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.     

A dark and constitutively active mutant of the tiger salamander UV pigment

A triple mutant (F86L/T93P/S118T; bovine rhodopsin numbering) of the tiger salamander UV cone pigment appears to be trapped in an open conformation that is metarhodopsin-II-like. The pigment is able to activate transducin in the dark, and the ligand-free apoprotein is also able to activate transducin constitutively. The pigment permits protons and chloride ions from solution access to the active site as it displays a pH- and NaCl-dependent absorption spectrum not observed with the wild-type pigment. However, the wild-type properties of light-dependent activity and a pH-independent absorption spectrum are recovered upon reconstitution of the triple mutant with 11-cis-9-demethyl retinal. These results suggest that binding the native chromophore cannot deactivate the protein because of steric interactions between the protein, possibly residue 118, and the 9-methyl group of the chromophore. Furthermore, the absorption spectrum of the 9-demethyl retinal regenerated pigment exhibits a band broader and with lower extinction at the absorption maximum than either the human blue or salamander UV wild-type pigments generated with the same retinal analogue. The broad spectrum appears to be comprised of two or more species and can be well-fit by a sum of scaled spectra of the two wild-type pigments. Binding the chromophore appears to trap the pigment in two or more conformations. The triple mutant reported here represents the first example of a dark-active cone pigment and constitutively active cone opsin.     

The molecular origin of the inhibition of transducin activation in rhodopsin lacking the 9-methyl group of the retinal chromophore: a UV-Vis and FTIR spectroscopic study

The formation of the active rhodopsin state metarhodopsin II (MII) is believed to be partially governed by specific steric constraints imposed onto the protein by the 9-methyl group of the retinal chromophore. We studied the properties of the synthetic pigment 9-demethyl rhodopsin (9dm-Rho), consisting of the rhodopsin apoprotein regenerated with synthetic retinal lacking the 9-methyl group, by UV-vis and Fourier transform infrared difference spectroscopy. Low activation rates of the visual G-protein transducin by the modified pigment reported in previous studies are shown to not be caused by the reduced activity of its MII state, but to be due to a dramatic equilibrium shift from MII to its immediate precursor, MI. The MII state of 9dm-Rho displays only a partial deprotonation of the retinal Schiff base, leading to the formation of two MII subspecies absorbing at 380 and 470 nm, both of which seem to be involved in transducin activation. The rate of MII formation is slowed by 2 orders of magnitude compared to rhodopsin. The dark state and the MI state of 9dm-Rho are distinctly different from their respective states in the native pigment, pointing to a more relaxed fit of the retinal chromophore in its binding pocket. The shifted equilibrium between MI and MII is therefore discussed in terms of an increased entropy of the 9dm-Rho MI state due to changed steric interactions.     

Signaling states of rhodopsin. Retinal provides a scaffold for activating proton transfer switches

The G-protein-coupled receptor rhodopsin is activated by photoconversion of its covalently bound ligand 11-cis-retinal to the agonist all-trans-retinal. After light-induced isomerization and early photointermediates, the receptor reaches a G-protein-dependent equilibrium between active and inactive conformations distinguished by the protonation of key opsin residues. In this report, we study the role of the 9-methyl group of retinal, one of the crucial steric determinants of light activation. We find that when this group is removed, the protonation equilibrium is strongly shifted to the inactive conformation. The residually formed active species is very similar to the active form of normal rhodopsin, metarhodopsin II. It has a deprotonated Schiff base, binds to the retinal G-protein transducin, and is favored at acidic pH. Our data show that the normal proton transfer reactions are inhibited in 9-demethyl rhodopsin but are still mandatory for receptor activation. We propose that retinal and its 9-methyl group act as a scaffold for opsin to adjust key proton donor and acceptor side chains for the proton transfer reactions that stabilize the active conformation. The mechanism may also be applicable to related receptors and may thus explain the partial agonism of certain ligands.     

