Coupling of protonation switches during rhodopsin activation

Recent studies of the activation mechanism of rhodopsin involving Fourier-transform infrared spectroscopy and a combination of chromophore modifications and site-directed mutagenesis reveal an allosteric coupling between two protonation switches. In particular, the ring and the 9-methyl group of the all-trans retinal chromophore serve to couple two proton-dependent activation steps: proton uptake by a cytoplasmic network between transmembrane (TM) helices 3 and 6 around the conserved ERY (Glu-Arg-Tyr) motif and disruption of a salt bridge between the retinal protonated Schiff base (PSB) and a protein counterion in the TM core of the receptor. Retinal analogs lacking the ring or 9-methyl group are only partial agonists--the conformational equilibrium between inactive Meta I and active Meta II photoproduct states is shifted to Meta I. An artificial pigment was engineered, in which the ring of retinal was removed and the PSB salt bridge was weakened by fluorination of C14 of the retinal polyene. These modifications abolished allosteric coupling of the proton switches and resulted in a stabilized Meta I state with a deprotonated Schiff base (Meta I(SB)). This state had a partial Meta II-like conformation due to disruption of the PSB salt bridge, but still lacked the cytoplasmic proton uptake reaction characteristic of the final transition to Meta II. As activation of native rhodopsin is known to involve deprotonation of the retinal Schiff base prior to formation of Meta II, this Meta I(SB) state may serve as a model for the structural characterization of a key transient species in the activation pathway of a prototypical G protein-coupled receptor.     

Retinal counterion switch mechanism in vision evaluated by molecular simulations

Photoisomerization of the retinylidene chromophore of rhodopsin is the starting point in the vision cascade. A counterion switch mechanism that stabilizes the retinal protonated Schiff base (PSB) has been proposed to be an essential step in rhodopsin activation. On the basis of vibrational and UV-visible spectroscopy, two counterion switch models have emerged. In the first model, the PSB is stabilized by Glu181 in the meta I state, while in the most recent proposal, it is stabilized by Glu113 as well as Glu181. We assess these models by conducting a pair of microsecond scale, all-atom molecular dynamics simulations of rhodopsin embedded in a 99-lipid bilayer of SDPC, SDPE, and cholesterol (2:2:1 ratio) varying the starting protonation state of Glu181. Theoretical simulations gave different orientations of retinal for the two counterion switch mechanisms, which were used to simulate experimental 2H NMR spectra for the C5, C9, and C13 methyl groups. Comparison of the simulated 2H NMR spectra with experimental data supports the complex-counterion mechanism. Hence, our results indicate that Glu113 and Glu181 stabilize the retinal PSB in the meta I state prior to activation of rhodopsin.     

Rhodopsin deactivation is affected by mutations of Tyrl91

The 9-methyl group of 11-cis retinal is important in the efficient formation of the active conformation of rhodopsin, Meta II. Here, Tyrl91 rhodopsin mutants were generated because of its proximity to that methyl group in the dark structure. If photoactivation results in movement of the 9-methyl group toward Tyrl91, the steric interactions involved with activation and/or deactivation might not be as tightly coupled in mutant proteins with smaller amino acids at that position. Tyrl91 mutations have no effect on the dark pigment. However, after photobleaching, the lifetime of Meta II is shorter; Meta II decays quickly into two inactive species: (1) a Meta III or Meta III-like species and (2) opsin and free retinal. The Meta III-like fraction maintains the covalent Schiff base linkage of the chromophore much longer than the wild type. On the other hand, the fast chromophore release is similar to cone pigments. Taken together, the data suggest that the role of the 9-methyl group after photo-isomerization is not only to form Meta II efficiently, but also to maintain its active conformation and allow for the timely hydrolysis of the Schiff base. Perturbation of this interaction effects changes in the hydrolysis of the Schiff base and for the case of the Y191A mutation the folded structure of the protein after photobleaching.     

