reverse transcriptase rhodopsin perspectivesbharath a, petrou m, editors. next generation...

sufficient brightness to stimulate the target cell layers. As

present virtual reality headsets typically have 40� fieldsof view and resolutions similar to modern mobile phone

displays, returned vision may be significantly improved

over electronic approaches. Alsowithout the requirement

for hours of surgery, it may be significantly cheaper than

electronic approaches.

Perspectives

In the coming years, retinal prostheses will become

increasingly capable. However, most in the community

accept that it is highly unlikely that present approaches

will return what is accepted as fully functional vision.

Presently, returned vision can be described as handful of

flashingdots (Dagnelie 2011). Simple high contrast objects

canbe perceived, but face recognition, normal reading, and

many normal daily visual tasks will still be very difficult

and perhaps best performed via assistive devices. Never-

theless, in these early years, if functional vision, sufficient

for navigation and simple tasks, canbe returned, the impact

on individual patients will be very high.

References

Bharath A, Petrou M, editors. Next generation artificial vision

systems: reverse engineering the human visual system. Bos-

ton: Artech House; 2008.

Dagnelie G, editor. Visual prosthetics – physiology, bioengi-

neering, Rehabilitation. New York: Springer; 2011.

Dowling J. Current and future prospects for optoelectronic reti-

nal prostheses. Eye. 2009;23:1999–2005.

Georg Nagel TS, HuhnW, Kateriya S, Adeishvili N, Berthold P,

Ollig D, Hegemann P, Bamberg E. Channelrhodopsin-2,

a directly light-gated cation-selective membrane channel.

Proc Natl Acad Sci USA. 2003;100(24):5.

Gr€usser O, Hagner M. On the history of deformation phosphenes

and the idea of internal light generated in the eye for the

purpose of vision. Doc Ophthalmol. 1990;74(1–2):57–85.

RNIB (2012). Vision 2020. From http://www.vision2020uk.org.uk.

Tombran-Tink J, Barnstable CJ, et al., editors. Visual prosthesis

and ophthalmic devices: new hope in sight. Totowa: Humana

Press; 2007.

Retinal Purple

▶Rhodopsin: Stability and Structural Organization in

Membranes

Reverse Transcriptase

▶HIV-1 Reverse Transcriptase – Computational

Studies on the Polymerase Active Site

Rhodopsin

▶Channelrhodopsin

▶ Sensory Rhodopsin I

▶ Sensory Rhodopsin II: Signal Development and

Transduction

▶Rhodopsin – Stability and Characterization of

Unfolded Structures


Membranes

Rhodopsin Activation Based onSolid-State NMR Spectroscopy

Michael F. Brown1 and Andrey V. Struts2,3

1Department of Chemistry and Biochemistry and

Department of Physics, University of Arizona, Tucson,

AZ, USA2Department of Chemistry and Biochemistry,

University of Arizona, Tucson, AZ, USA3Division of Medical Physics, St. Petersburg State

Medical University, St. Petersburg, Russia

Abbreviations

DARR Dipolar-assisted rotational resonance

DOPE 1,2-dioleoyl-sn-glycero-3-

phosphoethanolamine

EFG Electric field gradient

EPR Electron paramagnetic spin resonance

FTIR Fourier transform infrared

GPCR G protein–coupled receptor

MAS Magic angle spinning

Meta I Metarhodopsin I

Meta II Metarhodopsin II

POPC 1-palmitoyl-2-oleoyl-sn-glycero-3-

phosphocholine

TM Transmembrane

Rhodopsin Activation Based on Solid-State NMR Spectroscopy 2231 R

R

Synonyms

Seven-transmembrane domain receptors; Solid-state

NMR

Introduction

Rhodopsin is responsible for the dark (scotopic) vision of

vertebrates, and is amember of the vast protein family of

G protein–coupled receptors (GPCRs) that regulate

many important functions of the human body. Although

they are the targets of more than 30% of known drugs

and pharmaceuticals, the structures of most GPCRs are

currently unknown.Conventional approaches are limited

by difficulties in crystallization for X-ray analysis, as

well as the requirement for detergent solubilization in

high-resolution NMR spectroscopy. Notably, rhodopsin

is one of the fewGPCRs forwhich theX-ray structures in

different states have been recently determined (Park et al.

2008; Scheerer et al. 2008; Choe et al. 2011; Standfuss

et al. 2011). Activation of rhodopsin has been studied by

Fourier transform infrared (FTIR) methods which detect

short-range local interactions (Mahalingam et al. 2008),

together with solid-state 13C NMR spectroscopy for

short distance restraints (Ahuja et al. 2009a), and by

site-directed spin-labeling experiments that are sensitive

to longer distances (Altenbach et al. 2008). This entry

reviews the application of solid-state NMR methods for

obtaining structural and dynamical information for the

retinal ligand of rhodopsin pertinent to its activation

mechanism (Struts et al. 2007, 2011a; Ahuja et al.

2009a). Solid-state NMR spectra provide knowledge of

the retinal structure whereas NMR relaxation methods

reveal information about its dynamics. Combining the

results of solid-state 2H NMR spectroscopy with com-

plementary FTIR, 13C NMR, and spin-labeling methods

yields a unified picture of the rhodopsin activation

mechanism (Struts et al. 2011b).

