cherezov V, Chien EYT, et al. 2008. The 2.6 Angstrom crystalstructure of a human A2A adenosine receptor bound to an antagonist. Science 322:1211–17

22. Jager F, Jager S, Krautle O, Friedman N, Sheves M, et al. 1994. Interactions of the β-ionone ring withthe protein in the visual pigment rhodopsin control the activation mechanism. An FTIR and fluorescencestudy on artificial vertebrate rhodopsins. Biochemistry 33:7389–97

23. Janz JM, Fay JF, Farrens DL. 2003. Stability of dark state rhodopsin is mediated by a conserved ion pairin intradiscal loop E-2. J. Biol. Chem. 278:16982–91

24. Jin SN, Cornwall MC, Oprian DD. 2003. Opsin activation as a cause of congenital night blindness.Nat. Neurosci. 6:731–35

25. Karnik SS, Khorana HG. 1990. Assembly of functional rhodopsin requires a disulfide bond betweencysteine residues 110 and 187. J. Biol. Chem. 265:17520–24

26. Kaushal S, Ridge KD, Khorana HG. 1994. Structure and function in rhodopsin: the role of asparagine-linked glycosylation. Proc. Natl. Acad. Sci. USA 91:4024–28

27. Knierim B, Hofmann KP, Ernst OP, Hubbell WL. 2007. Sequence of late molecular events in theactivation of rhodopsin. Proc. Natl. Acad. Sci. USA 104:20290–95

28. Kochendoerfer GG, Verdegem PJE, Van Der Hoef I, Lugtenburg J, Mathies RA. 1996. Retinal analogstudy of the role of steric interactions in the excited state isomerisation dynamics of rhodopsin. Biochemistry35:16230–40

29. Lagerstrom MC, Schioth HB. 2008. Structural diversity of G protein-coupled receptors and significancefor drug discovery. Nat. Rev. Drug Discov. 7:339–57

30. Lewis JW, Szundi I, Kazmi MA, Sakmar TP, Kliger DS. 2006. Proton movement and photointermediatekinetics in rhodopsin mutants. Biochemistry 45:5430–39

31. Li J, Edwards PC, Burghammer M, Villa C, Schertler GFX. 2004. Structure of bovine rhodopsin in atrigonal crystal form. J. Mol. Biol. 343:1409–38

32. Liu W, Eilers M, Patel AB, Smith SO. 2004. Helix packing moments reveal diversity and conservation inmembrane protein structure. J. Mol. Biol. 337:713–29

326 Smith

Ann

u. R

ev. B

ioph

ys. 2

010.

39:3

09-3

28. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by W

IB60

13 -

Fre

ie U

nive

rsita

et B

erlin

- F

U B

erlin

on

11/2

2/11

. For

per

sona

l use

onl

y.

ANRV411-BB39-16 ARI 2 April 2010 11:11

33. Mahalingam M, Martinez-Mayorga K, Brown MF, Vogel R. 2008. Two protonation switches controlrhodopsin activation in membranes. Proc. Natl. Acad. Sci. USA 105:17795–800

34. Matsumoto H, Yoshizawa T. 1978. Recognition of opsin to longitudinal length of retinal isomers information of rhodopsin. Vision Res. 18:607–9

35. Mitchell DC, Litman BJ. 2000. Effect of ethanol and osmotic stress on receptor conformation—Reducedwater activity amplifies the effect of ethanol on metarhodopsin II formation. J. Biol. Chem. 275:5355–60

36. Nakamichi H, Okada T. 2006. Crystallographic analysis of primary visual photochemistry. Angew. Chem.Int. Ed. Engl. 45:4270–73

37. Nakamichi H, Okada T. 2006. Local peptide movement in the photoreaction intermediate of rhodopsin.Proc. Natl. Acad. Sci. USA 103:12729–34

38. Nakayama TA, Khorana HG. 1991. Mapping of the amino acids in membrane-embedded helices thatinteract with the retinal chromophore in bovine rhodopsin. J. Biol. Chem. 266:4269–75

39. Ohguro H, Rudnicka-Nawrot M, Buczylko J, Zhao X, Taylor JA, et al. 1996. Structural and enzymaticaspects of rhodopsin phosphorylation. J. Biol. Chem. 271:5215–24

40. Okada T, Sugihara M, Bondar AN, Elstner M, Entel P, Buss V. 2004. The retinal conformation and itsenvironment in rhodopsin in light of a new 2.2 A crystal structure. J. Mol. Biol. 342:571–83

41. Palczewski K. 2006. G protein-coupled receptor rhodopsin. Annu. Rev. Biochem. 75:743–6742. Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, et al. 2000. Crystal structure of rhodopsin:

a G protein-coupled receptor. Science 289:739–4543. Park JH, Scheerer P, Hofmann KP, Choe HW, Ernst OP. 2008. Crystal structure of the ligand-free

G-protein-coupled receptor opsin. Nature 454:183–8744. Park PSH, Sapra KT, Jastrzebska B, Maeda T, Maeda A, et al. 2009. Modulation of molecular interactions

and function by rhodopsin palmitylation. Biochemistry 48:4294–30445. Patel AB, Crocker E, Reeves PJ, Getmanova EV, Eilers M, et al. 2005. Changes in interhelical hydrogen

bonding upon rhodopsin activation. J. Mol. Biol. 347:803–1246. Rao VR, Oprian DD. 1996. Activating mutations of rhodopsin and other G protein-coupled receptors.