Rhodopsin Activation Switches in a Native Membrane Environment†

The elucidation of structure–function relationships of membrane proteins still poses a considerable challenge due to the sometimes profound influence of the lipid bilayer on the functional properties of the protein. The visual pigment rhodopsin is a prototype of the family of G protein‐coupled transmembrane receptors and a considerable part of our knowledge on its activation mechanisms has been derived from studies on detergent‐solubilized proteins. This includes in particular the events associated with the conformational transitions of the receptor from the still inactive Meta I to the Meta II photoproduct states, which are involved in signaling. These events involve disruption of an internal salt bridge of the retinal protonated Schiff base, movement of helices and proton uptake from the solvent by the conserved cytoplasmic E(D)RY network around Glu134. As the equilibria associated with these events are considerably altered by the detergent environment, we set out to investigate these equilibria in the native membrane environment and to develop a coherent thermodynamic model of these activating steps using UV–visible and Fourier‐transform infrared spectroscopy as complementary techniques. Particular emphasis is put on the role of protonation of Glu134 from the solvent, which is a thermodynamic prerequisite for full receptor activation in membranes, but not in detergent. In view of the conservation of this carboxylate group in family A G protein‐coupled receptors, it may also play a similar role in the activation of other family members.     

Alpha-retinals as rhodopsin chromophores--preference for the 9-Z configuration and partial agonist activity

The visual pigment rhodopsin, the photosensory element of the rod photoreceptor cell in the vertebrate retina, shows in combination with an endogenous ligand, 11-Z retinal, an astonishing photochemical performance. It exhibits an unprecedented quantum yield (0.67) in a highly defined and ultrafast photoisomerization process. This triggers the conformational changes leading to the active state Meta(rhodopsin) II. Retinal is covalently bound to Lys-296 of the protein opsin in a protonated Schiff base. The resulting positive charge delocalization over the terminal part of the polyene chain of retinal creates a conjugation defect that upon photoexcitation moves to the opposite end of the polyene. Shortening the polyene as in 4,5-dehydro,5,6-dihydro (alpha), 5,6-dihydro or 7,8-dihydro-analogs might facilitate photoisomerization of a 9-Z and a 11-Z bond. Here we describe pigment analogs generated with bovine opsin and 11-Z or 9-Z 4,5-dehydro,5,6-dihydro-retinal that were further characterized by UV-Vis and FTIR spectroscopy. The preference of opsin for native 11-Z retinal over the 9-Z isomer is reversed in 4,5-dehydro,5,6-dihydro-retinal. 9-Z 4,5-dehydro,5,6-dihydro-retinal readily generated a photosensitive pigment. This modification has no effect on the quantum yield, but affects the Batho<-->blueshifted intermediate (BSI) equilibrium and leads to a strong decrease in the G-protein activation rate because of a downshift of the pK(a) of the Meta I<-->Meta II equilibrium.     

Absorption studies of neutral retinal Schiff base chromophores

The neutral retinal Schiff base is connected to opsin in UV sensing pigments and in the blue-shifted meta-II signaling state of the rhodopsin photocycle. We have designed and synthesized two model systems for this neutral chromophore and have measured their gas-phase absorption spectra in the electrostatic storage ring ELISA with a photofragmentation technique. By comparison to the absorption spectrum of the protonated retinal Schiff base in vacuo, we found that the blue shift caused by deprotonation of the Schiff base is more than 200 nm. The absorption properties of the UV absorbing proteins are thus largely determined by the intrinsic properties of the chromophore. The effect of approaching a positive charge to the Schiff base was also studied, as well as the susceptibility of the protonated and unprotonated chromophores to experience spectral shifts in different solvents.     

Interaction of chromophore, 11-<Emphasis Type="Italic">cis</Emphasis>-retinal,with amino acid residues of the visual pigment rhodopsin in the region of protonated Schiff base: A molecular dynamics study

A molecular dynamics study of the dark adapted visual pigment rhodopsin molecule was carried out. The interaction of the chromophore group, 11-cis-retinal, with the nearest amino acid residues in the chromophore center of the molecule, namely, in the region of the protonated Schiff base linkage, was analyzed. Most likely, the interaction of the CH=NH bond with the negatively charged amino acid residue Glu113 cannot be described as a simple electrostatic interaction of two oppositely charged groups. One can propose that not only Glu113 but also Glu181 and Ser186 are involved in stabilization of the protonated Schiff base linkage. Accord-ing to calculations, Glu181 interacts, as the counter-ion, with the Schiff base indirectly via Ser186. The intramolecular mechanisms of protonated Schiff base stabilization in rhodopsin are discussed. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 1, pp. 19–27, January, 2007.     

