Equilibrium between metarhodopsin-I and metarhodopsin-II is dependent on the conformation of the third cytoplasmic loop

Rhodopsin is a G-protein-coupled receptor (GPCR) that is the light detector in the rod cells of the eye. Rhodopsin is the best understood member of the large GPCR superfamily and is the only GPCR for which atomic resolution structures have been determined. However, these structures are for the inactive, dark-adapted form. Characterization of the conformational changes in rhodopsin caused by light-induced activation is of wide importance, because the metarhodopsin-II photoproduct is analogous to the agonist-occupied conformation of other GPCRs, and metarhodopsin-I may be similar to antagonist-occupied GPCR conformations. In this work we characterize the interaction of antibody K42-41L with the metarhodopsin photoproducts. K42-41L is shown to inhibit formation of metarhodopsin-II while it stabilizes the metarhodopsin-I state. Thus, K42-41L recognizes an epitope accessible in dark-adapted rhodopsin and metarhodopsin-I that is lost upon formation of metarhodopsin-II. Previous work has shown that the peptide TGALQERSK is able to mimic the K42-41L epitope, and we have now determined the structure of the K42-41L-peptide complex. The structure demonstrates a central role for elements of the rhodopsin C3 loop, particularly Gln238 and Glu239, in the interaction with K42-41L. Geometric constraints taken from the antibody-bound peptide were used to model the epitope on the rhodopsin surface. The resulting model suggests that K42-41L locks the C3 loop into an extended conformation that is intermediate between two compact conformations seen in crystal structures of dark-adapted rhodopsin. Together, the structural and functional data strongly suggest that the equilibrium between metarhodopsin-I and metarhodopsin-II is dependent upon the conformation of the C3 loop. The biological implications of this model and its possible relations to dimeric and multimeric complexes of rhodopsin are discussed.     

Suramin affects coupling of rhodopsin to transducin.

Suramin, a polysulfonated naphthylurea, is under investigation for the treatment of several cancers. It interferes with signal transduction through G(s), G(i), and G(o), but structural and kinetic aspects of the molecular mechanism are not well understood. Here, we have investigated the influence of suramin on coupling of bovine rhodopsin to G(t), where G-protein activation and receptor structure can be monitored by spectroscopic in vitro assays. G(t) fluorescence changes in response to rhodopsin-catalyzed nucleotide exchange reveal that suramin inhibits G(t) activation by slowing down the rate of complex formation between metarhodopsin-II and G(t). The metarhodopsin-I/-II photoproduct equilibrium, GTPase activity, and nucleotide uptake by G(t) are unaffected. Attenuated total reflection Fourier transform infrared spectroscopy shows that the structure of rhodopsin, metarhodopsin-II, and the metarhodopsin-II G(t) complex is also not altered. Instead, suramin dissociates G(t) from disk membranes in the dark, whereas metarhodopsin-II G(t) complexes are stable. F?rster resonance energy transfer suggests a suramin-binding site near Trp(207) on the G(t alpha) subunit (K(d) approximately 0.5 microM). The kinetic analyses and the structural data are consistent with a specific perturbation by suramin of the membrane attachment site on G(t alpha). Disruption of membrane anchoring may contribute to some of the effects of suramin exerted on other G-proteins.     

Flash photolysis and low temperature photochemistry of bovine rhodopsin with a fixed 11-ene.

Nonbleachable rhodopsins containing retinal moieties with fixed 11-ene structures have been prepared. When the nonbleachable rhodopsin analogue corresponding to the natural pigment was flash-photolysed at 20.8 degrees C, no absorption changes occurred at the monitoring wavelengths of 380, 480, and 580 nm for the time range of 2 microseconds--10 s. This observation is in contrast to that of natural rhodopsin which showed the formation of metarhodopsin I and its decay to meta II. Irradiation of the artificial rhodopsin, 77 K, with light of 460 and 540 nm, also gave no spectral changes; in the case of natural rhodopsin, however, the irradiation leads to formation of the red-shifted intermediate bathorhodopsin. The absence of photochemistry in the artificial pigment shows that an 11-cis to trans photoisomerization of the retinal moiety is a crucial step in inducing the chain of events in te photolysis of rhodopsin.     

Effect of phospholipid removal on the kinetics of the metarhodopsin I to metarhodopsin II reaction.

