Opsin/all-trans-retinal complex activates transducin by different mechanisms than photolyzed rhodopsin

In rhodopsin, the 11-cis-retinal chromophore forms a complex with Lys296 of opsin via a protonated Schiff base. Absorption of light initiates the activation of rhodopsin by cis/trans photoisomerization of retinal. Thermal relaxation through different intermediates leads into the metarhodopsin states which bind and activate transducin (Gt) and rhodopsin kinase (RK). all-trans-Retinal also recombines with opsin independent of light, forming activating species of the receptor. In this study, we examined the mechanism by which all-trans-retinal activates opsin. To exclude other amines except active site Lys296 from formation of Schiff bases, we reductively methylated rhodopsin (PM-rhodopsin), which we then bleached to generate PM-opsin. Using spectroscopic methods and a Gt activation assay, we found that all-trans-retinal interacted with PM-opsin, producing a noncovalent complex that activated Gt. The residual nucleotide exchange in Gt catalyzed by opsin was approximately 1/250 lower relative to that of photoactivated rhodopsin (pH 8.0, 23 degrees C). Addition of equimolar all-trans-retinal led to an occupancy of one-tenth of the putative retinal binding site(s) of opsin and enhanced the Gt activation rate 2-fold. When the concentration of all-trans-retinal was increased to saturation, the Gt activation rate of the opsin/all-trans-retinal complex was approximately 1/33 lower compared to that of photoactivated rhodopsin. We conclude that all-trans-retinal can form a noncovalent complex with opsin that activates Gt by different mechanisms than photolyzed rhodopsin.     

Characterization of the mutant visual pigment responsible for congenital night blindness: a biochemical and Fourier-transform infrared spectroscopy study

A mutation in the gene for the rod photoreceptor molecule rhodopsin causes congenital night blindness. The mutation results in a replacement of Gly90 by an aspartic acid residue. Two molecular mechanisms have been proposed to explain the physiology of affected rod cells. One involves constitutive activity of the G90D mutant opsin [Rao, V. R., Cohen, G. B., & Oprian, D. D. (1994) Nature 367, 639-642]. A second involves increased photoreceptor noise caused by thermal isomerization of the G90D pigment chromophore [Sieving, P. A., Richards, J. E., Naarendorp F., Bingham, E. L., Scott, K., & Alpern, M. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 880-884]. Based on existing models of rhodopsin and in vitro biochemical studies of site-directed mutants, it appears likely that Gly90 is in the immediate proximity of the Schiff base chromophore linkage. We have studied in detail the mutant pigments G90D and G90D/E113A using biochemical and Fourier-transform infrared (FTIR) spectroscopic methods. The photoproduct of mutant pigment G90D, which absorbs maximally at 468 nm and contains a protonated Schiff base linkage, can activate transducin. However, the active photoproduct decays rapidly to opsin and free all-trans-retinal. FTIR studies of mutant G90D show that the dark state of the pigment has several structural features of metarhodopsin II, the active form of rhodopsin. These include a protonated carboxylic acid group at position Glu113 and increased hydrogen-bond strength of Asp83. Additional results, which relate to the structure of the active G90D photoproduct, are also reported. Taken together, these results may be relevant to understanding the molecular mechanism of congenital night blindness caused by the G90D mutation in human rhodopsin.     

Constitutive activation of opsin by mutation of methionine 257 on transmembrane helix 6

Rhodopsin is a member of the large family of G protein-coupled receptors (GPCR's). Constitutive activity of GPCR's, defined as ligand-independent signaling, has been recognized as an important feature of receptor function and has also been implicated in the molecular pathophysiology of a number of human diseases. Rhodopsin has evolved a unique mechanism to minimize receptor basal activity. The chromophore 11-cis-retinal, which acts as an inverse agonist in rhodopsin, is covalently bound to the receptor to ensure extremely low receptor signaling in the dark. In this study, we replaced Met257 in TM helix 6 of opsin with each of the remaining 19 amino acids. Only mutant opsin M257R failed to be expressed in COS-cell membranes. Each of the remaining 18 mutant opsins, with the exception of M257L, was significantly constitutively active. Two mutants in particular, M257Y and M257N, displayed very high levels of constitutive activity. In addition, the double-site mutants with substitutions of both Met257 and Glu113 in TM helix 3 tended to be much more constitutively active than the sums of the activities of the individual single-site mutants. Based on existing structural models of rhodopsin, we conclude that Met257 may form an important and specific interhelical interaction with a highly conserved NPXXY motif in TM helix 7, which stabilizes the inactive receptor conformation by preventing TM helix 6 movement in the absence of all-trans-retinal. Furthermore, we are able to show that the pharmacological properties of the large number (approximately 50) of mutant opsins that we have characterized to date support the two-state model of GPCR function. These results suggest that rhodopsin and other GPCR's share a common mechanism of receptor activation that involves specific changes in helix-helix interactions.     