Retinal Structure in Rhodopsin IsDetermined Using Angular and DistanceRestraints from Solid-State 2H and 13C NMR

Studies of aligned bilayers containing rhodopsin with

selectively deuterated retinal (Fig. 1a) allow one to

determine the orientation of the quadrupolar coupling

tensor of deuterium nuclei (Struts et al. 2007)

with respect to the membrane normal (vector n0 in

Fig. 1b). Orientations of the deuterated methyl groups

calculated in this way can be used for modeling of the

retinal structure in the binding pocket of rhodopsin.

For rapidly spinning methyl groups, the principal axis

of the motionally averaged quadrupolar tensor coin-

cides with the rotation axis (symmetry axis) of the

methyl group M, and thus determines the average

value of the angle OMD between M and n0 (Fig. 1).

Solid-state 2H NMR spectra of rhodopsin and

metarhodopsin I obtained for the different methyl

groups are presented in Fig. 2a, b (Struts et al. 2007).

The fitting parameters used in the simulations of the

theoretical 2H NMR spectra include the C–C2H3

methyl bond orientation, the width of the orientational

distribution of the local membrane normals (mosaic

spread), the residual deuterium quadrupolar coupling,

and the NMR line broadening. Reduction of the

quadrupolar interactions of deuterium nuclei with the

electric field gradient (EFG) in the molecule occurs

due to the fast spinning of the methyl groups and

reorientation of their symmetry axes. The 2H NMR

line shape is calculated by randomly generating the

angle of rotation of the rhodopsin molecule and angle

of deviation of the membrane normal from the average

value, and subsequently averaging the spectra over all

possible orientations (Struts et al. 2007). The above

method has been applied to aligned membranes

containing rhodopsin (Brown et al. 2010) or bacterio-

rhodopsin (Brown et al. 2007), as well as to aligned

DNA fibers (Nevzorov et al. 1999). For rhodopsin,

analysis of the angular-dependent, solid-state 2H

NMR spectra gives the following orientations of the

retinal C5-, C9-, and C13-methyl groups with respect

to the protein long axis: 70� 3, 52� 3, and 68� 2� inthe dark state, and 72 � 4, 53 � 3, and 59 � 3� in the

Meta I state (Fig. 1) (Struts et al. 2007).

Solid-state 2H NMR spectroscopy is complemen-

tary to 13C rotational resonance distance measure-

ments (Verdegem et al. 1999) in that orientational

restraints are provided, together with distance restraints

that allow site-specific structural information to be

obtained analogous to solution NMR. Magic angle spin-

ning (MAS) rotational resonance NMR utilizes dipolar

recoupling or magnetization transfer between nuclear

spins that arises when the rotation speed of the sample

matches the difference for the isotropic shifts of two

chemically nonequivalent nuclei. Both the NMR line

splitting and magnetization exchange depend on the

R 2232 Rhodopsin Activation Based on Solid-State NMR Spectroscopy

internuclear distance. One can then determine the dis-

tance either by calibration with similar reference com-

pounds, or by simulation of NMR spectra or exchange

plots with known NMR parameters (chemical shielding

tensor, scalar coupling constants, zero-quantum spin-

spin relaxation time).

Using retinals doubly 13C labeled at the

C8–C16/C17, C8–C18, C10–C20, and C11–C20

positions, the spatial structure of the C10–C11¼C12–

C13–C20 motif and rotation of the b-ionone ring with

respect to the polyene chain have been examined in the

dark and Meta I states. Distances between carbons

C10–C20 and C11–C20 calculated from 13C rotational

resonance NMR were 0.304 � 0.015 and 0.293 �0.015 nm, correspondingly, in the dark state, and

>0.435 and 0.283�0.015 nm in the Meta I state

Rhodopsin Activation Based on Solid-State NMRSpectroscopy, Fig. 1 Site-specific 2HNMRspectroscopy inves-

tigates the structure and functional dynamics of retinal within the

binding pocket of rhodopsin in a native-like membrane environ-

ment. (a) Chemical structure of 11-cis retinal chromophore indicat-

ing numbering scheme for b-ionone ring and polyene chain.

(b) Theoretical analysis of geometry and ps–ns mobility of

retinylidene methyl groups within the binding cavity of rhodopsin.

Rotational dynamics of methyl groups are treated as axial threefold

hops or continuous rotational diffusion. Euler angles OPI indicate

orientation of the C–2H bond (where P denotes principal axis of

EFG) within the internal frame of the methyl threefold axis (I).AnglesOIM(t) describe time dependence of fluctuations of theC–2H

bond relative to the average methyl rotor axis system (M). Angles

OMD are for average methyl axis to membrane normal n0 (D is the

membrane system), or alternatively OML for the methyl axis to

laboratory frame (L). Finally the angles ODL are for the membrane

to laboratory frame as defined by the main magnetic field B0

Rhodopsin Activation Based on Solid-State NMRSpectroscopy, Fig. 2 Solid-state 2H NMR spectroscopy char-

acterizes the structure of the retinal ligand bound to rhodopsin.