Solid-state 2H NMR spectroscopy of retinal proteins in aligned membranes

Solid-state 2H NMR spectroscopy gives a powerful avenue to investigating the structures of ligands and cofactors bound to integral membrane proteins. For bacteriorhodopsin (bR) and rhodopsin, retinal was site-specifically labeled by deuteration of the methyl groups followed by regeneration of the apoprotein. 2H NMR studies of aligned membrane samples were conducted under conditions where rotational and translational diffusion of the protein were absent on the NMR time scale. The theoretical lineshape treatment involved a static axial distribution of rotating C-C2H3 groups about the local membrane frame, together with the static axial distribution of the local normal relative to the average normal. Simulation of solid-state 2H NMR lineshapes gave both the methyl group orientations and the alignment disorder (mosaic spread) of the membrane stack. The methyl bond orientations provided the angular restraints for structural analysis. In the case of bR the retinal chromophore is nearly planar in the dark- and all-trans light-adapted states, as well upon isomerization to 13-cis in the M state. The C13-methyl group at the "business end" of the chromophore changes its orientation to the membrane upon photon absorption, moving towards W182 and thus driving the proton pump in energy conservation. Moreover, rhodopsin was studied as a prototype for G protein-coupled receptors (GPCRs) implicated in many biological responses in humans. In contrast to bR, the retinal chromophore of rhodopsin has an 11-cis conformation and is highly twisted in the dark state. Three sites of interaction affect the torsional deformation of retinal, viz. the protonated Schiff base with its carboxylate counterion; the C9-methyl group of the polyene; and the beta-ionone ring within its hydrophobic pocket. For rhodopsin, the strain energy and dynamics of retinal as established by 2H NMR are implicated in substituent control of activation. Retinal is locked in a conformation that is twisted in the direction of the photoisomerization, which explains the dark stability of rhodopsin and allows for ultra-fast isomerization upon absorption of a photon. Torsional strain is relaxed in the meta I state that precedes subsequent receptor activation. Comparison of the two retinal proteins using solid-state 2H NMR is thus illuminating in terms of their different biological functions.     

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.     

Deactivation and proton transfer in light-induced metarhodopsin II/metarhodopsin III conversion: a time-resolved fourier transform infrared spectroscopic study

Vertebrate rhodopsin shares with other retinal proteins the 11-cis-retinal chromophore and the light-induced 11-cis/trans isomerization triggering its activation pathway. However, only in rhodopsin the retinylidene Schiff base bond to the apoprotein is eventually hydrolyzed, making a complex regeneration pathway necessary. Metabolic regeneration cannot be short-cut, and light absorption in the active metarhodopsin (Meta) II intermediate causes anti/syn isomerization around the retinylidene linkage rather than reversed trans/cis isomerization. A new deactivating pathway is thereby triggered, which ends in the Meta III "retinal storage" product. Using time-resolved Fourier transform infrared spectroscopy, we show that the identified steps of receptor activation, including Schiff base deprotonation, protein structural changes, and proton uptake by the apoprotein, are all reversed. However, Schiff base reprotonation is much faster than the activating deprotonation, whereas the protein structural changes are slower. The final proton release occurs with pK approximately 4.5, similar to the pK of a free Glu residue and to the pK at which the isolated opsin apoprotein becomes active. A forced deprotonation, equivalent to the forced protonation in the activating pathway, which occurs against the unfavorable pH of the medium, is not observed. This explains properties of the final Meta III product, which displays much higher residual activity and is less stable than rhodopsin arising from regeneration with 11-cis-retinal. We propose that the anti/syn conversion can only induce a fast reorientation and distance change of the Schiff base but fails to build up the full set of dark ground state constraints, presumably involving the Glu(134)/Arg(135) cluster.     