Transmembrane signaling mediated by water in bovine rhodopsin

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 Glu113, 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 shfit 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 Glu113 affect water molecules, the peptide backbone, Asp83 and Glu122 in helices B and C through consecutive photochemical processes to metarhodopsin II.     

The last phase of the reprotonation switch in bacteriorhodopsin: the transition between the M-type and the N-type protein conformation depends on hydration

In order to elucidate the mechanism of the reprotonation switch of bacteriorhodopsin, the protein conformation of the M intermediate of the D96N mutant was examined at various hydration conditions by X-ray diffraction and FTIR spectroscopy. We observed two distinct protein conformations at different levels of hydration. One is like in the N photointermediate, although in this case with an unprotonated Schiff base. It is stabilized in highly hydrated samples. The other is a protein conformation identical to that in the normal M intermediate of wild-type bacteriorhodopsin, which is stabilized in partially dehydrated samples. The hydration dependence of the structural transition between the M-type and the N-type conformations suggests that there is a change in the binding of water at the cytoplasmic surface. Thus, more water molecules bind in the N-type structure than in the M-type. This is consistent with the idea that the conformational change from the M-type to the N-type corresponds to the opening of the proton channel to the cytoplasmic surface by tilt of the cytoplasmic end of helix F, and that this is required for proton transfer from Asp-96 to the retinal Schiff base.     

Mechanisms of spectral tuning in blue cone visual pigments. Visible and raman spectroscopy of blue-shifted rhodopsin mutants

Spectral tuning by visual pigments involves the modulation of the physical properties of the chromophore (11-cis-retinal) by amino acid side chains that compose the chromophore-binding pocket. We identified 12 amino acid residues in the human blue cone pigment that might induce the required green-to-blue opsin shift. The simultaneous substitution of nine of these sites in rhodopsin (M86L, G90S, A117G, E122L, A124T, W265Y, A292S, A295S, and A299C) shifted the absorption maximum from 500 to 438 nm, accounting for 2,830 cm-1, or 80%, of the opsin shift between rhodopsin and the blue cone pigment. Raman spectroscopy of mutant pigments shows that the dielectric character and architecture of the chromophore-binding pocket are specifically altered. An increase in the number of dipolar side chains near the protonated Schiff base of retinal increases the ground-excited state energy gap via long range dipole-dipole Coulomb interaction. In addition, the W265Y substitution causes a decrease in solvent polarizability near the chromophore ring structure. Finally, two substitutions on transmembrane helix 3 (A117G and E122L) act in combination with the other substitutions to alter the binding-pocket structure, resulting in stronger interaction of the protonated Schiff base group with the surrounding dipolar groups and the counterion. Taken together, these results identify the amino acid side chains and the underlying physical mechanisms responsible for a majority of the opsin shift in blue visual pigments.     

A comparison of the efficiency of G protein activation by ligand-free and light-activated forms of rhodopsin

Activation of the photoreceptor G protein transducin (Gt) by opsin, the ligand-free form of rhodopsin, was measured using rod outer segment membranes with densities of opsin and Gt similar to those found in rod cells. When GTPgammaS was used as the activating nucleotide, opsin catalyzed transducin activation with an exponential time course with a rate constant k(act) on the order of 2 x 10(-3)s(-1). Comparison under these conditions to activation by flash-generated metarhodopsin II (MII) revealed that opsin- and R*-catalyzed activation showed similar kinetics when MII was present at a surface density approximately 10(-6) lower than that of opsin. Thus, in contrast to some previous reports, we find that the catalytic potency of opsin is only approximately 10(-6) that of MII. In the presence of residual retinaldehyde-derived species present in membranes treated with hydroxylamine after bleaching, the apparent k(act) observed was much higher than that for opsin, suggesting a possible explanation for previous reports of more efficient activation by opsin. These results are important for considering the possible role of opsin in the diverse phenomena in which it has been suggested to play a key role, such as bleaching desensitization and retinal degeneration induced by continuous light or vitamin A deprivation.     