Examples of solid-state 2H NMR spectra for dark (a) and Meta I

(b) states of rhodopsin regenerated with 11-cis-retinal specifically2H-labeled at C5-, C9-, and C13-methyl positions in aligned

POPC membranes (1:50 molar ratio). The spectra are measured

for the orientation of the membrane normal parallel to the mag-

netic field at temperatures of�150 �C and�100 �C for rhodopsin

and Meta I, respectively. (c) The solid-state NMR structure pro-

posed for retinal in the dark state of rhodopsin. (d) The solid-stateNMR structure proposed for retinal in the Meta I state


R

(Verdegem et al. 1999). Distances for carbons C8–

C16/C17 and C8–C18 were reported to be 0.405 �0.025 and 0.295 � 0.015 nm in the dark state, and

effectively unchanged in the Meta I state (Spooner

et al. 2003). The twist for the C12–C13 bond was

estimated to be 44� 10� in the dark state and a relaxedall-trans retinal structure was observed in the Meta

I state (Verdegem et al. 1999). It has been established

that the C5¼C6–C7¼C8 torsion angle characterizing

the b-ionone ring orientation was�28� 7� in the darkstate, and a similar orientation was revealed in the

Meta I state (Spooner et al. 2003).

Investigation of the 3D retinal structure was carried

out within the framework of a three-plane model

(Struts et al. 2007) (Fig. 2c). Within each of the three

planes (A, B, and C) deviation of the torsion angles

from the ideal 11-cis or all-trans configuration was

neglected (the polyene chain was assumed to be flat

within the planes). Only torsion angles between the

different planes were taken into account (Fig. 2c).

Comparison of the 2H NMR structure with crystallo-

graphic data indicates that such a model corresponds

well to the X-ray structure of retinal within the binding

pocket of rhodopsin in the dark state (Struts

et al. 2007). Notably the methyl group orientations

are obtained directly from the NMR line shape; they

are not related to the above model (“model-free”), and

hence can be compared directly with the crystallo-

graphic results. Such a comparison indicates a very

good agreement between the 2H NMR (Struts

et al. 2007) and the X-ray data (Okada et al. 2004).

Referring to Fig. 2 we see that the three planes used

for the retinal modeling (Struts et al. 2007) encompass

the b-ionone ring (plane A) and the polyene chain on

both sides of the C12–C13 bond (planes B and C). The

methyl group orientations calculated from the orienta-

tion-dependent 2H NMR line shapes provide a set of

angular restraints that allow one to determine the

dihedral angles between the planes, together with the

orientation of the retinal ligand with respect to rhodop-

sin. The torsion angle wi;k between two planes with

common bond Ci–Ck is given by (Struts et al. 2007):

wi;k ¼ cos�1 cos yi cos yði;kÞB � cos yðiÞB

sin yi sin yði;kÞB

!

� cos�1 cos yk cos yði;kÞB � cos yðkÞB

sin yk sin yði;kÞB

! (1)

where yðiÞB and yðkÞB are the orientations of the individual

methyl group axes with respect to the membrane nor-

mal, and yi and yk are angles of the two methyl axes

relative to the Ci–Ck bond. The Ci–Ck bond angle to the

membrane normal is y ði;kÞB , and is calculated from the

C9-methyl orientation, together with the transition

dipole (Struts et al. 2007).

Orientations of the methyl groups obtained from 2H

NMR and the orientation of the electronic transition

dipolemoment of the retinal are used as angular restraints

(Struts et al. 2007). The distances between carbons C10–

C20 and carbons C11–C20 calculated from 13C NMR

(Verdegem et al. 1999) are used as additional geometri-

cal constraints in the extended model. Using this

approach, various solutions were obtained for the torsion

angles between the planes A, B, and C, giving a total of

64 combinations for the relative orientations of the

planes, and 128 configurations and orientations for the

overall orientation of retinal (Struts et al. 2007) within

the ligand-binding cavity. However, most of these solu-

tions can be discarded, as they do not satisfy the 13C

NMR distance constraints between the atoms C8–C18,

C8–C16/C17 (Spooner et al. 2003), C10–C20, and

C11–C20 (Verdegem et al. 1999), and also the signs of

the torsion angles obtained from circular dichroism (CD)

(Fujimoto et al. 2002). The resulting 2H NMR retinal

structure in the dark state is shown in Fig. 2c (Struts et al.

2007). One of its peculiarities is the twist around the

C11¼C12 bond. Note that attempting to calculate the

retinal structure without the twist failed, since the retinal

molecule does not fit into the binding cavity of the crystal

structure. This proposal can explain the ultrafast isomer-

ization of retinal within the binding pocket. Overall,

the 2H NMR structure of retinal and its orientation in

the binding pocket are in very good agreement with the

crystallographic data (Okada et al. 2004).

NMR Structure of Retinal in the PreactivatedMeta I State Shows Relaxation of TorsionalTwisting

In metarhodopsin I, the possible solutions for the torsion

angles are:�32� 7 and�57� 7� for the angle betweenplanes A and B (the b-ionone ring and the polyene

chain), and �173 � 4� for the angle between planes

B and C (Struts et al. 2007). Thus eight combinations

of the angles and the corresponding retinal structures are

conceivable. Fitting of the retinal into the rhodopsin


binding pocket in the dark state demonstrates that most

of the eight possible structures have the b-ionone ring

position different from the dark state (Struts et al. 2007).

On the other hand, according to electron crystallography

data, theb-ionone ring does not change its position in theMeta I state versus the dark state (Ruprecht et al. 2004).

Only two of the possible solutions for the 2H NMR

retinal structure in the Meta I state have the b-iononering in a position similar to the dark state: One is shown

in Fig. 2d with the torsion angles between the planes

A–B and B–C of �32 and +173�, respectively. The

other has corresponding torsion angles of 32 � 7 and

�173 � 4�, which can be obtained from the first struc-

ture as a mirror image reflected in a vertical plane

(Struts et al. 2007).