Characterization of rhodopsin congenital night blindness mutant T94I

The Thr94 --> Ile mutation in the second transmembrane segment of rhodopsin has been reported to be associated with a congenital night blindness phenotype in a large Irish pedigree. Previously, two other known rhodopsin mutants that cause congenital night blindness, A292E and G90D, have been shown in vitro to constitutively activate the G protein transducin in the absence of a chromophore. The proposed mechanism of constitutive activation of these two mutants is an electrostatic disruption of the active site salt bridge between Glu113 and Lys296 that contributes to stabilization of the protein in the inactive state. Here, the T94I rhodopsin mutant is characterized and compared to the two other known rhodopsin night blindness mutants. The T94I mutant opsin is shown also to constitutively activate transducin. The T94I mutant pigment (with a bound 11-cis-retinal chromophore), like the other known rhodopsin night blindness mutants, is not active in the dark and has wild-type activity upon exposure to light. Similar to the Gly90 --> Asp substitution, position 94 is close enough to the Schiff base nitrogen that an Asp at this position can functionally substitute for the Glu113 counterion. However, in contrast to the other night blindness mutants, the T94I MII intermediate decays with a half-life that is approximately 8-fold slower than in the wild-type MII intermediate. Thus, the one phenotype shared by all congenital night blindness mutants that is different from the wild-type protein is constitutive activation of the apoprotein.     

Rhodopsin and 9-demethyl-retinal analog: effect of a partial agonist on displacement of transmembrane helix 6 in class A G protein-coupled receptors

Rhodopsin is the visual pigment of rod cells and a prototypical G protein-coupled receptor. It is activated by cis-->trans photoisomerization of the covalently bound chromophore 11-cis-retinal, which acts in the cis configuration as an inverse agonist. Light-induced formation of the full agonist all-trans-retinal in situ triggers conformational changes in the protein moiety. Partial agonists of rhodopsin include a retinal analog lacking the methyl group at C-9, termed 9-demethyl-retinal (9-dm-retinal). Rhodopsin reconstituted with this retinal (9-dm-rhodopsin) activates G protein poorly. Here we investigated the molecular nature of the partial agonism in 9-dm-rhodopsin using site-directed spin labeling. Earlier site-directed spin labeling studies of rhodopsin identified a rigid-body tilt of the cytoplasmic segment of [corrected] transmembrane helix 6 (TM6) by approximately 6A as a central event in rhodopsin activation. Data presented here provide additional evidence for this mechanism. Only a small fraction of photoexcited 9-dm pigments reaches the TM6-tilted conformation. This fraction can be increased by increasing proton concentration or [corrected] by anticipation of the activating protonation step by the mutation E134Q in 9-dm-rhodopsin. These results on protein conformation are in complete accord with previous findings regarding the biological activity of the 9-dm pigments. When the proton concentration is further increased, a new state arises in 9-dm pigments that is linked to direct proton uptake at the retinal Schiff base. This state apparently has a conformation distinguishable from the active state.     

Role of the 9-methyl group of retinal in cone visual pigments

In rhodopsin, the 9-methyl group of retinal has previously been identified as being critical in linking the ligand isomerization with the subsequent protein conformational changes that result in the activation of its G protein, transducin. Here, we report studies on the role of this methyl group in the salamander rod and cone pigments. Pigments were generated by combining proteins expressed in COS cells with 11-cis 9-demethyl retinal, where the 9-methyl group on the polyene chain has been deleted. The absorption spectra of all pigments were blue-shifted. The red cone and blue cone/green rod pigments were unstable to hydroxylamine; whereas, the rhodopsin and UV cone pigments were stable. The lack of the 9-methyl group of the chromophore did not affect the ability of the red cone and blue cone/green rod pigments to activate transducin. On the other hand, with the rhodopsin and UV cone pigments, activation was diminished. Interestingly, the red cone pigment containing the retinal analogue remained active longer than the native pigment. Thus, the 9-methyl group of retinal is not important in the activation pathway of the red cone and blue cone/green rod pigments. However, for the red cone pigment, the 9-methyl group of retinal appears to be critical in the deactivation pathway.     

Molecular dynamics simulation of dark-adapted rhodopsin and free opsin

Molecular dynamics simulation was carried out for the rhodopsin protein to investigate its conformational changes in respect to inclusion of 11-cis retinal chromophore. Molecular dynamics calculations were performed within the time frame 3000 ps. Totally, 3 X 10(6) configurations ofrhodopsin and free opsin were analyzed and compared. It has been shown that the 11-cis retinal rearrangement (adaptation) in opsin strongly affects the surrounding amino acid residues of protein binding pocket and the protein cytoplasmic region. The extracellular part, however, shows comparatively little changes. On basis of the simulation results obtained we propose a molecular mechanism for the rhodopsin protein function as a G-protein-coupled receptor in the state of darkness. We discuss the role of the retinal chromophore as a ligand-antagonist stabilizing the inactive conformation of rhodopsin.     