Reduction of all-trans-retinal limits regeneration of visual pigment in mice

Absorption of photons by pigments in photoreceptor cells results in photoisomerization of the chromophore, 11-cis-retinal, to all-trans-retinal and activation of opsin. Photolysed chromophore is converted back to the 11-cis-configuration via several enzymatic steps in photoreceptor and retinal pigment epithelial cells. We investigated the levels of retinoids in mouse retina during constant illumination and regeneration in the dark as a means of obtaining more information about the rate-limiting step of the visual cycle and about cycle intermediates that could be responsible for desensitization of the visual system. All-trans-retinal accumulated in the retinas during constant illumination and following flash illumination. Decay of all-trans-retinal in the dark following constant illumination occurred without substantial accumulation of all-trans-retinal, generated by constant approximately equal to visual pigment regeneration (t1/2 approximately 5 and t1/2 approximately 7 min, respectively). All-trans-retinal, generated by constant illumination, decayed approximately 3 times more rapidly than that generated by a flash and, as shown previously, the rate of rhodopsin regeneration following a flash was approximately 4 times slower than after constant illumination. The retinyl ester pool (> 95% all-trans-retinyl ester) did not show a statistically significant change in size or composition during illumination. In addition, constant illumination increased the amount of photoreceptor membrane-associated arrestin. The results suggest that the rate-limiting step of the visual cycle is the reduction of all-trans-retinal to all-trans-retinol by all-trans-retinol dehydrogenase. The accumulation of all-trans-retinal during illumination may be responsible, in part, for the reduction in sensitivity of the visual system that accompanies photobleaching and may contribute to the development of retinal pathology associated with light damage and aging.     

Mechanism-based inactivation of a yeast methylamine oxidase mutant: implications for the functional role of the consensus sequence surrounding topaquinone

The copper-containing yeast methylamine oxidase E406N mutant has an altered consensus sequence surrounding the topaquinone cofactor (residue 405). The mutation has no effect on the final yield of the active-site topaquinone cofactor during biogenesis but causes the enzyme to be inactivated by substrate methylamine [Cai, D., and Klinman, J. P. (1994) Biochemistry 33, 7674-7653]. In this study we show that the inactivation leads to the formation of a covalent adduct, which has a UV/vis spectrum very similar to that of a product Schiff base, an intermediate of topaquinone-catalyzed amine oxidation reactions. The kinetic isotope effects on the second-order rate constant for the inactivation and catalytic turnover are identical, indicating that the two processes share a common intermediate that follows C_H bond cleavage. Resonance Raman spectroscopy provides direct evidence for the accumulation of a neutral product Schiff base species. Removal of excess methylamine leads to recovery of both activity and the native absorption spectrum for E406N, indicating that the cofactor in the inactivated enzyme is chemically competent for hydrolysis. The rate of the reactivation is slow, however; the shortest half-life of the inhibited E406N at 25 degrees C is 5.9 min at pH 6.15. pH effect experiments show that the inactivation and reactivation steps are controlled by a single ionizable group with a pKa of 6.9-7.1; under basic conditions, when this residue is deprotonated, the inactivation is the fastest and the half-life of the inhibited enzyme is the longest. On the basis of the available crystal structures of copper amine oxidases, we propose that a histidine residue in the dimer interface is responsible for the observed ionization. In the wild-type enzyme this histidine is kept protonated by virtue of Glu at position 406. Unlike methylamine, the larger substrates ethylamine and benzylamine give normal turnover with E406N. Disruption of structure at the subunit interface in E406N may allow a rotation of the relatively small topa-product Schiff base complex (formed from methylamine) away from the active-site base to a conformation that is incompetent toward hydrolysis.     