The kinetics of the metarhodopsin (meta) I → metarhodopsin II reaction have been studied by flash photolysis in two different types of preparations of bovine rhodopsin: (i) digitonin-solubilized rod outer segment (ROS) membranes with a molar ratio of phospholipid to rhodopsin of approximately 90, and (ii) digitonin-solubilized phospholipid-free rhodopsin with a molar ratio of phospholipid to rhodopsin of less than 0.2. At 20 °C the kinetics in both preparations are multiexponential, but four terms are required to fit the data with the solubilized membranes, whereas only two are required with the phospholipid-free preparation. Thus, phospholipid removal simplifies the kinetics of the meta I → meta II reaction, but the resulting preparation still does not show first-order kinetics. The ratio of the time constants of these two components with detergent-solubilized phospholipid-free rhodopsin was nearly equal to the values found with ROS particles, rhodopsin-phospholipid recombinants and intact rabbit eyes. This suggests a common origin for these two components in all these preparations and appears to exclude heterogeneity in bound phospholipid as the basis of these two-component kinetics.     

Studies on vision. The nature of the retinal–opsin linkage

1. Convenient methods for the preparation of tritiated 11-cis-retinol, 11-cis-retinal and rhodopsin are described. Irradiation of labelled rhodopsin in the presence of sodium borohydride resulted in the irreversible binding of the retinyl moiety to the active site. Degradative studies established that the retinyl moiety in this reduced derivative of rhodopsin was attached to the in-amino group of lysine. In connexion with this investigation the synthesis of a number of N-retinylidene- and N-retinyl-amino acids was achieved. Derivatives of lysine with the retinyl moiety attached either to the alpha-amino group or to the in-amino group were also synthesized and characterized. 2. It is suggested that the involvement of a charge-transfer interaction between the retinylidene chromophore and a suitable group -X or -X.H on the opsin best explains the spectroscopic properties of rhodopsin and other visual proteins.     

Transient light-induced conformational changes in rhodopsin

Arguments are presented which support the possibility that the unfolding of the rhodopsin molecule during photolysis up to the stage of metarhodopsin II is followed by a spontaneous refolding of the protein, once the isomerized retinaldehyde has left its original binding site. Such a transient conformational change might imply a very similar conformation for rhodopsin and opsin, apart from the presence of the chromophore.Presented at the EMBO-Workshop on Transduction Mechanism of Photoreceptors, Jülich, Germany, October 4–8, 1976     

Photoelectric signals generated by bovine rod outer segment disk membranes attached to a lecithin bilayer.

Purified bovine rod outer segment disk membranes were attached to a lecithin bilayer membrane. After photoexcitation with a 500-nm flash delivered by a dye laser, a negative photovoltage was observed on the bilayer under normal ionic strengths (100 mM KCl), which had a rise phase of 1-3 ms at 20 degrees C. The photoresponse was obviously due to bleaching of rhodopsin as it decreased for successive flashes of light. It originated most probably during the metarhodopsin-I metarhodopsin-II (meta-I-II) transition of rhodopsin because it was pH dependent at 2 degrees C but not at 20 degrees C. At 10 mM KCl, i.e., under hypotonic conditions, a positive photovoltage with slower kinetics than at high salt was observed. As the disk membranes were merely attached to the bilayer membrane, the photovoltage was apparently due to a light-induced transmembrane potential change in the disk membranes. Possible electrogenic mechanisms underlying the photosignal will be discussed.     

Ligand channeling within a G-protein-coupled receptor. The entry and exit of retinals in native opsin

Deactivation of light-activated rhodopsin (metarhodopsin II) involves, after rhodopsin kinase and arrestin interactions, the hydrolysis of the covalent bond of all-trans-retinal to the apoprotein. Although the long-lived storage form metarhodopsin III is transiently formed, all-trans-retinal is eventually released from the active site. Here we address the question of whether the release results in a retinal that is freely diffusible in the lipid phase of the photoreceptor membrane. The release reaction is accompanied by an increase in intrinsic protein fluorescence (release signal), which arises from the relief of the fluorescence quenching imposed by the retinal in the active site. An analogous fluorescence decrease (uptake signal) was evoked by exogenous retinoids when they non-covalently bound to native opsin membranes. Uptake of 11-cis-retinal was faster than formation of the retinylidene linkage to the apoprotein. Endogenous all-trans-retinal released from the active site during metarhodopsin II decay did not generate the uptake signal. The data show that in addition to the retinylidene pocket (site I) there are two other retinoidbinding sites within opsin. Site II involved in the uptake signal is an entrance site, while the exit site (site III) is occupied when retinal remains bound after its release from site I. Support for a retinal channeling mechanism comes from the rhodopsin crystal structure, which unveiled two putative hydrophobic binding sites. This mechanism enables a unidirectional process for the release of photoisomerized chromophore and the uptake of newly synthesized 11-cis-retinal for the regeneration of rhodopsin.     