The protonated Schiff base of halorhodopsin from Natronobacterium pharaonis is hydrolyzed at elevated temperatures

Halorhodopsin from Natronobacterium pharaonis (pHR) is a light-driven chloride pump in which photoisomerzation of a retinal chromophore triggers a photocycle which leads to a chloride anion transport across the plasma membrane. Similarly to other retinal proteins the protonated Schiff base (PSB), which covalently links the retinal to the protein, does not experience hydrolysis reaction at room temperature even though several water molecules are located in the protonated Schiff base (PSB) vicinity. In the present studies we have revealed that in contrast to other studied archaeal rhodopsins, temperature increase to about 70 degrees C hydrolyses the PSB linkage of pHR. The rate of the reaction is affected by Cl-concentration and reveals an anion binding site (in addition to the Cl- in the SB vicinity) with a binding constant of 100mM (measured at 70 degrees C). We suggest that this binding site is located on the extracellular side and its possible role in the Cl-pumping mechanism is discussed. The rate of the hydrolysis reaction is affected by the nature of the anion bound to pHR. Substitution of the Cl- anion by Br-, I- and SCN- exhibits similar behavior to that of CI- in the region of 100mM but higher concentrations are needed for N3-, HCOO- and NO2-to achieve similar behavior. Steady state pigment illumination accelerates the reaction and reduces the energy of activation and the frequency factor. Adjusting the sample temperature to 25 degrees C following the hydrolysis reaction led to about 80% pigment recovery. However, the newly reformed pigment is different from the mother pigment and has different characteristics. It is concluded that the apo-membrane adopts a modified conformation and/or aggregated state which rebinds the retinal to give a new conformation of the pHR pigment.     

Structure, spectroscopy, and spectral tuning of the gas-phase retinal chromophore: the beta-ionone "handle" and alkyl group effect

The low-lying singlet states (i.e. S0, S1, and S2) of the chromophore of rhodopsin, the protonated Schiff base of 11-cis-retinal (PSB11), and of its all-trans photoproduct have been studied in isolated conditions by using ab initio multiconfigurational second-order perturbation theory. The computed spectroscopic features include the vertical excitation, the band origin, and the fluorescence maximum of both isomers. On the basis of the S0-->S1 vertical excitation, the gas-phase absorption maximum of PSB11 is predicted to be 545 nm (2.28 eV). Thus, the predicted absorption maximum appears to be closer to that of the rhodopsin pigment (2.48 eV) and considerably red-shifted with respect to that measured in solution (2.82 eV in methanol). In addition, the absorption maxima associated with the blue, green, and red cone visual pigments are tentatively rationalized in terms of the spectral changes computed for PSB11 structures featuring differently twisted beta-ionone rings. More specifically, a blue-shifted absorption maximum is explained in terms of a large twisting of the beta-ionone ring (with respect to the main conjugated chain) in the visual S-cone (blue) pigment chromophore. In contrast, the chromophore of the visual L-cone (red) pigment is expected to have a nearly coplanar beta-ionone ring yielding a six double bond fully conjugated framework. Finally, the M-cone (green) chromophore is expected to feature a twisting angle between 10 and 60 degrees. The spectroscopic effects of the alkyl substituents on the PSB11 spectroscopic properties have also been investigated. It is found that they have a not negligible stabilizing effect on the S1-S0 energy gap (and, thus, cause a red shift of the absorption maximum) only when the double bond of the beta-ionone ring conjugates significantly with the rest of the conjugated chain.     

Primary events in dim light vision: a chemical and spectroscopic approach toward understanding protein/chromophore interactions in rhodopsin

The visual pigment rhodopsin (bovine) is a 40 kDa protein consisting of 348 amino acids, and is a prototypical member of the subfamily A of G protein-coupled receptors (GPCRs). This remarkably efficient light-activated protein (quantum yield = 0.67) binds the chromophore 11-cis-retinal covalently by attachment to Lys296 through a protonated Schiff base. The 11-cis geometry of the retinylidene chromophore keeps the partially active opsin protein locked in its inactive state (inverse agonist). Several retinal analogs with defined configurations and stereochemistry have been incorporated into the apoprotein to give rhodopsin analogs. These incorporation results along with the spectroscopic properties of the rhodopsin analogs clarify the mode of entry of the chromophore into the apoprotein and the biologically relevant conformation of the chromophore in the rhodopsin binding site. In addition, difference UV, CD, and photoaffinity labeling studies with a 3-diazo-4-oxo analog of 11-cis-retinal have been used to chart the movement of the retinylidene chromophore through the various intermediate stages of visual transduction.     