Connectivity of the retinal Schiff base to Asp85 and Asp96 during the bacteriorhodopsin photocycle: the local-access model

In the recently proposed local-access model for proton transfers in the bacteriorhodopsin transport cycle (Brown et al. 1998. Biochemistry. 37:3982-3993), connection between the retinal Schiff base and Asp85 (in the extracellular direction) and Asp96 (in the cytoplasmic direction)is maintained as long as the retinal is in its photoisomerized state. The directionality of the proton translocation is determined by influences in the protein that make Asp85 a proton acceptor and, subsequently, Asp96 a proton donor. The idea of concurrent local access of the Schiff base in the two directions is now put to a test in the photocycle of the D115N/D96N mutant. The kinetics had suggested that there is a single sequence of intermediates, L<-->M1<-->M2<-->N, and the M2-->M1 reaction depends on whether a proton is released to the extracellular surface. This is now confirmed. We find that at pH 5, where proton release does not occur, but not at higher pH, the photostationary state created by illumination with yellow light contains not only the M1 and M2 states, but also the L and the N intermediates. Because the L and M1 states decay rapidly, they can be present only if they are in equilibrium with later intermediates of the photocycle. Perturbation of this mixture with a blue flash caused depletion of the M intermediate, followed by its partial recovery at the expense of the L state. The change in the amplitude of the C=O stretch band at 1759 cm-1 demonstrated protonation of Asp85 in this process. Thus, during the reequilibration the Schiff base lost its proton to Asp85. Because the N state, also present in the mixture, arises by protonation of the Schiff base from the cytoplasmic surface, these results fulfill the expectation that under the conditions tested the extracellular access of the Schiff base would not be lost at the time when there is access in the cytoplasmic direction. Instead, the connectivity of the Schiff base flickers rapidly (with the time constant of the M1<-->M2 equilibration) between the two directions during the entire L-to-N segment of the photocycle.     

Sulfhydryl oxidation of mutants with cysteine in place of acidic residues in the lactose permease

To examine further the role of charge-pair interactions in the structure and function of lactose permease, Asp237 (helix VII), Asp240 (helix VII), Glu126 (cytoplasmic loop IV/V), Glu269 (helix VIII), and Glu325 (helix X) were replaced individually with Cys in a functional mutant devoid of Cys residues. Each mutant was then oxidized with H2O2 in order to generate a sulfinic and/or sulfonic acid at these positions. Due to the isosteric relationship between aspartate and sulfinate, in particular, and the lower pKa of the sulfinic and sulfonic acid side chains, oxidized derivatives of Cys are useful probes for examining the role of carboxylates. Asp237-->Cys or Asp240-->Cys permease is inactive, as shown previously, but H2O2 oxidation restores activity to an extent similar to that observed when a negative charge is reintroduced by other means. Glu126-->Cys, Glu269-->Cys, or Glu325-->Cys permease is inactive, but oxidation does not restore active lactose transport. The data are consistent with previous observations indicating that Asp237 and Asp240 are not critical for active lactose transport, while Glu126, Glu269, and Glu325 are irreplaceable. Although Glu269-->Cys permease does not transport lactose, the oxidized mutant exhibits significant transport of beta,D-galactosylpyranosyl 1-thio-beta,D-galactopyranoside, a property observed with Glu269-->Asp permease. The observation supports the idea that an acidic residue at position 269 is important for substrate recognition. Finally, oxidized Glu325-->Cys permease catalyzes equilibrium exchange with an apparent pKa of about 6.5, more than a pH unit lower than that observed with Glu325-->Asp permease, thereby providing strong confirmatory evidence that a negative charge at position 325 determines the rate of translocation of the ternary complex between the permease, substrate, and H+.     

Regulatory arrestin cycle secures the fidelity and maintenance of the fly photoreceptor cell

