Activity switches of rhodopsin

Rhodopsin, the visual pigment of the rod photoreceptor cell contains as its light-sensitive cofactor 11-cis retinal, which is bound by a protonated Schiff base between its aldehyde group and the Lys296 side chain of the apoprotein. Light activation is achieved by 11-cis to all-trans isomerization and subsequent thermal relaxation into the active, G protein-binding metarhodopsin II state. Metarhodopsin II decays via two parallel pathways, which both involve hydrolysis of the Schiff base eventually to opsin and released all-trans retinal. Subsequently, rhodopsin's dark state is regenerated by a complicated retinal metabolism, termed the retinoid cycle. Unlike other retinal proteins, such as bacteriorhodopsin, this regeneration cycle cannot be short cut by light, because blue illumination of active metarhodopsin II does not lead back to the ground state but to the formation of largely inactive metarhodopsin III. In this review, mechanistic details of activating and deactivating pathways of rhodopsin, particularly concerning the roles of the retinal, are compared. Based on static and time-resolved UV/Vis and FTIR spectroscopic data, we discuss a model of the light-induced deactivation. We describe properties and photoreactions of metarhodopsin III and suggest potential roles of this intermediate for vision.     

THE INVOLVEMENT OF WATER AT THE RETINAL BINDING SITE IN RHODOPSIN AND EARLY LIGHT-INDUCED INTRAMOLECULAR PROTON TRANSFER

Abstract Extensive dehydration of air-dried films of bovine rod outer segment membranes induces fully reversible changes in the absorption spectrum of rhodopsin, indicative of deprotonation of the retinylidene Schiff base in more than 50% of the rhodopsin molecules in the sample. This suggests that water is involved at the site of the Schiff base protonation in rhodopsin. In contrast, the spectrum of metarhodopsin I is resistant to similar dehydrating conditions, implying a significant difference in the mechanism for protonation in metarhodopsin I. The photochemistry of dehydrated membranes was also explored. Photoexcitation of deprotonated rhodopsin (λ_max 390 nm) induces a large bathochromic shift of the chromophore. The major photoproduct at room temperature was spectrally similar to metarhodopsin I (λ_max, 478 nm). These findings suggest that intramolecular proton transfer involving the Schiff base proton may occur in the earlier stages of the visual cycle, prior to or during the formation of metarhodopsin I.     

MAGIC ANGLE SPINNING NMR STUDIES ON THE METARHODOPSIN II INTERMEDIATE OF BOVINE RHODOPSIN: EVIDENCE FOR AN UNPROTONATED SCHIFF BASE

Magic angle spinning (MAS)13C-NMR spectra of the metarhodopsin II intermediate have been obtained using bovine rhodopsin regenerated with retinal 13C-labeled at the C-13 and C-15 positions to investigate the protonation state of the retinal Schiff base linkage. The 13C-labeled rhodopsin was reconstituted into 1,2-dipalmitoleoylphosphatidylcholine bilayers to increase the amount of meta II trapped at low temperature. Both the 13C-15 (159.2 ppm) and 13C-13 (144.0 ppm) isotropic chemical shifts are characteristic of an unprotonated Schiff base, while the 13C-15 shift is significantly different from that of retinal (191 ppm) or a tetrahedral carbinolamine group (70-90 ppm) previously proposed as an intermediate in the hydrolysis of the Schiff base at the meta II stage. This rules out the possibility that meta II non-covalently binds retinal or is a carbinolamine intermediate and provides convincing evidence that Schiff base deprotonation occurs in the meta I-meta II transition, an event that is likely to be important in triggering the activation of transducin.     

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.     

PHOTOCHEMISTRY OF METHYLATED RHODOPSINS

Abstract— Rhodopsin, in which the active-site Schiff-base lysine has been chemically modified by monomethylation, is unable to form the deprotonated Schiff base bleaching intermediate, rnetarhodop-sin II. The photochemistry of the methylated Schiff base rhodopsin stops at the metarhodopsin I stage, which then slowly decays to all-trans retinal and opsin. Methylation of the non active-site lysines does not block the photochemical transformation but does speed up the formation and decay of the metarhodopsins.     