7,8-dihydro retinals outperform the native retinals in conferring photosensitivity to visual opsin

The visual pigment rhodopsin presents an astonishing photochemical performance. It exhibits an unprecedented quantum yield (0.67) in a highly defined and ultrafast photoisomerization process. This triggers the conformational changes leading to the active state Meta II of this G protein-coupled receptor. The responsible ligand, retinal, is covalently bound to Lys-296 of the protein in a protonated Schiff base. The resulting positive charge delocalization over the terminal part of the polyene chain of retinal creates a conjugation defect that upon photoexcitation moves to the opposite end of the polyene. Shortening the polyene as in 5,6-dihydro- or 7,8-dihydro analogues might facilitate photoisomerization of a 9-Z and an 11-Z bond. Here we describe pigment analogues generated with bovine opsin and 11-Z 7,8-dihydro retinal or 9-Z 7,8-dihydro retinal. Both isomers readily generate photosensitive pigments that differ remarkably in spectral properties from the native pigments. In addition, in spite of the more flexible 7,8 single bond, both analogue pigments exhibit strikingly efficient photoisomerization while largely maintaining the activity toward the G-protein. These results bear upon the activation of ligand-gated signal transducers such as G protein-coupled receptors.     

pH-dependent transitions in xanthorhodopsin

Xanthorhodopsin (XR), the light-driven proton pump of the halophilic eubacterium Salinibacter ruber, exhibits substantial homology to bacteriorhodopsin (BR) of archaea and proteorhodopsin (PR) of marine bacteria, but unlike them contains a light-harvesting carotenoid antenna, salinixanthin, as well as retinal. We report here the pH-dependent properties of XR. The pKa of the retinal Schiff base is as high as in BR, i.e. > or =12.4. Deprotonation of the Schiff base and the ensuing alkaline denaturation cause large changes in the absorption bands of the carotenoid antenna, which lose intensity and become broader, making the spectrum similar to that of salinixanthin not bound to XR. A small redshift of the retinal chromophore band and increase of its extinction, as well as the pH-dependent amplitude of the M intermediate indicate that in detergent-solubilized XR the pKa of the Schiff base counterion and proton acceptor is about 6 (compared to 2.6 in BR, and 7.5 in PR). The protonation of the counterion is accompanied by a small blueshift of the carotenoid absorption bands. The pigment is stable in the dark upon acidification to pH 2. At pH < 2 a transition to a blueshifted species absorbing around 440 nm occurs, accompanied by loss of resolution of the carotenoid absorption bands. At pH < 3 illumination of XR with continuous light causes accumulation of long-lived photoproduct(s) with an absorption maximum around 400 nm. The photocycle of XR was examined between pH 4 and 10 in solubilized samples. The pH dependence of recovery of the initial state slows at both acid and alkaline pH, with pKas of 6.0 and 9.3. The decrease in the rates with pKa 6.0 is apparently caused by protonation of the counterion and proton acceptor, and that at high pH reflects the pKa of the internal proton donor, Glu94, at the times in the photocycle when this group equilibrates with the bulk.     

Mechanism of G-protein activation by rhodopsin

Rhodopsin is a member of the family of G-protein-coupled receptors (GPCRs), and is an excellent molecular switch for converting light signals into electrical response of the rod photoreceptor cells. Light initiates cis-trans isomerization of the retinal chromophore of rhodopsin and leads to the formation of several thermolabile intermediates during the bleaching process. Recent investigations have identified spectrally distinguishable two intermediate states that can interact with the retinal G-protein, transducin, and have elucidated the functional sharing of these intermediates. The initial contact with GDP-bound G-protein occurs in the meta-Ib intermediate state, which has a protonated Schiff base as its chromophore. The meta-Ib intermediate in the complex with the G-protein converts to the meta-II intermediate with releasing GDP from the alpha-subunit of the G protein. Meta-II has a de-protonated Schiff base chromophore and induces binding of GTP to the alpha-subunit of the G-protein. Thus, the GDP-GTP exchange reaction, namely G-protein activation, by rhodopsin proceeds through at least two steps, with conformational changes in both rhodopsin and the G-protein.     