Light-dependent conformational change at rhodopsin's cytoplasmic surface detected by increased susceptibility to proteolysis

Rhodopsin in bovine photoreceptor disk membranes was subjected to limited proteolysis by thermolysin, removing twelve amino acids from rhodopsin's carboxyl terminus. (1) The rate of proteolysis is significantly faster with rhodopsin following exposure to light than with unbleached rhodopsin, provided that the incubation conditions (pH, temperature) favor the formation of metarhodopsin II. (2) If the disk membranes are illuminated under conditions in which metarhodopsin I is the predominant photoproduct (pH 8.5, 0°C), no increase in the rate of proteolysis is observed compared to unilluminated membranes. (3) The light-induced increase in the rate of proteolysis is transient: it slowly decays in the dark to the original rate found for unbleached rhodopsin. The enhanced susceptibility to proteolysis appears to measure a conformational change at rhodopsin's cytoplasmic surface which is first exhibited at the metarhodopsin II stage. This and possibly other light-dependent changes may allow rhodopsin to mediate its signal as a light-receptor protein by binding to and activating certain rod cell enzymes.     

Interaction of bovine rhodopsin with calcium ions. I: the metarhodopsin I--II reaction and the regeneration of rhodopsin.

The formation of metarhodopsin II in various bovine rhodopsin preparations (rod outer segment (ROS) suspensions and rhodopsin-detergent solutions) was measured by means of flash spectrophotometry. The half-lifetime and formation of metarhodopsin II in ROS did not depend on the calcium concentration in the range of less than 10(-9) M (using EGTA ro EDTA) to 15 x 10(-3) M calcium at pH values of 5.0, 7.1, and 9.0 (Table 1). The regeneration of rhodopsin from opsin by adding 11-cis retinal to ROS-suspensions and rhodopsin digitonin solutions was measured spectrophotometrically. It was not substantially different in either saline, one containing less than 10(-7) M calcium (by adding EGTA), the other containing 10(-3) M calcium (Table 2).     

Light-induced conformational changes of rhodopsin probed by fluorescent alexa594 immobilized on the cytoplasmic surface

A novel fluorescence method has been developed for detecting the light-induced conformational changes of rhodopsin and for monitoring the interaction between photolyzed rhodopsin and G-protein or arrestin. Rhodopsin in native membranes was selectively modified with fluorescent Alexa594-maleimide at the Cys(316) position, with a large excess of the reagent Cys(140) that was also derivatized. Modification with Alexa594 allowed the monitoring of fluorescence changes at a red excitation light wavelength of 605 nm, thus avoiding significant rhodopsin bleaching. Upon absorption of a photon by rhodopsin, the fluorescence intensity increased as much as 20% at acidic pH with an apparent pK(a) of approximately 6.8 at 4 degrees C, and was sensitive to the presence of hydroxylamine. These findings indicated that the increase in fluorescence is specific for metarhodopsin II. In the presence of transducin, a significant increase in fluorescence was observed. This increase of fluorescence emission intensity was reduced by addition of GTP, in agreement with the fact that transducin enhances the formation of metarhodopsin II. Under conditions that favored the formation of a metarhodopsin II-Alexa594 complex, transducin slightly decreased the fluorescence. In the presence of arrestin, under conditions that favored the formation of metarhodopsin I or II, a phosphorylated, photolyzed rhodopsin-Alexa594 complex only slightly decreased the fluorescence intensity, suggesting that the cytoplasmic surface structure of metarhodopsin II is different in the complex with arrestin and transducin. These results demonstrate the application of Alexa594-modified rhodopsin (Alexa594-rhodopsin) to continuously monitor the conformational changes in rhodopsin during light-induced transformations and its interactions with other proteins.     