A LINEAR DICHROISM STUDY OF THE ORIENTATION OF AROMATIC PROTEIN RESIDUES IN MAGNETICALLY ORIENTED BOVINE ROD OUTER SEGMENTS

Abstract— Linear dichroism measurements using magnetic field oriented bovine visual rod outer segments have been made in the UV and visible spectral regions. The results indicate that the planes of the aromatic amino acid residues of rhodopsin tend to be oriented normal to the membrane plane both before and after bleaching. In contrast, the retinal chromophore which tends to be oriented with its absorption oscillator parallel to the membrane plane before bleaching is randomly oriented about 10min after bleaching whereas the membranes remain oriented. Estimates of the anisotropy in the diamagnetic susceptibility of rhodopsin aromatic residues indicate that the anisotropic magnetic properties of these protein residues are sufficient to account for the observed orientation of visual rod outer segments in a homogenous magnetic field.     

Transducin activation by molecular species of rhodopsin other than metarhodopsin II

Decay of metarhodopsin II was accelerated by hydroxylamine treatment or dark incubation of metarhodopsin II at 30 degrees C. The products thus obtained after decay of metarhodopsin II induced GTPase activity on transducin as well as metarhodopsin II suggesting that rhodopsin could activate transducin after the decay of metarhodopsin II intermediate. After urea-treated bovine rod outer segment membrane was completely bleached, rhodopsin in the membrane was regenerated by the addition of 11-cis retinal at various temperatures between 0 and 37 degrees C. The capacity to induce GTPase activity on transducin and phosphate incorporating capacity catalyzed by rhodopsin kinase were measured on such rhodopsins. The results showed that: (1) Regeneration of alpha band of rhodopsin was complete regardless of regeneration temperature; (2) When regenerated at temperatures below 10 degrees C, rhodopsins induced a GTPase activity on transducin in the dark even after treatment with hydroxylamine, whereas rhodopsins after regeneration at temperatures above 13 degrees C did not; (3) When regenerated at 0 degrees C, rhodopsin was phosphorylated if incubated with rhodopsin kinase and ATP in the dark, whereas the spectrally regenerated rhodopsin at 30 degrees C was not. The complete quenching of functions of photoactivated rhodopsin was achieved by recombination with 11-cis retinal at temperatures above 13 degrees C but not below 10 degrees C suggesting the existence of a low temperature intermediate upon regeneration.     

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.     

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.     

A COMPARISON OF LIGHT-TRIGGERED SPECTRAL CHANGES IN RHODOPSIN AND ISORHODOPSIN

Abstract— This report describes spectral changes associated with the transformation of metarhodopsin I to metarhodopsin II following light excitation of isorhodopsin and rhodopsin. Irradiated isorhodopsin gives rise to an equilibrium mixture of metarhodopsin I and metarhodopsin II which at 2°C and pH 6.8 favors the former. Isorhodopsin and rhodopsin are converted to metarhodopsin II via metarhodopsin I at very similar rates and activation parameters for the conversions are essentially identical. It is concluded that the initial cis to trans isomerization erases all differences in the two pigments.     

Dissection of Environmental Changes at the Cytoplasmic Surface of Light‐activated Bacteriorhodopsin and Visual Rhodopsin: Sequence of Spectrally Silent Steps†

The physico‐chemical properties as well as the conformation of the cytoplasmic surface of the 7‐helix retinal proteins bacteriorhodopsin (bR) and visual rhodopsin change upon light activation. A recent study found evidence for a transient softening of bR in its key intermediate M [Pieper et al. (2008) Phys. Rev. Lett. 100 , 228103] as a direct proof for the functional significance of protein flexibility. In this report we compare environmental and flexibility changes at the cytoplasmic surface of light‐activated bR and rhodopsin detected by time‐resolved fluorescence spectroscopy. The changes in fluorescence of covalently bound fluorescent probes and protein real‐time dynamics were investigated. We found that in fluorescently labeled bR and rhodopsin the intensity of fluorescein and Atto647 increased upon formation of the key intermediates M and metarhodopsin‐II, respectively, suggesting different surface properties compared to the dark state. Furthermore, time‐resolved fluorescence anisotropy experiments reveal an increase in steric restriction of loop flexibility because of changes in the surrounding protein environment in both the M‐intermediate as well as the active metarhodopsin‐II state. The kinetics of the fluorescence changes at the rhodopsin surface uncover multiple transitions, suggesting metarhodopsin‐II substates with different surface properties. Proton uptake from the aqueous bulk phase correlates with the first transition, while late proton release seems to parallel the second transition. The last transition between states of different surface properties correlates with metarhodopsin‐II decay.     

