Internal water molecules of pharaonis phoborhodopsin studied by low-temperature infrared spectroscopy.

In the Schiff base region of bacteriorhodopsin (BR), a light-driven proton-pump protein, three internal water molecules are involved in a pentagonal cluster structure. These water molecules constitute a hydrogen-bonding network consisting of two positively charged groups, the Schiff base and Arg82, and two negatively charged groups, Asp85 and Asp212. Previous infrared spectroscopy of BR revealed stretching vibrations of such water molecules under strong hydrogen-bonding conditions using spectral differences in D2O and D2(18O) [Kandori and Shichida (2000) J. Am. Chem. Soc. 122, 11745-11746]. The present study extends the infrared analysis to another archaeal rhodopsin, pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin-II, psR-II), involved in the negative phototaxis of Natronobacterium pharaonis. Despite functional differences between ppR and BR, similar spectral features of water bands were observed before and after photoisomerization of the retinal chromophore at 77 K. This implies that the structure and the structural changes of internal water molecules are similar between ppR and BR. Higher stretching frequencies of the bridged water in ppR suggest that the water-containing pentagonal cluster structure is considerably distorted in ppR. These observations are consistent with the crystallographic structures of ppR and BR. The water structure and structural changes upon photoisomerization of ppR are discussed here on the basis of their infrared spectra.

Photoisomerization in rhodopsin.

This article reviews the primary reaction processes in rhodopsin, a photoreceptive pigment for twilight vision. Rhodopsin has an 11-cis retinal as the chromophore, which binds covalently with a lysine residue through a protonated Schiff base linkage. Absorption of a photon by rhodopsin initiates the primary photochemical reaction in the chromophore. Picosecond time-resolved spectroscopy of 11-cis locked rhodopsin analogs revealed that the cis-trans isomerization of the chromophore is the primary reaction in rhodopsin. Then, generation of femtosecond laser pulses in the 1990s made it possible to follow the process of isomerization in real time. Formation of photorhodopsin within 200 fsec was observed by a transient absorption (pump-probe) experiment, which also revealed that the photoisomerization in rhodopsin is a vibrationally coherent process. Femtosecond fluorescence spectroscopy directly captured excited-state dynamics of rhodopsin, so that both coherent reaction process and unreacted excited state were observed. Faster photoreaction of the chromophore in rhodopsin than that in solution implies that the protein environment facilitates the efficient isomerization process. Such contributions of the protein residues have been monitored by infrared spectroscopy of rhodopsin, bathorhodopsin, and isorhodopsin (9-cis rhodopsin) at low temperatures. The crystal structure of bovine rhodopsin recently reported will lead to better understanding of the mechanism in future.

Structural changes of pharaonis phoborhodopsin upon photoisomerization of the retinal chromophore: infrared spectral comparison with bacteriorhodopsin.

Archaeal rhodopsins possess a retinal molecule as their chromophores, and their light energy and light signal conversions are triggered by all-trans to 13-cis isomerization of the retinal chromophore. Relaxation through structural changes of the protein then leads to functional processes, proton pump in bacteriorhodopsin and transducer activation in sensory rhodopsins. In the present paper, low-temperature Fourier transform infrared spectroscopy is applied to phoborhodopsin from Natronobacterium pharaonis (ppR), a photoreceptor for the negative phototaxis of the bacteria, and infrared spectral changes before and after photoisomerization are compared with those of bacteriorhodopsin (BR) at 77 K. Spectral comparison of the C--C stretching vibrations of the retinal chromophore shows that chromophore conformation of the polyene chain is similar between ppR and BR. This fact implies that the unique chromophore-protein interaction in ppR, such as the blue-shifted absorption spectrum with vibrational fine structure, originates from both ends, the beta-ionone ring and the Schiff base regions. In fact, less planer ring structure and stronger hydrogen bond of the Schiff base were suggested for ppR. Similar frequency changes upon photoisomerization are observed for the C==N stretch of the retinal Schiff base and the stretch of the neighboring threonine side chain (Thr79 in ppR and Thr89 in BR), suggesting that photoisomerization in ppR is driven by the motion of the Schiff base like BR. Nevertheless, the structure of the K state after photoisomerization is different between ppR and BR. In BR, chromophore distortion is localized in the Schiff base region, as shown in its hydrogen out-of-plane vibrations. In contrast, more extended structural changes take place in ppR in view of chromophore distortion and protein structural changes. Such structure of the K intermediate of ppR is probably correlated with its high thermal stability. In fact, almost identical infrared spectra are obtained between 77 and 170 K in ppR. Unique chromophore-protein interaction and photoisomerization processes in ppR are discussed on the basis of the present infrared spectral comparison with BR.