Excitation of fly photoreceptor cells is initiated by photoisomerization of rhodopsin to the active form of metarhodopsin. Fly metarhodopsin is thermostable, does not bleach, and does not regenerate spontaneously to rhodopsin. For this reason, the activity of metarhodopsin must be stopped by an effective termination reaction. On the other hand, there is also a need to restore the inactivated photopigment to an excitable state in order to keep a sufficient number of photopigment molecules available for excitation. The following findings reveal how these demands are met. The photopigment undergoes rapid phosphorylation upon photoconversion of rhodopsin to metarhodopsin and an efficient Ca2+ dependent dephosphorylation upon regeneration of metarhodopsin to rhodopsin. Phosphorylation decreases the ability of metarhodopsin to activate the guanine nucleotide-binding protein. Binding of 49-kDa arrestin further quenches the activity of metarhodopsin and protects it from dephosphorylation. Light-dependent binding and release of 49-kDa arrestin from metarhodopsin- and rhodopsin-containing membranes, respectively, directs the dephosphorylation reaction toward rhodopsin. This ensures the return of phosphorylated metarhodopsin to the rhodopsin pool without initiating transduction in the dark. Assays of rhodopsin dephosphorylation in the Drosophila retinal degeneration C (rdgC) mutant, a mutant in a gene previously cloned and predicted to encode a serine/threonine protein phosphatase, reveal that phosphorylated rhodopsin is a major substrate for the rdgC phosphatase. We propose that mutations resulting in either a decrease or an improper regulation of rhodopsin phosphatase activity bring about degeneration of the fly photoreceptor cells.     

Spectroscopic evidence for interaction between transmembrane helices 3 and 5 in rhodopsin

Recent molecular models of rhodopsin (Rho) propose a specific interaction between transmembrane (TM) helices 3 and 5, which appears to be mediated by amino acid residues Glu122 and His211 on TM helices 3 and 5, respectively. To test this proposed interaction, four single-site histidine replacement mutants (H100N, H152N, H211N, and H211F), two single-site glutamic acid replacement mutants (E122Q and E122A), and three double-site replacement mutants (E122Q/H211F, E122Q/H211N, and E122A/H211F) of Rho were prepared. The expressed mutant pigments reconstituted into membranes were studied by FTIR difference spectroscopy addressing especially the transition to metarhodopsin I (MI). It is shown that the lipid environment influences bands typical of the MI state. Spectra of mutants with substituted Glu122 allowed assignments of the C=O stretch of protonated Glu122 in the dark state and in MI of Rho. Mutation of His211, but not of other histidine residues, affects these vibrational modes assigned to Glu122. In addition, replacements of His211 affect protein modes that are proposed to arise from a third, hydroxyl-bearing group, which also interacts with Glu122. These modes are influenced as well when Glu122 is replaced by Ala in mutant E122A but not when it is replaced by Gln in mutant E122Q. These results provide direct experimental evidence for an interaction between TM helices 3 and 5 in Rho, which is mediated by Glu122 and His211.     

Structure and function in rhodopsin: rhodopsin mutants with a neutral amino acid at E134 have a partially activated conformation in the dark state

The Glu-134-Arg-135 residues in rhodopsin, located near the cytoplasmic end of the C helix, are involved in G protein binding, or activation, or both. Furthermore, the charge-neutralizing mutation Glu-134 to Gln-134 produces hyperactivity in the activated state and produces constitutive activity in opsin. The Glu/Asp-Arg charge pair is highly conserved in equivalent positions in other G protein-coupled receptors. To investigate the structural consequences of charge-neutralizing mutations at Glu-134 and Arg-135 in rhodopsin, single spin-labeled side chains were introduced at sites in the cytoplasmic domains of helices C (140), E (227), F (250), or G (316) to serve as "molecular sensors" of the local helix bundle conformation. In each of the spin-labeled rhodopsins, a Gln substitution was introduced at either Glu-134 or Arg-135, and the electron paramagnetic resonance spectrum of the spin label was used to monitor the structural response of the helix bundle. The results indicate that a Gln substitution at Glu-134 induces a photoactivated conformation around helices C and G even in the dark state, an observation of potential relevance to the hyperactivity and constitutive activity of the mutant. In contrast, little change is induced in helix F, which has been shown to undergo a dominant motion upon photoactivation. This result implies that the multiple helix motions accompanying photoactivation are not strongly coupled and can be induced to take place independently. Gln substitution at Arg-135 produces only minor structural changes in the dark- or light-activated conformation, suggesting that this residue is not a determinant of structure in the regions investigated, although it may be functionally important.     