Transmembrane Signaling Mediated by Water in Bovine Rhodopsin

Abstract— Unhydrated air-dried films of rhodopsin from bovine rod outer segment membranes do not produce its active state, metarhodopsin II. In order to reveal requirements for its formation, we studied changes in H-bonding of water, peptide carbonyl and carboxylic acid in the photochemical reactions by means of difference Fourier transform infrared spectroscopy, under both hydrated and unhydrated conditions. A water molecule near Glull3, which undergoes H-bonding change in bathorhodopsin, remained in the unhydrated film, but with a weaker H-bonding state than in the hydrated film. The other water molecules, which shift in lumirhodopsin and metarhodopsin I as well as in bathorhodopsin of the hydrated film, were not observed in the unhydrated film. Effects of the dehydration were detected in all the C=O stretching vibrations of the peptide backbone and of Asp83 in the formation of bathorhodopsin. The C=O stretching band of Asp83 of lumirhodopsin and metarhodopsin I is intensified in the unhydrated film. We propose that structural changes at the intradiscal site in the interaction between the Schiff base and Glull3 affect water molecules, the peptide backbone, Asp83 and Glul22 in helices B and C through consecutive photochemical processes to metarhodopsin II.     

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.     

RETINAL-OPSIN LINKAGE OF CEPHALOPOD RHODOPSIN

Abstract— The pK_a of the Schiff base of N-retinylidene butylamine was determined in anionic (sodium dodecylsulfate), cationic (cetyltrimethylammonium bromide) and nonionic (polyoxyethylene (9) lauryl ether) detergent solutions. The pK_a of the Schiff base was raised from 6.4 to 9.9 by the effect of the neighboring anion. The rise of the pK_a was affected by the ion strength. Squid metarhodopsin behaved in a manner similar to the model Schiff base in the anionic detergent solution. The cationic group showed the opposite effect on the pK_a of the Schiff base. The retinal Schiff base in rhodopsin might be heavily influenced by adjacent anionic groups. The nature of the interaction is discussed.     

取代芳基席夫碱类化合物的室温研磨合成及其紫外可见吸收光谱的研究

研究了取代芳基单席夫碱及双席夫碱的室温研磨固态缩合反应,测定了其在环己烷和甲醇中的紫外可见吸收光谱,并探讨了其电子吸收光谱与分子结构的关系,以及溶剂对电子吸收光谱的影响.     

Chromophore interaction in xanthorhodopsin--retinal dependence of salinixanthin binding

Xanthorhodopsin is a light-driven proton pump in the extremely halophilic bacterium Salinibacter ruber. Its unique feature is that besides retinal it has a carotenoid, salinixanthin, with a light harvesting function. Tight and specific binding of the carotenoid antenna is controlled by binding of the retinal. Addition of all-trans retinal to xanthorhodopsin bleached with hydroxylamine restores not only the retinal chromophore absorption band, but causes sharpening of the salinixanthin bands reflecting its rigid binding by the protein. In this report we examine the correlation of the changes in the two chromophores during bleaching and reconstitution with native all-trans retinal, artificial retinal analogs and retinol. Bleaching and reconstitution both appear to be multistage processes. The carotenoid absorption changes during bleaching occurred not only upon hydrolysis of the Schiff base but continued while the retinal was leaving its binding site. In the case of reconstitution, the 13-desmethyl analog formed the protonated Schiff base slower than retinal, and provided the opportunity to observe changes in carotenoid binding at various stages. The characteristic sharpening of the carotenoid bands, indicative of its reduced conformational heterogeneity in the binding site, occurs when the retinal occupies the binding site but the covalent bond to Lys-240 via a Schiff base is not yet formed. This is confirmed by the results for retinol reconstitution, where the Schiff base does not form but the carotenoid exhibits its characteristic spectral change from the binding.     