Accessibility of the iodopsin chromophore.

Iodopsin can replace its chromophore (11-cis retinal) by added 9-cis retinal, resulting in the formation of isoiodopsin. NaBH4 bleaches iodopsin in the dark. In a relatively low concentration of digitonin, the scotopsin (the protein moiety of chicken rhodopsin) removes 11-cis retinal from iodopsin in the dark. These facts suggest that the linkage of the chromophore to opsin in the iodopsin molecule (presumably a Schiff-base linkage) is accessible to these reagents, which is different from the situation in rhodopsin.     

Solid-state NMR analysis of ligand--receptor interactions reveals an induced misfit in the binding site of isorhodopsin

Rhodopsin is the photosensitive protein of the rod photoreceptor in the vertebrate retina and is a paradigm for the superfamily of G-protein-coupled receptors (GPCRs). Natural rhodopsin contains an 11-cis-retinylidene chromophore. We have prepared the 9-cis analogue isorhodopsin in a natural membrane environment using uniformly (13)C-enriched 9-cis retinal. Subsequently, we have determined the complete (1)H and (13)C assignments with ultra-high field solid-state magic angle spinning NMR. The 9-cis substrate conforms to the opsin binding pocket in isorhodopsin in a manner very similar to that of the 11-cis form in rhodopsin, but the NMR data reveal an improper fit of the 9-cis chromophore in this binding site. We introduce the term "induced misfit" to describe this event. Downfield proton NMR ligation shifts (Deltasigma(lig)(H) > 1 ppm) are observed for the 16,17,19-H and nearby protons of the ionone ring and for the 9-methyl protons. They provide converging evidence for global, nonspecific steric interactions between the chromophore and protein, and contrast with the specific interactions over the entire ionone ring and its substituents detected for rhodopsin. The Deltasigma(lig)(C) pattern of the polyene chain confirms the positive charge delocalization in the polyene associated with the protonation of the Schiff base nitrogen. In line with the misalignment of the ionone ring, an additional and anomalous perturbation of the (13)C response is detected in the region of the 9-cis bond. This provides evidence for strain in the isomerization region of the polyene and supports the hypothesis that perturbation of the conjugation around the cis bond induced by the protein environment assists the selective photoisomerization.     

Comparative study on the chromophore binding sites of rod and red-sensitive cone visual pigments by use of synthetic retinal isomers and analogues

A comparative study on the chromophore (retinal) binding sites of the opsin (R-photopsin) from chicken red-sensitive cone visual pigment (iodopsin) and that scotopsin) from bovine rod pigment (rhodopsin) was made by the aid of geometric isomers of retinal (all-trans, 13-cis, 11-cis, 9-cis, and 7-cis) and retinal analogues including fluorinated (14-F, 12-F, 10-F, and 8-F) and methylated (12-methyl) 11-cis-retinals. The stereoselectivity of R-photopsin for the retinal isomers and analogues was almost identical with that of scotopsin, indicating that the shapes of the chromophore binding sites of both opsins are similar, although the former appears to be somewhat more restricted than the latter. The rates of pigment formation from R-photopsin were considerably greater than those from scotopsin. In addition, all the iodopsin isomers and analogues were more susceptible to hydroxylamine than were the rhodopsin ones. These observations suggest that the retinal binding site of iodopsin is located near the protein surface. On the basis of the spectral properties of fluorinated analogues, a polar group in the chromophore binding site of iodopsin as well as rhodopsin was estimated to be located near the hydrogen atom at the C10 position of the retinylidene chromophore. A large difference in wavelength between the absorption maxima of iodopsin and rhodopsin was significantly reduced in the 9-cis and 7-cis pigments. On the assumption that the retinylidene chromophore is anchored rigidly at the alpha-carbon of the lysine residue and loosely at the cyclohexenyl ring, each of the two isomers would have the Schiff-base nitrogen at a position altered from that of the 11-cis pigments.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The all-trans-15-syn-retinal chromophore of metarhodopsin III is a partial agonist and not an inverse agonist