The role of the beta-ionone ring in the photochemical reaction of rhodopsin

Time-dependent density functional theory (TDDFT) calculations on the photoabsorption process of the 11-cis retinal protonated Schiff base (PSB) chromophore show that the Franck-Condon relaxation of the first excited state of the chromophore involves a torsional twist motion of the beta-ionone ring relative to the conjugated retinyl chain. For the ground state, the beta-ionone ring and the retinyl chain of the free retinal PSB chromophore form a -40 degrees dihedral angle as compared to -94 degrees for the first excited state. The double bonds of the retinal are shorter for the fully optimized structure of the excited state than for the ground state suggesting a higher cis-trans isomerization barrier for the excited state than for the ground state. According to the present TDDFT calculations, the excitation of the retinal PSB chromophore does not primarily lead to a reaction along the cis-trans torsional coordinate at the C11-C12 bond. The activation of the isomerization center seems to occur at a later stage of the photo reaction. The results obtained at the TDDFT level are supported by second-order M?ller-Plesset (MP2) and approximate singles and doubles-coupled cluster (CC2) calculations on retinal chromophore models; the MP2 and CC2 calculations yield for them qualitatively the same ground state and excited-state structures as obtained in the density functional theory and TDDFT calculations.     

ORIENTATION OF AROMATIC RESIDUES IN RHODOPSIN. ROTATION OF ONE TRYPTOPHAN UPON THE META I→META II TRANSITION AFTER ILLUMINATION

Abstract— Linear dichroism measurements have been performed in the visible and the UV on suspensions of intact retinal rod outer segments, isolated from frog and from cattle retinas and oriented by a magnetic field. In the UV a sharp double peaked signal is observed around 290 nm. It is characteristic of a tryptophan residue having its ¹L_b transition oriented preferentially perpendicular to the membrane plane. Only one tryptophan residue per rhodopsin molecule seems to be involved, and this is insufficient to account for the diamagnetic anisotropy of the rods which causes their orientation. Upon bleaching one observes a rotation of this tryptophan coupled to the Meta I →Meta II transition, and the rotation is reversed in the next step Meta II → Meta III. The correlation with other spectral changes suggests that this tryptophan is in close relation with the retinal chromophore.     

Constraints of opsin structure on the ligand-binding site: studies with ring-fused retinals

Ring-fused retinal analogs were designed to examine the hula-twist mode of the photoisomerization of the 9-cis retinylidene chromophore. Two 9-cis retinal analogs, the C11-C13 five-membered ring-fused and the C12-C14 five-membered ring-fused retinal derivatives, formed the pigments with opsin. The C11-C13 ring-fused analog was isomerized to a relaxed all-trans chromophore (lambda(max) > 400 nm) at even -269 degrees C and the Schiff base was kept protonated at 0 degrees C. The C12-C14 ring-fused analog was converted photochemically to a bathorhodopsin-like chromophore (lambda(max) = 583 nm) at -196 degrees C, which was further converted to the deprotonated Schiff base at 0 degrees C. The model-building study suggested that the analogs do not form pigments in the retinal-binding site of rhodopsin but form pigments with opsin structures, which have larger binding space generated by the movement of transmembrane helices. The molecular dynamics simulation of the isomerization of the analog chromophores provided a twisted C11-C12 double bond for the C12-C14 ring-fused analog and all relaxed double bonds with a highly twisted C10-C11 bond for the C11-C13 ring-fused analog. The structural model of the C11-C13 ring-fused analog chromophore showed a characteristic flip of the cyclohexenyl moiety toward transmembrane segments 3 and 4. The structural models suggested that hula twist is a primary process for the photoisomerization of the analog chromophores.     