The opsin shift and mechanism of spectral tuning in rhodopsin

Molecular dynamics simulations and combined quantum mechanical and molecular mechanical calculations have been performed to investigate the mechanism of the opsin shift and spectral tuning in rhodopsin. A red shift of -980 cm(-1) was estimated in the transfer of the chromophore from methanol solution environment to the protonated Schiff base (PSB)-binding site of the opsin. The conformational change from a 6-s-cis-all-trans configuration in solution to the 6-s-cis-11-cis conformer contributes additional -200 cm(-1), and the remaining effects were attributed to dispersion interactions with the aromatic residues in the binding site. An opsin shift of 2100 cm(-1) was obtained, in reasonable accord with experiment (2730 cm(-1)). Dynamics simulations revealed that the 6-s-cis bond can occupy two main conformations for the β-ionone ring, resulting in a weighted average dihedral angle of about -50°, which may be compared with the experimental estimate of -28° from solid-state NMR and Raman data. We investigated a series of four single mutations, including E113D, A292S, T118A, and A269T, which are located near the PSB, along the polyene chain of retinal and close to the ionone ring. The computational results on absorption energy shift provided insights into the mechanism of spectral tuning, which involves all means of electronic structural effects, including the stabilization or destabilization of either the ground or the electronically excited state of the retinal PSB.     

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.     

KINETICS OF RHODOPSIN PHOTOLYSIS INTERMEDIATES IN RETINAL ROD DISK MEMBRANES—I. TEMPERATURE DEPENDENCE OF LUMIRHODOPSIN AND METARHODOPSIN I KINETICS

Abstract— Kinetic measurements have been carried out on rhodopsin photolysis intermediates in retinal rod membrane suspensions on a millisecond time scale over a wide spectral range at 10, 20 and 36°C. To adequately account for the data we find that a three exponential fit is required at most wavelengths and temperatures investigated. The fastest component at 380, 420, 480, 515 and 540 nm is due to the lumirhodopsin → metarhodopsin I transition. The slowest process is not isochromic with the larger amplitude process found on the metarhodopsin I → metarhodopsin II time scale. The properties of the larger amplitude slow component are identical with the classical metarhodopsin I → metarhodopsin II process. Effects of various experimental conditions are discussed. It is shown that scattered light, in particular, can significantly affect the measured kinetics. For example, sonication, low salt and refractive index matching reduce light scattering and increase the contribution of the lumirhodopsin → metarhodopsin I reaction to the absorption transients. Care must also be taken in the analysis because the isosbestic wavelengths in the spectral transients are highly temperature dependent. For example, the lumirhodopsin–metarhodopsin I isosbestic is 490–500 nm at 10°C, 480–490 nm at 20°C and to the blue of 470 nm at 36°C. Activation energies of 77.8, 130.9 and 101.3 kJ/mol were found for the lumirhodopsin → metarhodopsin I, the metarhodopsin I → metarhodopsin II and the slow millisecond processes, respectively. All three processes contribute to the signals at lower temperatures. The amplitude of the slowest component decreases as the temperature is raised, and at physiological temperature its amplitude is essentially negligible compared to the metarhodopsin I → metarhodopsin II reaction. The lumirhodopsin → metarhodopsin I reaction makes a large contribution to the amplitude of the signals at most wavelengths observed from 380–540 nm, especially at physiological temperatures. At physiological temperatures the decay rates of lumirhodopsin and metarhodopsin I are within a factor of three of each other. Thus, lumirhodopsin decay may be much more important for visual transduction than suggested by low temperature studies. In contrast to reports of several other laboratories we have no evidence for kinetic complexity in the metarhodopsin I → metarhodopsin II reaction.     

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.