Photochemical functionality of rhodopsin-phospholipid recombinant membranes

Purified rhodopsin was incorporated into phospholipid bilayers to give recombinant membranes. The photochemical functionality in these systems was examined by low-temperature spectroscopy and by kinetic spectrophotometry. Changes in the absorption spectra of glycerol-water mixtures of rhodopsin-egg phosphatidylcholine and rhodopsin-asolectin recombinants were monitored after the sample was cooled to -196 degrees C, presented with light of wavelength greater than 440 nm, and then warmed gradually to room temperature. Absorption characteristics indicative of the spectral intermediates prelumirhodopsin, lumirhodopsin, metarhodopsin I, and metarhodopsin II were observed. The kinetics of the metarhodopsin I -o metarhodopsin II transition in these recombinants was studied by flash photolytic observation of the decay of meta I and the formation of meta II. Recombinants prepared from unsaturated phospholipids, e.g., asolectin, egg phosphatidylcholine, egg phosphatidylethanolamine, and dioleoylphosphatidylcholine, showed first-order kinetics for the transition with rates comparable to that of rod outer segment membranes. Recombinants prepared from saturated phosphatidylcholines have a retarded rate of conversion from meta I to meta II and are considered to be nonfunctional. The photochemical functionality of rhodopsin-phospholipid recombinants is dependent upon the presence of phospholipid unsaturation and the fluidity of the phospholipid hydrocarbon chains, and is independent of the polar head group of the phospholipid.     

Effects of lipid environment on the light-induced conformational changes of rhodopsin. 1. Absence of metarhodopsin II production in dimyristoylphosphatidylcholine recombinant membranes

Photolysis of bovine rhodopsin in dimyristoylphosphatidylcholine recombinant membranes results in the production of a relatively stable metarhodopsin I like photointermediate that decays slowly to a species with a broad absorbance maximum centered at about 380 nm [O'Brien, D. F., Costa, L. F., & Ott, R. A. (1977) Biochemistry 16, 1295-1303]. On the basis of the results of a variety of chemical and spectroscopic tests, we show that this process corresponds to the production of free retinal plus opsin and not to the slow production of metarhodopsin II. Electron spin resonance studies using a novel disulfide spin-label that is covalently linked to rhodopsin indicate that the apparent arrest of the protein at the metarhodopsin I stage is not due to simple aggregation of the protein in this short-chain, saturated lipid bilayer but must be understood in terms of the effect of the lipid host on the conformational energies of individual protein molecules. Limited production of metarhodopsin II is observed under acidic conditions. Thus, the rhodopsin-dimyristoylphosphatidylcholine recombinants offer a unique system for the study of the effect of the phospholipid bilayer environment on the conformation of an intrinsic membrane protein.     

Metarhodopsin control by arrestin,light-filtering screening pigments,and visual pigment turnover in invertebrate microvillar photoreceptors

The visual pigments of most invertebrate photoreceptors have two thermostable photo-interconvertible states, the ground state rhodopsin and photo-activated metarhodopsin, which triggers the phototransduction cascade until it binds arrestin. The ratio of the two states in photoequilibrium is determined by their absorbance spectra and the effective spectral distribution of illumination. Calculations indicate that metarhodopsin levels in fly photoreceptors are maintained below ~35% in normal diurnal environments, due to the combination of a blue-green rhodopsin, an orange-absorbing metarhodopsin and red transparent screening pigments. Slow metarhodopsin degradation and rhodopsin regeneration processes further subserve visual pigment maintenance. In most insect eyes, where the majority of photoreceptors have green-absorbing rhodopsins and blue-absorbing metarhodopsins, natural illuminants are predicted to create metarhodopsin levels greater than 60% at high intensities. However, fast metarhodopsin decay and rhodopsin regeneration also play an important role in controlling metarhodopsin in green receptors, resulting in a high rhodopsin content at low light intensities and a reduced overall visual pigment content in bright light. A simple model for the visual pigment–arrestin cycle is used to illustrate the dependence of the visual pigment population states on light intensity, arrestin levels and pigment turnover.     

Photolysis intermediates of human rhodopsin.