Electron crystallography does not reveal any

noticeable displacements for the seven transmembrane

helices of rhodopsin in theMeta I state compared to the

dark state, and hence the shape of the retinal binding

pocket should be essentially the same. In that case, the

second of the above retinal structures seems unlikely

due to multiple steric clashes within the binding

C15

Ser186

ppm

60

65

Tyr178

C14 Meta II C14 Rho

C15Meta II

C15Rho

70

ppm

155

160

165

170

160 140 130 125ppm ppm

13C Chemical Shift

C14

3-Ser, 4′-Tyr / C14, C15

H7H5 H7

H6 H3

H5

His211Phe212

Tyr268

Tyr191

Tyr178Tyr268

Tyr191 Tyr178 Ser188

Phe212

His211

Ser186

b

a

3-Ser, 4′-Tyr / C14, C15

Rhodopsin Activation Based on Solid-State NMRSpectroscopy, Fig. 3 Solid-state 13C NMR evaluates distance

restraints that change upon light activation of rhodopsin. (a) Twoviews of the retinal binding pocket illustrating the distance

range of the DARR NMR experiment (Patel et al. 2004). The

spheres around the 13C-labeled atoms on tyrosine and serine

with a 5.5-A radius indicate the approximate detection limit of

the DARR experiment. The 13C-labeled retinal methyl carbons

are shown by black spheres. (b) Representative expanded

regions of 2D DARR NMR spectra of rhodopsin (black) andMeta II (red) showing cross peaks between the retinal and

protein 13C labels (Patel et al. 2004). The protein and retinal

labels are: 40-13C tyrosine, 3-13C serine, and 14,15-13C retinal.

The spectra were obtained at �80 �C. (Adapted with permis-

sion from Patel et al. (2004). Copyright 2004 National Acad-

emy of Sciences, USA)


R

pocket. That leaves us with the only possible retinal

structure in the Meta I state presented in Fig. 2d. This

structure indicates only a single steric clash with

the side chain of Trp265. As further discussed below,

the main changes in the retinal conformation in the

Meta I state include rotation of the polyene chain

adjacent to the C13-methyl group toward the C9-

methyl group, straightening and elongation of the

polyene chain, and a resulting displacement of the

b-ionone ring toward helix H5. The displacement of

the b-ionone ring, which is also observed by X-ray

crystallography and 13C NMR (Nakamichi and

Okada 2006; Ahuja et al. 2009a) in different rhodopsin

photointermediates, is assumed to play a crucial role in

the receptor activation. The rotation of the (proximal)

part of the polyene chain containing the C13-methyl

group may contribute to destabilization of the ionic

lock involving the protonated Schiff base, and thus

facilitate its deprotonation.

Conformational Changes in the RetinalBinding Pocket Are Revealed by RotationalResonance 13C NMR

Another solid-state NMR technique that has proved to

be quite useful for structural studies of membrane pro-

teins is 2D dipolar-assisted rotational resonance

(DARR) 13C NMR spectroscopy (Ahuja et al.

2009b). It allows one to determine close contacts

between selectively labeled carbon atoms in the pro-

tein. If the distance between the labeled atoms is 5 A or

less (Fig. 3a) one can observe a cross peak in 2D

DARR NMR spectra between 13C-labels (Fig. 3b);

for larger distances a cross peak does not appear in

the spectrum. A number of experiments have mapped

distance changes between the labeled carbons in the

retinal and amino acids in the binding pocket in

metarhodopsin II with respect to the dark state.

Labeled amino acids included tyrosine, serine, cyste-

ine, glycine, threonine, and others. The active Meta II

state was stabilized by solubilizing rhodopsin in deter-

gent micelles (1% n-dodecyl maltoside solution). The

DARR NMR spectra revealed displacement of the

retinal b-ionone ring in the rhodopsin binding pocket

toward helix H5 (Ahuja et al. 2009a) and the E2 loop

toward the extracellular side of the membrane (Ahuja

et al. 2009b), in agreement with 2H NMR data (Brown

et al. 2010).

Retinal 2H NMR Relaxation Times ShowSite-Specific Differences in Mobility in theRhodopsin Dark State

Utilization of solid-state NMR relaxation is useful for

membrane protein studies, because it provides infor-

mation on local dynamics and intramolecular interac-

tions of biomolecules that is generally unavailable

using other methods (Struts et al. 2011a, b). Dynamics

of the methyl groups of the retinal in the dark, Meta I,

and Meta II states of rhodopsin have been studied by

solid-state 2H NMR relaxation. Experimental temper-

ature dependences of the deuterium relaxation times of

Zeeman (T1Z) and quadrupolar (T1Q) order are summa-

rized in Fig. 4. Relaxation times T1Z and T1Q are

characterized by the spectral densities of thermal fluc-

tuations of the quadrupolar coupling tensor near the

resonance frequency:

1=T1Z ¼ 3

4p2w2Q½J1ðo0Þ þ 4J2ð2o0Þ� (2)

1=T1Q ¼ 9

4p2w2QJ1ðo0Þ (3)

Here wQ is the quadrupolar coupling constant, Jm(mo0)

denotes the spectral density (m ¼ 1,2), and o0 is the

nuclear resonance (Larmor) frequency. In terms of

generalized model-free analysis (Brown 1982), the

spectral densities Jm(mo0) are given by:

Jm oð Þ ¼Xr;q

��Dð2Þ0r ðOPIÞ

��2hD��Dð2Þrq ðOIMÞ

��2E

��DDð2Þ

rq ðOIMÞE��2dr0dq0i jð2Þrq ðoÞ

��Dð2ÞqmðOMLÞ

��2 (4)

where o ¼ mo0 and m ¼ 1,2 (Brown 1982). The

quantities in square brackets are the mean-square ampli-

tudes of motion, and the corresponding reduced spectral

densities are designated as jð2Þrq ðoÞ. In (4)D(j)(Oij) denotes

theWigner rotationmatrix of rank j (Brown 1996), where

Oij � ðaij; bij; gijÞ are the Euler angles describing the

relative spatial orientations of coordinate systems

i, j ¼ P,I,M,L. Referring to Fig. 1b, the label P means

the principal axis system of the EFG tensor for an 2H

nucleus (principal z-axis is parallel to C–2H bond); I is for

an intermediate coordinate frame for methyl rotation (z-axis is C3 symmetry axis of C–C2H3 bond);M represents

the coordinate frame for the average methyl orientation

(z-axis is averageC–C2H3 bond direction); andL signifies


the laboratory frame (z-axis is external magnetic field).

For unoriented (powder-type) samples, averaging

over all the methyl orientations is performed

(Nevzorov et al. 1998). Assuming exponential relaxation,

the reduced spectral densities are given by:

jð2Þrq ðoÞ ¼ 2trq=ð1þ o2trq2Þ where trq are the (rota-

tional) correlation times for the Wigner rotation matrix

elements.

Further analysis requires implementation of

a motional model to describe the molecular fluctuations

(Brown 1982). In the case of N-fold hops about a single

axis, the correlation times read (Brown 1982; Torchia and

Szabo 1982): 1=trq ! 1=tr ¼ 4ksin2ðpr=NÞ (axial

averaging is assumed) and yield 1/tr ¼ 3k for a methyl

group. For continuous diffusion in a potential of mean

torque (Trouard et al. 1994), the rotational correlation

times are written as (Brown 1982):

1

trq¼ mrq

Dð2Þrq OIMð Þ

�� 2� �� D

ð2Þrq OIMð Þ

D E�� 2dr0dq0 D?

þ Djj � D?� �

r2

(5)

Rhodopsin Activation Based on Solid-State NMRSpectroscopy, Fig. 4 Solid-state 2H NMR relaxation times

reveal site-specific differences in the mobility of retinal within

the binding cavity of rhodopsin. Spin-lattice (T1Z) and

quadrupolar-order (T1Q) relaxation times are plotted against

inverse temperature for rhodopsin in POPC membranes (1:50

molar ratio) in the dark state with retinal 2H-labeled at (a, b) the

C5-methyl, (c) the C9-methyl, and (d) the C13-methyl groups.

The experimentally determined T1Z and T1Q times were fit

simultaneously using analytical models for molecular dynamics

(Brown 1982) (see text): axial threefold jump model (rate con-

stant k, solid lines); continuous rotational diffusion model (coef-

ficients D|| and D⊥ with solid lines for D⊥ ¼ 0 and dashed linesfor D⊥ ¼ D||)


R

Here the diffusion coefficients D|| and D? quantify

axial and off-axial rotations of a methyl group. The

moments mrq and the mean-squared moduli

h|D(2)rq(OIM)|

2i depend on the second- and fourth-rank

order parameters (Trouard et al. 1994). In a strong-

collision approximation, the orientation changes by

any amount, and the correlation times correspond to

those of a rigid rotor (Brown 1982):

1=trq ! 1=tr ¼ 6D? þ ðDjj � D?Þr2. For either

a threefold jump or continuous diffusion model, the

rotational correlation times are inversely related to

A exp(�Ea/RT), where A is the preexponential factor

(k0 or D0), and Ea is the activation energy (barrier)

for methyl rotation (Struts et al. 2011a). Both models

have been used to fit experimental relaxation data

(Struts et al. 2011a). In the dark, Meta I, and Meta II

states the temperature dependences of the T1Z relaxa-tion times can be fit using either the jump or continuous

diffusion models. Measurement of both Zeeman and

quadrupolar-order relaxation allows one to obtain

information on the anisotropy of the local molecular

motion (Struts et al. 2011a).

Rotational correlation times are found in the range of

2–12 ps at 30 �C and 3–45 ps at �60 �C, depending on

the position of the methyl group. The T1Z and T1Qtemperature dependences correspond to the activation

energy for methyl rotation and are related to the height

of the rotational potential barriers. The unexpectedly

low activation energy for the C9-methyl group

can be explained by compensation of intra-retinal 1–6

Rhodopsin Activation Based on Solid-State NMRSpectroscopy, Fig. 5 Deuterium NMR relaxation illuminates

changes in dynamics of the retinylidene methyl groups upon

light activation of rhodopsin. Spin-lattice relaxation times (T1Z)are plotted against inverse temperature for rhodopsin in POPC

membranes (1:50 molar ratio) with retinal 2H-labeled at the C5-,

C9-, or C13-methyl groups, respectively in (a, c) the Meta I, and

(b, d) the Meta II states. The relaxation data are fit both with an

axial threefold jumpmodel (rate constant k) as well as a diffusionmodel (axial coefficient D|| with D⊥ ¼ 0). For the C5-methyl

group in Meta I either different rotational diffusion constants

were assumed (D|| 6¼ D⊥, not shown) or different conformers

(Struts et al. 2011b). Following 11-cis to trans isomerization,

a reduction occurs from three distinct methyl environments in

the dark state to � two environments in Meta I and Meta II,

suggesting a more planar retinal conformation


interactions with hydrogens H7 and H11 (Fig. 2c), since

the corresponding rotational potentials are offset with

respect to one another by about 180�. Higher activationenergies for the C5- and C13-methyl groups are the

result of intra-retinal 1–7 interactions with the H8 and

H10 hydrogens, respectively. Lastly, the high activation

energy for the C5-methyl group indicates a 6-s-cis con-

formation of the b-ionone ring. Contributions due to

interactions with the side chains in the binding pocket

of rhodopsin in the dark state are secondary due to

large distances from the methyl groups (>4 A)

(Fig. 5).