The second cytoplasmic loop of metabotropic glutamate receptor functions at the third loop position of rhodopsin.

G protein-coupled receptors identified so far are classified into at least three major families based on their amino acid sequences. For the family of receptors homologous to rhodopsin (family 1), the G protein activation mechanism has been investigated in detail, but much less for the receptors of other families. To functionally compare the G protein activation mechanism between rhodopsin and metabotropic glutamate receptor (mGluR), which belong to distinct families, we prepared a set of bovine rhodopsin mutants whose second or third cytoplasmic loop was replaced with either the second or third loop of Gi/Go- or Gq-coupled mGluR (mGluR6 or mGluR1). Among these mutants, the mutants in which the second or third loop was replaced with the corresponding loop of mGluR exhibited no G protein activation ability. In contrast, the mutant whose third loop was replaced with the second loop of Gi/Go-coupled mGluR6 efficiently activated Gi but not Gt: this activation profile is almost identical with those of the mutant rhodopsins whose third loop was replaced with those of the Gi/Go-coupled receptors in family 1 [Yamashita et al. (2000) J. Biol. Chem. 275, 34272-34279]. The mutant whose third loop was replaced with the second loop of Gq-coupled mGluR1 partially retained the Gi coupling ability of rhodopsin, which is in contrast to the fact that all the rhodopsin mutants having the third loops of Gq-coupled receptors in family 1 exhibit no detectable Gi activation. These results strongly suggest that the molecular architectures of rhodopsin and mGluR are different, although the G protein activation mechanism involving the cytoplasmic loops is common.

O-Glycosylation of G-protein-coupled receptor, octopus rhodopsin. Direct analysis by FAB mass spectrometry.

In addition to the N-glycan that is evidently conserved in G-protein-coupled receptors (GPCRs), O-glycans in the N-terminus of GPCRs have been suggested. Using a combination of enzymatic and manual Edman degradation in conjunction with G-protein coupled receptor mass spectrometry, the structure and sites of O-glycans in octopus rhodopsin are determined. Two N-acetylgalactosamine residues are O-linked to Thr4 and Thr5 in the N-terminus of octopus rhodopsin. Further, we found chicken iodopsin, but not bovine rhodopsin, contains N-acetylgalactosamine. This is the first direct evidence to determine the structure and sites of O-glycans in GPCRs.

Demonstration of a rhodopsin-retinochrome system in the stalk eye of a marine gastropod, Onchidium, by immunohistochemistry.

The stalk eye of Onchidium sp. (Gastropoda, Mollusca) is the principal photoreceptor in a multiple photoreceptive system that consists of the stalk and dorsal eyes, dermal photoreceptor cells, and photosensitive neurons. To examine the localization of photopigments, the stalk eyes were immunostained with specific antibodies to rhodopsin, retinochrome, and retinal-binding protein (RALBP), which had been generated against squid retinal proteins. The retina of the stalk eye was divided into villous, pigmented, somatic, and neural layers. It was comprised mainly of two types of visual and pigmented supportive cells. The type 1 visual (VC1) cell was characterized by well-developed microvilli on its apical protrusion and photic vesicles in the cytoplasm. The photic vesicles were specifically blackened by prolonged osmification. The type 2 visual (VC2) cell had less numerous, shorter microvilli on its concave apical surface and lacked photic vesicles. The anti-squid rhodopsin antiserum was localized specifically to the villous layer that corresponded to the VC1 microvilli. With the anti-retinochrome peptide antibody, the somatic layer showed specific but patchy, positive staining that corresponded to the cytoplasm of the VC1 cells. Because the photic vesicles are known to contain retinochrome, these results indicate that this retinochrome is localized in the VC1 cytoplasm. Anti-RALBP antibody stained the supranuclear cytoplasm to the distal cytoplasm of VC1 cells. This is the first demonstration of the localization of RALBP in the Gastropoda Onchidium stalk eye. In squid retina that were immunostained as positive controls, the anti-rhodopsin antibody stained rhabdomeric microvilli, the anti-retinochrome antibody stained the inner segment and the basal region of the outer segment, and the anti-RALBP antibody stained the outer and inner segments, respectively. These results suggest that the rhodopsin-retinochrome system that has been established in cephalopod eyes is present in the Onchidium stalk eye.