Photochemical studies were conducted on human rhodopsin at 20 degrees C to characterize the intermediates which precede the formation of metarhodopsin II, the trigger for the enzyme cascade mechanism of visual transduction. Human rhodopsin was prepared from eyes which had previously been used for corneal donations. Time resolved absorption spectra collected from 10(-8) to 10(-6) s after photolysis of human rhodopsin in detergent suspensions displayed biexponential decay kinetics. The apparent lifetimes obtained from the data are 65 +/- 20 and 292 +/- 25 ns, almost a factor of 2 slower than the corresponding rates in bovine rhodopsin. The spectra can be fit well using a model in which human bathorhodopsin decays toward equilibrium with a blue-shifted intermediate (BSI) which then decays to lumirhodopsin. Spectra and kinetic rate constants were determined for all these intermediates using a global analysis which showed that the spectra of the human intermediates are remarkably similar to bovine intermediates. Microscopic rate constants derived from this model are 7.4 x 10(6) s-1 for bathorhodopsin decay and 7.5 x 10(6) s-1 and 4.6 x 10(6) s-1 for the forward and reverse reactions of BSI, respectively. Decay of lumirhodopsin to later intermediates was studied from 10(-6) to 10(-1) s after photolysis of rhodopsin in human disk membrane suspensions. The human metarhodopsin I in equilibrium metarhodopsin II equilibrium appears to be more forward shifted than in comparable bovine studies.     

Analogue pigment studies of chromophore-protein interactions in metarhodopsins

Several analogue pigments have been prepared containing retinals altered at the cyclohexyl ring or proximal to the aldehyde group in order to examine the role of the chromophore in the formation of the metarhodopsin I and II states of visual pigments. Deletion of the 13-methyl group on the isoprenoid chain did not affect metarhodopsin formation. However, analogue pigments containing chromophores with modified rings did not show the typical absorption changes associated with the metarhodopsin transitions of native or regenerated rhodopsins. In particular, 4-hydroxyretinal pigments did not show clear transitions between the metarhodopsin I and metarhodopsin II states. Pigment formed with an acyclic retinal showed no evidence by absorption spectroscopy of metarhodopsin formation. A retinal altered by substitution of a five-membered ring containing a nitroxide required a more acidic pH than the native pigment for formation of the metarhodopsin II state. ESR data suggest that the ring remains buried within the protein through the metarhodopsin II state. However, the Schiff base linkage is susceptible to hydrolysis of hydroxylamine in the metarhodopsin II state. These data indicate that (1), in the transition from rhodopsin to metarhodopsin II, major protein conformational changes are occurring near the lysine-retinal linkage whereas the ring portion of the chromophore remains deeply buried within the protein and (2) pigment absorptions characteristic of the metarhodopsin I and II states may be due to specific protein-chromophore interactions near the region of the chromophore ring.     

Octopus photoreceptor membranes. Surface charge density and pK of the Schiff base of the pigments.

The chromophore of octopus rhodopsin is 11-cis retinal, linked via a protonated Schiff base to the protein backbone. Its stable photoproduct, metarhodopsin, has all-trans retinal as its chromphore. The Schiff base of acid metarhodopsin (lambda max = 510 nm) is protonated, whereas that of alkaline metarhodopsin (lambda max = 376 nm) is unprotonated. Metarhodopsin in photoreceptor membranes was titrated and the apparent pK of the Schiff base was measured at different ionic strengths. From these salt-dependent pKs the surface charge density of the octopus photoreceptor membranes and the intrinsic Schiff base pK of metarhodopsin were obtained. The surface charge density is sigma = -1.6 +/- 0.1 electronic charges per 1,000 A2. Comparison of the measured surface charge density with values from octopus rhodopsin model structures suggests that the measured value is for the extracellular surface and so the Schiff base in metarhodopsin is freely accessible to protons from the extracellular side of the membrane. The intrinsic Schiff base pK of metarhodopsin is 8.44 +/- 0.12, whereas that of rhodopsin is found to be 10.65 +/- 0.10 in 4.0 M KCl. These pK values are significantly higher than the pK value around 7.0 for a retinal Schiff base in a polar solvent; we suggest that a plausible mechanism to increase the pK of the retinal pigments is the preorganization of their chromophore-binding sites. The preorganized site stabilizes the protonated Schiff base with respect to the unprotonated one. The difference in the pK for the octopus rhodopsin compared with metarhodopsin is attributed to the relative freedom of the latter's chromophore-binding site to rearrange itself after deprotonation of the Schiff base.     