Light-Induced Changes in Retinal DynamicsOccur in the Meta I and Meta II States

Noticeable changes in the dynamics of the retinal methyl

groups are observed in the Meta I and the Meta II states

after photoisomerization (Struts et al. 2011a). The Meta

I and Meta II states were quantitatively cryotrapped in

POPC and POPC:DOPE (3:1 ratio) bilayers, respectively

(Struts et al. 2011a). The largest photoinduced changes

take place for the C9-methyl group, whose activation

energy Ea increases sequentially from the dark to the

Meta I state, and then to Meta II. At the same time, the

Ea value for the C13-methyl group decreases progres-

sively in the Meta I and the Meta II states. Consequently,

after isomerization to yield trans retinal the activation

energies and spin-lattice relaxation times become com-

parable for both the C13- and C9-methyl groups, which

appears logical because both groups are now on the same

side of the molecule, and have similar rotational poten-

tials (Fig. 2d). The decrease of the activation energy for

the C13-methyl group can be explained analogously to

the low Ea value for the C9-methyl group in the dark

state, viz. by the compensation of intra-retinal 1–6 inter-

actions with hydrogens H11 and H15, whose rotational

potentials are in antiphase (180� phase shift; see above).The larger activation energy for the C9-methyl group and

decrease in its mobility after retinal isomerization can be

attributed to the changes in the retinal conformation, and

possible interactions with the surrounding side chains

(e.g., Tyr268 or Thr118) in the binding pocket. Note

that the observed Ea values do not indicate any steric

clashes of the C9-methyl and the C13-methyl groups in

the Meta I and Meta II states. For the C5-methyl group

some interesting and unusual changes are observed in the

T1Z temperature dependence in the Meta I state

(Struts et al. 2011a). Overall, the activation energy

for C5-methyl rotation does not change much in the

transition from the dark to active Meta II state.

Consequently, one can conclude that the conforma-

tion and the environment of the b-ionone ring remain

similar after rhodopsin activation (Struts et al. 2011a).

Rhodopsin Activation Is Illuminated byCombining Crystallography with MagneticResonance Methods

Rhodopsin activation is the result of retinal conforma-

tional changes that disrupt a system of hydrogen-

bonding networks stabilizing the inactive state, first

unlocking the receptor around the retinal, and then in

other more distal domains. The receptor is transformed

into its intrinsic “natural” active state, which has

been proposed to resemble the ligand-free opsin struc-

ture, with the formation of new hydrogen-bonding

networks. Transformation of the photonic energy

into thermal motions that include helical fluctuations

facilitates transitions over the barriers separating

the inactive, preactivated, and active states. However,

a detailed picture of the activation mechanism is more

complicated, and not completely understood at present

(Patel et al. 2004; Vogel et al. 2005; Nakamichi and

Okada 2006; Struts et al. 2007, 2011a; Altenbach et al.

2008; Mahalingam et al. 2008; Ahuja et al. 2009b;

Choe et al. 2011; Standfuss et al. 2011). Figure 6

illustrates changes in the retinal binding pocket in the

activation process. In Fig. 6a an electron density map

of the constitutively active rhodopsin mutant E113Q in

complex with a peptide derived from the carboxyl

terminus of the a-subunit of the G protein transducin

(the GaCT peptide) is presented (Standfuss et al.

2011). Figure 6b shows the 3D structure of E113Q

(PDB code: 2X72) superimposed with ground-state rho-

dopsin (PDB code: 1GZM). However a different retinal

conformation was observed for opsin regenerated with

all-trans-retinal to form the putative active Meta II state

(Choe et al. 2011). Superposition of this retinal structure

(PDB code: 3PQR) with the ligand conformation in

rhodopsin (PDB code: 1U19) (Okada et al. 2004) is

presented in Fig. 6c. Last of all Fig. 6d shows the 2H

NMR structure of retinal in the Meta I state overlapped

with the binding pocket in the dark state (Struts et al.

2007, 2011b). A relaxed all-trans conformation of

the ligand and displacement of its b-ionone ring are


R

clearly observed in the Meta I (Fig. 6d) and Meta II

(Fig. 6b, c) states.

Data for the 2H NMR retinal structure and dynamics

in different states can be combined with the results of

other structural and biophysical studies (Fujimoto et al.

2002; Patel et al. 2004; Vogel et al. 2005; Nakamichi

and Okada 2006; Altenbach et al. 2008; Mahalingam

et al. 2008; Ahuja et al. 2009a; Choe et al. 2011;

Standfuss et al. 2011) to yield the following proposed

mechanism of rhodopsin activation (Struts et al. 2011b).

Photoisomerization of retinal leads to the rotation of the

C13-methyl group and the adjacent part of the retinal

polyene chain toward the b4-sheet on the extracellular

loop E2 (Nakamichi and Okada 2006; Struts et al. 2007).