Difference in molecular structure of rod and cone visual pigments studied by Fourier transform infrared spectroscopy.

To investigate the local structure that causes the differences in molecular properties between rod and cone visual pigments, we have measured the difference infrared spectra between chicken green and its photoproduct at 77 K and compared them with those from bovine and chicken rhodopsins. In contrast to the similarity of the vibrational bands of the chromophore, those of the protein part were notably different between chicken green and the rhodopsins. Like the rhodopsins, chicken green has an aspartic acid at position 83 (D83) but exhibited no signals due to the protonated carboxyl of D83 in the C=O stretching region, suggesting that the molecular contact between D83 and G120 through water molecule evidenced in bovine rhodopsin is absent in chicken green. A pair of positive and negative bands due to the peptide backbone (amide I) was prominent in chicken green, while the rhodopsins exhibited only small bands in this region. Furthermore, chicken green exhibited characteristic paired bands around 1480 cm(-1), which were identified as the imide bands of P189 using site-directed mutagenesis. P189, situated in the putative second extracellular loop, is conserved in all the known cone visual pigments but not in rhodopsins. Thus, some region of the second extracellular loop including P189 is situated near the chromophore and changes its environment upon formation of the batho-intermediate. The results noted above indicate that differences in the protein parts between chicken green and the rhodopsins alter the changes seen in the protein upon photoisomerization of the chromophore. Some of these changes appear to be the pathway from the chromophore to cytoplasmic surface of the pigment and thus could affect the activation process of transducin.

Tight Asp-85--Thr-89 association during the pump switch of bacteriorhodopsin.

Unidirectional proton transport in bacteriorhodopsin is enforced by the switching machinery of the active site. Threonine 89 is located in this region, with its O--H group forming a hydrogen bond with Asp-85, the acceptor for proton transfer from the Schiff base of the retinal chromophore. Previous IR spectroscopy of [3-(18)O]threonine-labeled bacteriorhodopsin showed that the hydrogen bond of the O--D group of Thr-89 in D(2)O is strengthened in the K photocycle intermediate. Here, we show that the strength and orientation of this hydrogen bond remains unchanged in the L intermediate and through the M intermediate. Furthermore, a strong interaction between Asp-85 and the O--H (O--D) group of Thr-89 in M is indicated by a shift in the C==O stretching vibration of the former because of (18)O substitution in the latter. Thus, the strong hydrogen bond between Asp-85 and Thr-89 in K persists through M, contrary to structural models based on x-ray crystallography of the photocycle intermediates. We propose that, upon photoisomerization of the chromophore, Thr-89 forms a tight, persistent complex with one of the side-chain oxygens of Asp-85 and is thereby precluded from participating in the switching process. On the other hand, the loss of hydrogen bonding at the other oxygen of Asp-85 in M may be related to the switching event.

Chloride effect on iodopsin studied by low-temperature visible and infrared spectroscopies.

To investigate the chloride effect on the spectral properties of iodopsin, we have prepared an anion-free iodopsin (iodopsin.free) by extensive dialysis of an iodopsin sample against a buffer containing no chloride, and visible and infrared difference spectra between iodopsin.free and its photoproduct at 77 K were recorded. The absorption maximum of iodopsin.free in L-alpha-phosphatidylcholine liposomes was 528 nm, which was almost identical with that of the nitrate-bound form of iodopsin (526 nm, iodopsin.NO(3)), but 43 nm blue-shifted from that of the chloride-bound form of iodopsin (iodopsin.Cl). The iod/batho visible difference spectrum obtained from iodopsin.free was similar in shape to that from iodopsin.NO(3), but not to that from iodopsin.Cl. FTIR spectroscopy revealed that the chromophore vibrational bands and the peptide bonds of the original state in iodopsin.free were identical with those in iodopsin.NO(3) and were also similar to those in iodopsin.Cl except for the ethylenic vibrations of the chromophore. In contrast, those of the batho state in iodopsin.free were similar to those in iodopsin.NO(3) but considerably different from those in iodopsin.Cl. These results suggested that the binding of chloride but not nitrate induces a conformational change in the protein and that the chloride binding site is situated in a position where it directly interacts with the chromophore when the chromophore is photoisomerized. FTIR spectroscopy also revealed that one of the four water bands observed in the batho/iod spectrum of iodospin.Cl is absent in the spectra of iodopsin.free and iodopsin.NO(3). Thus, in contrast to nitrate, a lyotropic anion, chloride would bind to the binding site with water molecule(s) which could form a hydrogen-bonding network with amino acid residue(s) near the chromophore, thereby resulting in the red shift of the absorption maximum of iodopsin.