Annu. Rev. Biophys. Biomol. Struct. 25:287–31447. Rattner A, Sun H, Nathans J. 1999. Molecular genetics of human retinal disease. Annu. Rev. Genet. 33:89–

13148. Robinson PR, Cohen GB, Zhukovsky EA, Oprian DD. 1992. Constitutively active mutants of rhodopsin.

Neuron 9:719–2549. Ruprecht JJ, Mielke T, Vogel R, Villa C, Schertler GFX. 2004. Electron crystallography reveals the

structure of metarhodopsin I. EMBO J. 23:3609–2050. Sakmar TP, Menon ST, Marin EP, Awad ES. 2002. Rhodopsin: insights from recent structural studies.

Annu. Rev. Biophys. Biomol. Struct. 31:443–8451. Scheerer P, Park JH, Hildebrand PW, Kim YJ, Krauss N, et al. 2008. Crystal structure of opsin in its

G-protein-interacting conformation. Nature 455:497–50252. Schoenlein RW, Peteanu LA, Mathies RA, Shank CV. 1991. The first step in vision: femtosecond iso-

merization of rhodopsin. Science 254:412–1553. Schreiber M, Sugihara M, Okada T, Buss V. 2006. Quantum mechanical studies on the crystallographic

model of bathorhodopsin. Angew. Chem. Int. Ed. Engl. 45:4274–7754. Shi L, Liapakis G, Xu R, Guarnieri F, Ballesteros JA, Javitch JA. 2002. β2 adrenergic receptor activation—

modulation of the proline kink in transmembrane 6 by a rotamer toggle switch. J. Biol. Chem. 277:40989–9655. Struts AV, Salgad GFJ, Tanaka K, Krane S, Nakanishi K, Brown MF. 2007. Structural analysis and dynam-

ics of retinal chromophore in dark and metal states of rhodopsin from 2H NMR of aligned membranes.J. Mol. Biol. 372:50–66

56. Stryer L. 1986. Cyclic GMP cascade of vision. Annu. Rev. Neurosci. 9:87–11957. Sugihara M, Hufen J, Buss V. 2006. Origin and consequences of steric strain in the rhodopsin binding

pocket. Biochemistry 45:801–1058. Sum CS, Tikhonova IG, Costanzi S, Gershengorn MC. 2009. Two arginine-glutamate ionic locks near

the extracellular surface of FFAR1 gate receptor activation. J. Biol. Chem. 284:3529–3659. Visiers I, Ballesteros JA, Weinstein H. 2002. Three-dimensional representations of G protein-coupled

receptor structures and mechanisms. Methods Enzymol. 343:329–71


Ann

u. R

ev. B

ioph

ys. 2

010.

39:3

09-3

28. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by W

IB60

13 -

Fre

ie U

nive

rsita

et B

erlin

- F

U B

erlin

on

11/2

2/11

. For

per

sona

l use

onl

y.

ANRV411-BB39-16 ARI 2 April 2010 11:11

60. Vogel R, Siebert F, Ludeke S, Hirshfeld A, Sheves M. 2005. Agonists and partial agonists of rhodopsin:retinals with ring modifications. Biochemistry 44:11684–99

61. Wang Q, Kochendoerfer GG, Schoenlein RW, Verdegem PJE, Lugtenburg J, et al. 1996. Femtosecondspectroscopy of a 13-demethylrhodopsin visual pigment analogue: the role of nonbonded interactions inthe isomerization process. J. Phys. Chem. 100:17388–94

62. Warne T, Serrano-Vega MJ, Baker JG, Moukhametzianov R, Edwards PC, et al. 2008. Structure of aβ1-adrenergic G-protein-coupled receptor. Nature 454:486–91

328 Smith

Ann

u. R

ev. B

ioph

ys. 2

010.

39:3

09-3

28. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by W

IB60

13 -

Fre

ie U

nive

rsita

et B

erlin

- F

U B

erlin

on

11/2

2/11

. For

per

sona

l use

onl

y.