In addition, the polyene chain of the retinal straightens

and elongates (Struts et al. 2007; Choe et al. 2011).

Straightening of the polyene chain leads to a steric

clash with Trp265, and its displacement toward the cyto-

plasmic end of the protein and toward helix H5. On the

other hand, retinal elongation leads to a translation of the

b-ionone ring toward helix H5. Rotation of the retinal

polyene chain with the C13-methyl group at the Schiff

Rhodopsin Activation Based on Solid-State NMRSpectroscopy, Fig. 6 Local changes in retinal initiate large-

scale protein rearrangements leading to receptor activation. (a)Electron density map and (b) PDB structure (accession code

2X72, blue) of constitutively active rhodopsin mutant E113Q

with the GaCT peptide (Standfuss et al. 2011) compared to

rhodopsin structure in dark state (PDB code 1GZM, green).Note that all-trans-retinal (yellow) is not covalently bound to

Lys296. (Adapted with permission from Standfuss et al. 2011.)

(c) Retinal conformation in the Meta II state (PDB code 3PQR,

blue, protein is shown in orange) compared with the dark state

(PDB code 1U19, red, protein is green) (Okada et al. 2004; Choeet al. 2011). (d) Data from 2H NMR spectroscopy (Struts et al.

2011a) and other biophysical measurements suggest the C13-

methyl and the C¼NH+ moieties of the protonated Schiff base

change their orientation upon ligand isomerization. The b-ionone ring with the C5-methyl group remains in its hydropho-

bic pocket, but its displacement due to elongation of retinal

triggers allosteric movements of helices H5 and H6 that lead to

rhodopsin activation (see text). (Figure produced with PyMOL

(http://pymol.sourceforge.net/))


base end of the ligand destabilizes the hydrogen-bonding

networks connecting the E2 loop, helices H1–H3, H6,

H7, and retinal in the inactive state (this includes the

ionic lock between the protonated Schiff base and its

counterion Glu113) (Okada et al. 2004; Vogel et al.

2004). Additionally, the ionic lock of the protonated

Schiff base with its (complex) counterion may be

destabilized by the 11-cis to trans retinal isomerization,

which changes the pKa of the protonated Schiff base

(Mahalingam et al. 2008). As a result, the hydrogen-

bonding network rearranges (Choe et al. 2011) and the

Schiff base deprotonates, breaking the ionic lock and

allowing rearrangement of the helical bundle. Displace-

ment of the b-ionone ring (whose C5-methyl group is in

the vicinity of Glu122) toward helix H5 is apparently

coupled to rearrangement of another hydrogen-bonding

network between Glu122 on H3 and His211 on H5

(Vogel et al. 2005; Struts et al. 2007; Choe et al. 2011),

which initially stabilizes the inactive conformation. It is

also coupled to rotation of the cytoplasmic end of the

helix H5 closer to H6 (Ahuja et al. 2009a). This motion

destabilizes the ionic lock between the helices H3 and

H6 involving Glu134, Arg135, and Glu247. Conse-

quently, the activating rotation of the helix H6 occurs

accompanied by transient opening of the transducin

binding site (Scheerer et al. 2008; Struts et al.

2011a, b). The displacement of Trp265 on helix H6 due

to a steric clash with the retinal polyene chain after

photoisomerization may facilitate H6 helical rotation in

the process of activation (Struts et al. 2007).

Molecular Aspects of Large-Scale FunctionalProtein Motions in Rhodopsin Activation

In the sequence of the events in rhodopsin activation,

the early photointermediates (photo-, batho-,

lumirhodopsin) are followed by the major reactions:

Meta I ⇌ Meta IIa ⇌ Meta IIb + H3O+ ⇌ Meta IIbH

+,

where Meta IIb and Meta IIbH+ are active conforma-

tional substates. All of the substates (from Meta I to

Meta IIbH+) are in thermodynamic equilibrium after

the last state in the activation is reached. The above

picture of rhodopsin activation naturally raises the

question: Are these the only substates of rhodopsin in

the activation process? An important aspect is how the

local isomerization of retinal (Struts et al. 2007) initi-

ates the large-scale helix fluctuations in the Meta

I–Meta II equilibrium. Induced fit of retinal to the

binding pocket gives a highly twisted conformation

of the ligand in the dark state (Struts et al. 2007). Our

MD simulations (Huber et al. 2004) clearly indicate

that in the dark state not only the cytoplasmic loops but

also the extracellular loops of rhodopsin undergo

ms–ms timescale motions. On the other hand, the TM

helical core is more stable (Huber et al. 2004). Large-

scale functional protein motions, such as the

Meta I–Meta II fluctuations of rhodopsin, have large

activation barriers; they occur infrequently due to their

collective nature because they involve displacements

of many atoms within the protein on the ms timescale.

Rhodopsin has been proposed to be partially unfolded

in the Meta II state (Brown 1994), so an ensemble of

conformers is expected.