Proteinase-treated photoreceptor discs. Photoelectric activity of the partially-digested rhodopsin and membrane orientation

Photoreceptor discs from rod outer segments of cattle retina were treated with (a) papain, (b) thermolysin or (c) trypsin, the procedures resulting in the cleavage of the rhodopsin polypeptide chain between (a) 323 and 324, 236 and 237, 241 and 242, (b) 327 and 328, 240 and 241, or (c) 339 and 340 amino acid residues, respectively. In all the cases, partially digested rhodopsins proved to be competent in generating photoelectric potential and increasing membrane conductance of the discs adsorbed onto phospholipid-impregnated collodion film. The kinetics of generation and dissipation of photopotential as well as of formation of metarhodopsin II and of the light-induced rhodopsin protonation were found to be the same in the partially digested preparations and in the intact one. Incubation of papain-treated or thermolysin-treated discs at pH 6.0 induced formation of inside-out vesicles which, when incorporated into the collodion film, generated an oppositely directed photopotential. Treatment of such vesicles with papain gave rise to further cleavages of the polypeptide localized between 30 and 31, 186 and 187 amino acid residues. One more proteinase-sensitive site, localized between 104 and 105 residues, has been discovered in the inside-out vesicles treated with thermolysin. This fact consistent with the scheme of the 'seven column' arrangement of the visual rhodopsin [Ovchinnikov, Yu. A. (1982) FEBS Lett. 148, 179-191]. Rhodopsin, when treated with papain on both sides, was deprived of sixty amino acid residues being split in two sites in the middle part of the polypeptide, but was still active as a photoelectric energy transducer. The main specific feature inherent in the photoelectric response of the papain-treated or thermolysin-treated rhodopsin and absent from the native protein is that the former survives addition of long trains of saturating flashes when the response of the intact preparation becomes negligible. This effect was shown to be due to conversion of partially digested rhodopsin to a photolytic product that at room temperature lived for minutes even in the presence of NH2OH. A 532-nm laser flash effectively converted this product back to rhodopsin.     

Conformations of the active and inactive states of opsin

The signaling state metarhodopsin II of the visual pigment rhodopsin decays to the apoprotein opsin and all-trans retinal, which are then regenerated to rhodopsin by the visual cycle. Opsin is known to have at neutral pH only a small residual constitutive activity toward its G protein transducin, which is thought to play a considerable role in light adaptation (bleaching desensitization). In this study we show with Fourier-transform infrared spectroscopy that after metarhodopsin II decay, opsin exists in two conformational states that are in a pH-dependent equilibrium at 30 degrees C with a pK of 4.1 in the presence of hydroxylamine scavenging the endogenous all-trans retinal. Despite the lack of the native agonist in its binding pocket, the low pH opsin conformation is very similar to that of metarhodopsin II and is likewise stabilized by peptides derived from rhodopsin's cognate G protein, transducin. The high pH form, on the other hand, has some conformational similarity to the inactive metarhodopsin I state. We therefore conclude that the opsin apoprotein displays intrinsic conformational states that are merely modulated by bound all-trans retinal.     

Location of Trp265 in metarhodopsin II: implications for the activation mechanism of the visual receptor rhodopsin

Isomerization of the 11-cis retinal chromophore in the visual pigment rhodopsin is coupled to motion of transmembrane helix H6 and receptor activation. We present solid-state magic angle spinning NMR measurements of rhodopsin and the metarhodopsin II intermediate that support the proposal that interaction of Trp265(6.48) with the retinal chromophore is responsible for stabilizing an inactive conformation in the dark, and that motion of the beta-ionone ring allows Trp265(6.48) and transmembrane helix H6 to adopt active conformations in the light. Two-dimensional dipolar-assisted rotational resonance NMR measurements are made between the C19 and C20-methyl groups of the retinal and uniformly 13C-labeled Trp265(6.48). The retinal C20-Trp265(6.48) contact present in the dark-state of rhodopsin is lost in metarhodopsin II, and a new contact is formed with the C19 methyl group. We have previously shown that the retinal translates 4-5 A toward H5 in metarhodopsin II. This motion, in conjunction with the Trp-C19 contact, implies that the Trp265(6.48) side-chain moves significantly upon rhodopsin activation. NMR measurements also show that a packing interaction in rhodopsin between Trp265(6.48) and Gly121(3.36) is lost in metarhodopsin II, consistent with H6 motion away from H3. However, a close contact between Gly120(3.35) on H3 and Met86(2.53) on H2 is observed in both rhodopsin and metarhodopsin II, suggesting that H3 does not change orientation significantly upon receptor activation.