AR411-FM ARI 2 April 2010 20:22

Annual Review ofBiophysics

Volume 39, 2010Contents

Adventures in Physical ChemistryHarden McConnell � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Global Dynamics of Proteins: Bridging Between Structureand FunctionIvet Bahar, Timothy R. Lezon, Lee-Wei Yang, and Eran Eyal � � � � � � � � � � � � � � � � � � � � � � � � � � � � �23

Simplified Models of Biological NetworksKim Sneppen, Sandeep Krishna, and Szabolcs Semsey � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �43

Compact Intermediates in RNA FoldingSarah A. Woodson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �61

Nanopore Analysis of Nucleic Acids Bound to Exonucleasesand PolymerasesDavid Deamer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �79

Actin Dynamics: From Nanoscale to MicroscaleAnders E. Carlsson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �91

Eukaryotic Mechanosensitive ChannelsJohanna Arnadottir and Martin Chalfie � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 111

Protein Crystallization Using Microfluidic Technologies Based onValves, Droplets, and SlipChipLiang Li and Rustem F. Ismagilov � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 139

Theoretical Perspectives on Protein FoldingD. Thirumalai, Edward P. O’Brien, Greg Morrison, and Changbong Hyeon � � � � � � � � � � � 159

Bacterial Microcompartment Organelles: Protein Shell Structureand EvolutionTodd O. Yeates, Christopher S. Crowley, and Shiho Tanaka � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 185

Phase Separation in Biological Membranes: Integration of Theoryand ExperimentElliot L. Elson, Eliot Fried, John E. Dolbow, and Guy M. Genin � � � � � � � � � � � � � � � � � � � � � � � � 207

v

Ann

u. R

ev. B

ioph

ys. 2

010.

39:3

09-3

28. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by W

IB60

13 -

Fre

ie U

nive

rsita

et B

erlin

- F

U B

erlin

on

11/2

2/11

. For

per

sona

l use

onl

y.

AR411-FM ARI 2 April 2010 20:22

Ribosome Structure and Dynamics During Translocationand TerminationJack A. Dunkle and Jamie H.D. Cate � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 227

Expanding Roles for Diverse Physical Phenomena During the Originof LifeItay Budin and Jack W. Szostak � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 245

Eukaryotic Chemotaxis: A Network of Signaling Pathways ControlsMotility, Directional Sensing, and PolarityKristen F. Swaney, Chuan-Hsiang Huang, and Peter N. Devreotes � � � � � � � � � � � � � � � � � � � � � 265

Protein Quantitation Using Isotope-Assisted Mass SpectrometryKelli G. Kline and Michael R. Sussman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 291

Structure and Activation of the Visual Pigment RhodopsinSteven O. Smith � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 309

Optical Control of Neuronal ActivityStephanie Szobota and Ehud Y. Isacoff � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 329

Biophysics of KnottingDario Meluzzi, Douglas E. Smith, and Gaurav Arya � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 349

Lessons Learned from UvrD Helicase: Mechanism forDirectional MovementWei Yang � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 367

Protein NMR Using Paramagnetic IonsGottfried Otting � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 387

The Distribution and Function of Phosphatidylserinein Cellular MembranesPeter A. Leventis and Sergio Grinstein � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 407

Single-Molecule Studies of the ReplisomeAntoine M. van Oijen and Joseph J. Loparo � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 429

Control of Actin Filament Treadmilling in Cell MotilityBeata Bugyi and Marie-France Carlier � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 449

Chromatin DynamicsMichael R. Hubner and David L. Spector � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 471

Single Ribosome Dynamics and the Mechanism of TranslationColin Echeverrıa Aitken, Alexey Petrov, and Joseph D. Puglisi � � � � � � � � � � � � � � � � � � � � � � � � � � 491

Rewiring Cells: Synthetic Biology as a Tool to Interrogate theOrganizational Principles of Living SystemsCaleb J. Bashor, Andrew A. Horwitz, Sergio G. Peisajovich, and Wendell A. Lim � � � � � 515

vi Contents

Ann

u. R

ev. B

ioph

ys. 2

010.

39:3

09-3

28. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by W

IB60

13 -

Fre

ie U

nive

rsita

et B

erlin

- F

U B

erlin

on

11/2

2/11

. For

per

sona

l use

onl

y.

AR411-FM ARI 2 April 2010 20:22

Structural and Functional Insights into the Myosin Motor MechanismH. Lee Sweeney and Anne Houdusse � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 539

Lipids and Cholesterol as Regulators of Traffic in theEndomembrane SystemJennifer Lippincott-Schwartz and Robert D. Phair � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 559

Index

Cumulative Index of Contributing Authors, Volumes 35–39 � � � � � � � � � � � � � � � � � � � � � � � � � � � 579

Errata

An online log of corrections to Annual Review of Biophysics articles may be found athttp://biophys.annualreviews.org/errata.shtml

Contents vii

Ann

u. R

ev. B

ioph

ys. 2

010.

39:3

09-3

28. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by W

IB60

13 -

Fre

ie U

nive

rsita

et B

erlin

- F

U B

erlin

on

11/2

2/11

. For

per

sona

l use

onl

y.

Structure and Activation of the Visual Pigment Rhodopsin

Documents

Transcript of Structure and Activation of the Visual Pigment Rhodopsin