Meta III is formed during the decay of rhodopsin's active receptor state at neutral to alkaline pH by thermal isomerization of the retinal Schiff base C15=N bond, converting the ligand from all-trans 15-anti to all-trans 15-syn. The thereby induced change of ligand geometry switches the receptor to an inactive conformation, such that the decay pathway to Meta III contributes to the deactivation of the signaling state at higher pH values. We have examined the conformation of Meta III over a wider pH range and found that Meta III exists in a pH-dependent conformational equilibrium between this inactive conformation at neutral to alkaline pH and an active conformation similar to that of Meta II, which, however, is assumed at very acidic pH only. The apparent pKa of this transition is around 5.1 and thus several units lower than that of the Meta I/Meta II photoproduct equilibrium with its all-trans 15-anti ligand, but still about 1 unit higher than that of the opsin conformational equilibrium in the absence of ligand. The all-trans-15-syn-retinal chromophore is therefore not an inverse agonist like 11-cis- or 9-cis-retinal, which lock the receptor in an inactive conformation, but a classical partial agonist, which is capable of activating the receptor, yet with an efficiency considerably lower than the full agonist all-trans 15-anti. As the Meta III chromophore differs structurally from this full agonist only in the isomeric state of the C15=N bond, this ligand represents an excellent model system to study principal mechanisms of partial agonism which are helpful to understand the partial agonist behavior of other ligands.     

Changes in interhelical hydrogen bonding upon rhodopsin activation

Hydrogen bonding interactions between transmembrane helices stabilize the visual pigment rhodopsin in an inactive conformation in the dark. The crystal structure of rhodopsin has previously revealed that Glu122 and Trp126 on transmembrane helix H3 form a complex hydrogen bonding network with Tyr206 and His211 on H5, while the indole nitrogen of Trp265 on H6 forms a water-mediated hydrogen bond with Asn302 on H7. Here, we use solid-state magic angle spinning NMR spectroscopy to probe the changes in hydrogen bonding upon rhodopsin activation. The NMR chemical shifts of 15N-labeled tryptophan are consistent with the indole nitrogens of Trp126 and Trp265 becoming more weakly hydrogen bonded between rhodopsin and metarhodopsin II. The NMR chemical shifts of 15N-labeled histidine show that His211 is neutral; the unprotonated imidazole nitrogen is not coordinated to zinc in rhodopsin and becomes more strongly hydrogen bonded in metarhodopsin II. Moreover, measurements of rhodopsin containing 13C-labeled histidine show that a strong hydrogen bond between the side-chain of Glu122 and the backbone carbonyl of His211 is disrupted in metarhodopsin II. The implications of these observations for the activation mechanism of rhodopsin are discussed.     

Cyclic nucleotide-gated ion channels in rod photoreceptors are protected from retinoid inhibition

In vertebrate rods, photoisomerization of the 11-cis retinal chromophore of rhodopsin to the all-trans conformation initiates a biochemical cascade that closes cGMP-gated channels and hyperpolarizes the cell. All-trans retinal is reduced to retinol and then removed to the pigment epithelium. The pigment epithelium supplies fresh 11-cis retinal to regenerate rhodopsin. The recent discovery that tens of nanomolar retinal inhibits cloned cGMP-gated channels at low [cGMP] raised the question of whether retinoid traffic across the plasma membrane of the rod might participate in the signaling of light. Native channels in excised patches from rods were very sensitive to retinoid inhibition. Perfusion of intact rods with exogenous 9- or 11-cis retinal closed cGMP-gated channels but required higher than expected concentrations. Channels reopened after perfusing the rod with cellular retinoid binding protein II. PDE activity, flash response kinetics, and relative sensitivity were unchanged, ruling out pharmacological activation of the phototransduction cascade. Bleaching of rhodopsin to create all-trans retinal and retinol inside the rod did not produce any measurable channel inhibition. Exposure of a bleached rod to 9- or 11-cis retinal did not elicit channel inhibition during the period of rhodopsin regeneration. Microspectrophotometric measurements showed that exogenous 9- or 11-cis retinal rapidly cross the plasma membrane of bleached rods and regenerate their rhodopsin. Although dark-adapted rods could also take up large quantities of 9-cis retinal, which they converted to retinol, the time course was slow. Apparently cGMP-gated channels in intact rods are protected from the inhibitory effects of retinoids that cross the plasma membrane by a large-capacity buffer. Opsin, with its chromophore binding pocket occupied (rhodopsin) or vacant, may be an important component. Exceptionally high retinoid levels, e.g., associated with some retinal degenerations, could overcome the buffer, however, and impair sensitivity or delay the recovery after exposure to bright light.     