According to the above activation mechanism,

allosteric interactions trigger concerted movements of

the TMhelices, togetherwith changes in the cytoplasmic

loops that expose the Gt recognition sites. Extension of

helix H5 (Park et al. 2008) and a rigid-body rotation of

H6 (Altenbach et al. 2008) lead to an overall elongation

of the protein (Botelho et al. 2006), accompanied by its

expansion within the lipid bilayer (Attwood and

Gutfreund 1980). Stemming from a highly locked recep-

tor core, activation of rhodopsin generates an opening of

the protein, whereby the collective, large-scale protein

fluctuations are paralleled by a gain in partial molar

volume (Attwood and Gutfreund 1980). A concomitant

loosening of the receptor core enables increased water

penetration to the ligand-binding cavity. These conclu-

sions differ from the conventional view of a single light-

activated receptor (R*) conformation. The notion of

a dynamically activated receptor explains how the

large-scale protein fluctuations in rhodopsin activation

are initiated by the local retinal dynamics.

The presence of multiple active substates in the

Meta I–Meta II equilibrium may explain the different

orientations of the retinal in the Meta II state reported

by different groups (Patel et al. 2004; Ahuja et al.

2009a; Choe et al. 2011; Standfuss et al. 2011). In the

Meta II X-ray structure obtained by soaking opsin

crystals with all-trans-retinal, the retinal is rotated by

�180� about its long axis compared to rhodopsin in the

dark state (Fig. 6c) (Choe et al. 2011). By contrast, the

distance constraints obtained from dipolar-assisted

rotational resonance NMR spectroscopy for Meta II

(Patel et al. 2004; Ahuja et al. 2009a) indicate an

opposite orientation of the retinal ligand, which is

supported by the X-ray crystal structure of the active


R

E113Q rhodopsin mutant (Standfuss et al. 2011). Sub-

stantial further work is needed to firmly establish the

nature of the activated state(s) of rhodopsin; however,

it is clear that solid-state NMR probes different tiers of

the protein energy landscape (Frauenfelder et al.

2009). Solid-state NMR has an important role to play

in combination with X-ray diffraction, because it is

exquisitely sensitive to dynamics (Struts et al. 2007).

The implementation of innovative new crystallogra-

phy and magnetic resonance approaches can be

expected to contribute to a more complete understand-

ing of the structure and functioning of rhodopsin and

other membrane proteins. These subjects are certain to

occupy membrane biophysicists for years to come.

Summary

Rhodopsin is a representative member of the G protein–

coupled receptors, which are responsible for signal trans-

duction across cellular membranes and are of great

pharmaceutical interest. However, many details of the

activation mechanism remain unknown or contradictory,

notably the positioning and orientation of the bound

retinal in the active Meta II state. Solid-state NMR spec-

troscopy allows investigation of the local structure and

molecular motion of the retinal ligand in different rho-

dopsin intermediates, and illuminates large-scale func-

tional protein dynamics. To characterize light-induced

changes in structure and mobility of rhodopsin, site-

specific 2H and 13C labels are introduced into the retinal

ligand. Solid-state NMR spectra and 2H NMR relaxation

times (spin-lattice, T1Z, and quadrupolar-order, T1Q) inthe dark, Meta I, and Meta II states of rhodopsin are

measured by cryotrapping the various states inmembrane

lipid bilayers. The solid-state NMR data together with

Fourier transform infrared and spin-labeling results sug-

gest that local changes in the retinal structure trigger

activating helical fluctuations of the receptor. The Meta

I–Meta II equilibrium of rhodopsin entails a complex

energy landscape with multiple substates rather than

a simple activation switch.

Cross-References

▶Membrane Proteins in Phospholipid Bilayers:

Structure Determination by Solid-State NMR

▶Rhodopsin


Membranes

▶ Solid-State NMR

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Rhodopsins – Intramembrane Signalingby Hydrogen Bonding

Hideki Kandori

Department of Frontier Materials, Nagoya Institute of

Technology, Showa-ku, Nagoya, Japan

Synonyms

FTIR spectroscopy; Hydrogen bonding; Light-signal

transduction

Definition

Rhodopsins are members of the seven-transmembrane

receptor family, in which a retinal molecule is embed-

ded as the chromophore (Sharma et al. 2006; Hofmann

et al. 2009). Rhodopsins convert light into energy or

a signal. Such a function is initiated by photoisome-

rization of the retinal chromophore, followed by struc-

tural changes of protein (Kandori 2011). There are two

types of rhodopsins, microbial (type 1) and visual

(type 2) rhodopsins. Microbial rhodopsins show wide

variety of functions, such as light-driven outward

proton and inward chloride pumps (light-energy con-

version), light-gated cation channels, and photosensors

that activate either transmembrane or soluble trans-

ducers (light-signal transduction) (Fig. 1). Visual rho-

dopsin is a member of the G protein-coupled receptor

family that has evolved into a photoreceptive protein in

visual cells of vertebrates and invertebrates.

There is no sequential homology between microbial

and visual rhodopsins, which possess an all-trans and11-cis retinal, respectively. Nevertheless, similar acti-

vation mechanism has been implicated. In microbial

and visual rhodopsins, a retinal molecule is commonly

attached to a lysine residue in the seventh helix via

protonated Schiff base linkage (Fig. 2). The protonated

Rhodopsins – Intramembrane Signaling by Hydrogen Bonding 2243 R

R

Gordon C. K. RobertsEditor

Encyclopedia of Biophysics

With 1597 Figures and 131 Tables

EditorGordon C. K. RobertsHonorary Professor of BiochemistryDepartment of BiochemistryUniversity of LeicesterLeicester, UK

ISBN 978-3-642-16711-9 ISBN 978-3-642-16712-6 (eBook)ISBN 978-3-642-16713-3 (print and electronic bundle)DOI 10.1007/978-3-642-16712-6Springer Heidelberg New York Dordrecht London

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