Aspartate substitutions establish the concerted action of P-region glutamates in repeats I and III in forming the protonation site of L-type Ca2+ channels

Hydrogen ions reduce ion flux through voltage-gated Ca2+ channels by binding to a single protonation site with an unusually high pKa. Recent evidence localizes the protonation site to the same locus that supports high affinity Ca2+ binding and selectivity, a set of four conserved glutamate residues near the external mouth of the pore. Remaining controversy concerns the question of whether the protonation site arises from a single glutamate, Glu-1086 (EIII), or a combination of Glu-1086 and Glu-334 (EI) working in concert. We tested these hypotheses with individual Glu --> Asp substitutions. The Glu --> Asp replacements in repeats I and III stood out in two ways. First, in both EID and EIIID, protonation was destabilized relative to wild type, whereas it was unchanged in EIID and stabilized in EIVD. The changes in affinity were entirely due to alterations in H+ off-rate. Second, the ratio of protonated conductance to deprotonated conductance was significantly closer to unity for EID and EIIID than for wild-type channels or other Asp mutants. Both results support the idea that EI and EIII act together to stabilize a single titratable H+ ion and behave nearly symmetrically in influencing pore conductance. Neutralization of EIII by alanine replacement clearly failed to abolish susceptibility to protonation, indicating that no single glutamate was absolutely required. Taken together, all the evidence supports a model in which multiple carboxylates work in concert to form a single high affinity protonation site.     

Reconstitution of bacteriorhodopsin from the apoprotein and retinal studied by Fourier-transform infrared spectroscopy

The reconstitution of the retinal-containing protein bacteriorhodopsin (BR) from the apoprotein and retinal has been studied by Fourier-transform infrared (FTIR) difference spectroscopy. 9-cis-Retinal which occupies the binding site but does not reconstitute the chromophore was used as "caged retinal". Photoisomerization to the all-trans isomer triggers the reconstitution reaction. Absorption bands in the FTIR difference spectra of the educt and product of the reaction could be assigned by comparison with a 9-cis-retinal FTIR spectrum or an FT-Raman spectrum of BR and due to band shifts observed upon deuterium exchange. Specific difference bands were assigned to the protonated carboxyl groups of D96 and D115 by use of the mutants D115N and D96N. Both aspartic acids are protonated also in the apoprotein with pKa values above 10 and undergo a frequency shift toward higher wavenumbers indicating a more hydrophobic environment in the reconstituted protein. No indication was found for protonation changes of carboxyl groups or other protonatable residues when carrying out the reaction at pH values between 4 and 10. The pH-dependent protonation changes reported earlier [Fischer & Oesterhelt (1980) Biophys. J. 31, 139-146] therefore may be caused by protons in a hydrogen-bonded network. Mutations of E204, but not of D38 or E9, cancel proton uptake during reconstitution at high pH as well as proton release at low pH. It is concluded, that E204, without changing its protonation state itself, is part of a protonatable hydrogen-bonded network which changes its pKa during reconstitution thereby causing the observed protonation changes.     

Chlamyrhodopsin represents a new type of sensory photoreceptor

In order to find optimal light conditions for photosynthetic growth, the green alga Chlamydomonas uses a visual system. An optical device, a rhodopsin photoreceptor and an electrical signal transduction chain that mediates between photoreceptor and flagella comprise this system. Here we present an improved strategy for the preparation of eyespot membranes. These membranes contain a retinal binding protein, which has been proposed to be the apoprotein of the phototaxis receptor. The retinal binding protein, which we named chlamyopsin, was purified and opsin-specific antibodies were raised. Using these antibodies, the opsin was localized in the eyespot region of whole cells during growth and cell division. The opsin cDNA was purified and sequenced. The sequence reveals that chlamyopsin is not a typical seven helix receptor. It shows some homology to invertebrate opsins but not to opsins from halobacteria. It contains many polar and charged residues and might function as a light-gated ion channel complex. It is likely that this lower plant rhodopsin diverged from animal opsins early in opsin evolution.     

Transducin-alpha C-terminal peptide binding site consists of C-D and E-F loops of rhodopsin

The binding of heterotrimeric GTP-binding proteins (G-proteins) to serpentine receptors involves several independent contacts. We have deduced the points of interaction between mutant bovine rhodopsins and alphat-(340-350), a peptide corresponding to the C terminus of the alpha subunit (alphat) of bovine retinal G-protein, transducin. Direct binding of alphat-(340-350) to rhodopsin stabilizes the activated metarhodopsin II state (M II), consequently uncoupling the rhodopsin-transducin interaction. This peptide action requires two segments on the cytoplasmic domain of rhodopsin: the Tyr136-Val137-Val138-Val139 sequence on the C-D loop and the Glu247-Lys248-Glu249-Val250-Thr251 sequence on the E-F loop. We propose that a tertiary interaction of these two loop regions forms a pocket for binding the alphat C terminus of the transducin during light transduction in vivo. In most G-proteins, the C termini of alpha subunits are important for interaction with receptors, and, in several serpentine receptors, regions similar to those in rhodopsin are essential for G-protein activation, indicating that the interaction described here may be a generally applicable mode of G-protein binding in signal transduction.     