Studies on cephalopod rhodopsin. Conformational changes in chromophore and protein during the photoregeneration process

The ultraviolet absorbance of squid and octopus rhodopsin changes reversibly at 234 nm and near 280 nm in the interconversion of rhodopsin and metarhodopsin. The absorbance change near 280 nm is ascribed to both protein and chromophore parts. Rhodopsin is photoregenerated from metarhodopsin via an intermediate, P380, on irradiation with yellow light (λ > 520 nm). The ultraviolet absorbance decreases in the change from rhodopsin to metarhodopsin and recovers in two steps; mostly in the process from metarhodopsin to P380 and to a lesser extent in the process from P380 to rhodopsin. P380 has a circular dichroism (CD) band at 380 nm and its magnitude is the same order as that of rhodopsin. Thus it is considered that the molecular structure of P380 is close to that of rhodopsin and that the chromophore is fixed to opsin as in rhodopsin. In the change from metarhodopsin to P380, the chromophore is isomerized from the all-trans to the 11-cis form, and the conformation of opsin changes to fit 11-cis retinal. In the change from P380 to rhodopsin, a small change in the conformation of the protein part and the protonation of the Schiff base, the primary retinal-opsin link, occur.     

Solid-State H NMR spectroscopy of retinal proteins in aligned membranes

Solid-state ²H NMR spectroscopy gives a powerful avenue to investigating the structures of ligands and cofactors bound to integral membrane proteins. For bacteriorhodopsin (bR) and rhodopsin, retinal was site-specifically labeled by deuteration of the methyl groups followed by regeneration of the apoprotein. ²H NMR studies of aligned membrane samples were conducted under conditions where rotational and translational diffusion of the protein were absent on the NMR time scale. The theoretical lineshape treatment involved a static axial distribution of rotating C-C²H₃ groups about the local membrane frame, together with the static axial distribution of the local normal relative to the average normal. Simulation of solid-state ²H NMR lineshapes gave both the methyl group orientations and the alignment disorder (mosaic spread) of the membrane stack. The methyl bond orientations provided the angular restraints for structural analysis. In the case of bR the retinal chromophore is nearly planar in the dark- and all-trans light-adapted states, as well upon isomerization to 13-cis in the M state. The C13-methyl group at the “business end” of the chromophore changes its orientation to the membrane upon photon absorption, moving towards W182 and thus driving the proton pump in energy conservation. Moreover, rhodopsin was studied as a prototype for G protein-coupled receptors (GPCRs) implicated in many biological responses in humans. In contrast to bR, the retinal chromophore of rhodopsin has an 11-cis conformation and is highly twisted in the dark state. Three sites of interaction affect the torsional deformation of retinal, viz. the protonated Schiff base with its carboxylate counterion; the C9-methyl group of the polyene; and the β-ionone ring within its hydrophobic pocket. For rhodopsin, the strain energy and dynamics of retinal as established by ²H NMR are implicated in substituent control of activation. Retinal is locked in a conformation that is twisted in the direction of the photoisomerization, which explains the dark stability of rhodopsin and allows for ultra-fast isomerization upon absorption of a photon. Torsional strain is relaxed in the meta I state that precedes subsequent receptor activation. Comparison of the two retinal proteins using solid-state ²H NMR is thus illuminating in terms of their different biological functions.