RESONANCE RAMAN STUDIES OF BACTERIORHODOPSIN ANALOGUES

Abstract— We present the results of resonance Raman measurements on a series of bacteriorhodopsin (bR) analogues formed from synthetic retinals which have replaced the native chromophore in the active site. Specifically, 5,6-dihydro-bR, 13-desmethyl-bR, 10-methyl-bR, 14-methyl-bR, and 10.14-dimethyl-bR have been studied. All five analogues bind and form Schiff base retinal-apoprotein linkages. While the Schiff base linkages of 5,6-dihydro-bR, 13-desmethyl-bR, and 10-methyl-bR are protonated, like the native chromophore, the 14-methyl-bR, and 10,14-dimethyl-bR Schiff bases are unprotonated. These results suggest that the binding site of bacteriorhodopsin near the Schiff base moiety is different from that of rhodopsin. The protonated Schiff base -C=NH- stretching frequency of 5.6-dihydro-bR lies at 1660 cm^-1 which is unusually high for a bacteriorhodopsin based pigment. The downward shift upon deuteration is 16 cm^-1, essentially identical to that measured for bacteriorhodopsin. This and the other analogue results strongly reinforce our previous arguments that the Schiff base stretching frequency is determined in large part by two factors, the C=N force constant and the stretch interaction with C=N-H bend. On the other hand, the deuterium isotope effect is determined primarily by the stretch-bend interaction.     

Effects of water re-location and cavity trimming on the CASPT2//CASSCF/AMBER excitation energy of Rhodopsin

We have used quantum-mechanics/molecular-mechanics computations based on ab initio multiconfigurational perturbation theory to determine and rationalize the effect of the re-location of one crystallographic water molecule on the vertical excitation energy of the visual pigment rhodopsin. It is found that the re-location of one water molecule to the opposite side of the 11-cis retinal chromophore leads to a large 0.7–0.8 Å contraction in the chromophore—counterion salt-bridge distance. In spite of this structural effect, the change in excitation energy is found to be limited (< 1.5 kcal mol^?1). Through an analysis of different rhodopsin models in terms of “components” (isolated chromophore, isolated chromophore—counterion ion-pair and models deprived of the counterion charges) we show that the limited change of the excitation energy can be related to a displacement of the retinal chromophore to a different spot of the protein cavity.     

A molecular spring for vision

Light absorption by the visual pigment rhodopsin leads to vision via a complex signal transduction pathway that is initiated by the ultrafast and highly efficient photoreaction of its chromophore, the retinal protonated Schiff base (RPSB). Here, we investigate this reaction in real time by means of unrestrained molecular dynamics simulations of the protein in a membrane mimetic environment, treating the chromophore at the density functional theory level. We demonstrate that a highly strained all-trans RPSB is formed starting from the 11-cis configuration (dark state) within approximately 100 fs by a minor rearrangement of the nuclei under preservation of the saltbridge with Glu113 and virtually no deformation of the binding pocket. Hence, the initial step of vision can be understood as the compression of a molecular spring by a minor change of the nuclear coordinates. This spring can then release its strain by altering the protein environment.     

Rhodopsin regeneration is accelerated via noncovalent 11-cis retinal-opsin complex--a role of retinal binding pocket of opsin

The regeneration of bovine rhodopsin from its apoprotein opsin and the prosthetic group 11-cis retinal involves the formation of a retinylidene Schiff base with the epsilon-amino group of the active lysine residue of opsin. The pH dependence of a Schiff base formation in solution follows a typical bell-shaped profile because of the pH dependence of the formation and the following dehydration of a 1-aminoethanol intermediate. Unexpectedly, however, we find that the formation of rhodopsin from 11-cis retinal and opsin does not depend on pH over a wide pH range. These results are interpreted by the Matsumoto and Yoshizawa (Nature 258 [1975] 523) model of rhodopsin regeneration in which the 11-cis retinal chromophore binds first to opsin through the beta-ionone ring, followed by the slow formation of the retinylidene Schiff base in a restricted space. We find the second-order rate constant of the rhodopsin formation is 6100+/-300 mol(-1) s(-1) at 25 degrees C over the pH range 5-10. The second-order rate constant is much greater than that of a model Schiff base in solution by a factor of more than 10(7). A previous report by Pajares and Rando (J Biol Chem 264 [1989] 6804) suggests that the lysyl epsilon-NH(2) group of opsin is protonated when the beta-ionone ring binding site is unoccupied. The acceleration of the Schiff base formation in rhodopsin is explained by stabilization of the deprotonated form of the lysyl epsilon-NH(2) group which might be induced when the beta-ionone ring binding site is occupied through the noncovalent binding of 11-cis retinal to opsin at the initial stage of rhodopsin regeneration, followed by the proximity and orientation effect rendered by the formation of noncovalent 11-cis retinal-opsin complex.