Interaction between Asp-85 and the proton-releasing group in bacteriorhodopsin. A study of an O-like photocycle intermediate

Upon light adaptation by continuous (or pulsed) illumination, the artificial bacteriorhodopsin (bR) pigments, I and II, derived from synthetic 14F retinal and a short polyenal, respectively produce a long-lived red-shifted species denoted O1. An analogous phenomenon was observed by Sonar, S., et al. [(1993) Biochemistry 32, 2263-2271], in the case of the Y185F mutant (pigment III). The nature of these O1 species was investigated by studying a series of effects, primarily their red light photoreversibility, the associated proton uptake and release processes, and the effects of pH on their relative amounts, which are interpreted in terms of pH-dependent acid-base equilibria. Experiments were also carried out with pigments I and II derived from the mutants D96A, E204Q, R82Q, and D85N. The O1 species of pigments I and II (and possibly also that of pigment III) are identified as an unusually long-lived (all-trans) intermediate of the photocycle of their 13-cis isomer. It is concluded that in O1, Asp-85 is protonated, a process associated with proton uptake from the extracellular side. Subsequent proton release (to the same side of the membrane) occurs from Glu-204 (or from a group closely interacting with it) prior to the decay of O1. At high pH (>9), O1 reversibly converts to a purple form, due to deprotonation of Asp-85, while at still higher pH (> 11), a blue-shifted species characterized by a deprotonated Schiff base is generated. These transitions constitute the first demonstration of the titration of a photocycle intermediate of a retinal protein. The respective pKa values are determined and discussed in relation to those pertaining to the unphotolyzed (dark-adapted) pigments. It appears that the pKa values are controlled by a hydrogen bond network involving water molecules, which binds the protonated Schiff base with Asp-85 and Glu-204. The disruption of this network in pigments I-III may also be responsible for the long lifetime of the O1 species, due to the inhibition of thermal trans-13-cis isomerization. The results are relevant to the molecular mechanism of the photocycles of both 13-cis- and all-trans-bR, primarily to the nature and to the deprotonation mechanism of the proton-releasing group.     

Evidence from normal and degenerating photoreceptors that two outer segment integral membrane proteins have separate transport pathways

Detachment of the neural retina from the retinal pigment epithelium induces photoreceptor degeneration. We studied the effects of this degeneration on the localization of two photoreceptor outer segment-specific integral membrane proteins, opsin and peripherin/rds, in rod photoreceptors. Results from laser scanning confocal microscopic and electron microscopic immunolocalization demonstrate that these two proteins, normally targeted to the newly-forming discs of the outer segments, accumulate in different sub-cellular compartments during photoreceptor degeneration: opsin immunolabeling increases throughout the photoreceptor cell's plasma membrane, while peripherin/rds immunolabeling occurs within cytoplasmic vesicles. The simplest hypothesis to explain our results is that these proteins are transported in different post-Golgi transport vesicles and separately inserted into the plasma membrane. More complex mechanisms involve having the two co-transported and then opsin finds its way into the plasma membrane but peripherin/rds does not, remaining behind in vesicles. Alternatively, both insert into the plasma membrane but peripherin/rds is recycled into cytoplasmic vesicles. We believe the data most strongly supports the first possibility. Although the transport pathways for these proteins have not been fully characterized, the presence of peripherin/rds-positive vesicles adjacent to the striated rootlet suggests a transport role for this cytoskeletal element. The accumulation of these proteins in photoreceptors with degenerated outer segments may also indicate that their rate of synthesis has exceeded the combined rates of their incorporation into newly forming outer segment disc membranes and their degradation. The accumulation may also provide a mechanism for rapid recovery of the outer segment following retinal reattachment and return of the photoreceptor cell to an environment favorable to outer segment regeneration.