Regulatory mechanism for the stability of the meta II intermediate of pinopsin.

Pinopsin is a chicken pineal photoreceptive molecule with a possible role in photoentrainment of the circadian clock. Sequence comparison among members of the rhodopsin family has suggested that pinopsin might have properties more similar to cone visual pigments than to rhodopsin, but the lifetime of the physiologically active intermediate (meta II) of pinopsin is rather similar to that of metarhodopsin II, which is far more stable than meta II intermediates of cone visual pigments [Nakamura, A. et al., (1999) Biochemistry 38, 14738-14745]. In the present study, we investigated the amino acid residue(s) contributing to this unique property of pinopsin by using site-directed mutagenesis to pinopsin-specific structural features, (i) Ser171, (ii) Asn184, and (iii) the second extracellular loop two-amino acids shorter than that of cone visual pigments. The meta II stability of the 171/184 double mutant of pinopsin (S171R/N184D) is almost the same as that of wild-type pinopsin. In contrast, the meta II lifetime is markedly shortened (one third) by introduction of the third mutation (replacement of a six-amino acid stretch, 188-193, by the corresponding eight residues of chicken green-sensitive cone pigment) to the 171/184 double mutant of pinopsin. Consistently, meta II of the green-sensitive pigment mutant, in which the eight-amino acid stretch is inversely replaced by the corresponding six residues of pinopsin, is more stable than meta II of the wild-type pigment. These results strongly suggest that the specific sequence and/or the number of residues at amino acids 188-193 in pinopsin play an important role in the stabilization of the meta II intermediate.

Highly conserved glutamic acid in the extracellular IV-V loop in rhodopsins acts as the counterion in retinochrome, a member of the rhodopsin family.

Retinochrome is a member of the rhodopsin family having a chromophore retinal and functioning as a retinal photoisomerase in squid photoreceptor cells. Unlike vertebrate rhodopsins, but like many invertebrate rhodopsins, retinochrome does not have a glutamic acid at position 113 to serve as a counterion for the protonated retinylidene Schiff base. Here we investigated possible counterions in retinochrome by site-specific mutagenesis. Our results showed that the counterion is the glutamic acid at position 181, at which almost all the pigments in the rhodopsin family, including vertebrate and invertebrate rhodopsins, have a glutamic or aspartic acid. The remarkable exceptions are the long-wavelength visual pigments that have a histidine that, together with a nearby lysine, serves as a chloride-binding site. Replacement of Glu-181 of bovine rhodopsin with Gln caused a 10-nm red-shift of absorption maximum. Because the position at 181 is in the extracellular loop connecting the transmembrane helices VI and V, these results demonstrate the importance of this loop to function for spectral tuning in the rhodopsin family.

Distinct roles of the second and third cytoplasmic loops of bovine rhodopsin in G protein activation.

In contrast to the extensive studies of light-induced conformational changes in rhodopsin, the cytoplasmic architecture of rhodopsin related to the G protein activation and the selective recognition of G protein subtype is still unclear. Here, we prepared a set of bovine rhodopsin mutants whose cytoplasmic loops were replaced by those of other ligand-binding receptors, and we compared their ability for G protein activation in order to obtain a clue to the roles of the second and third cytoplasmic loops of rhodopsin. The mutants bearing the third loop of four other G(o)-coupled receptors belonging to the rhodopsin superfamily showed significant G(o) activation, indicating that the third loop of rhodopsin possibly has a putative site(s) related to the interaction of G protein and that it is simply exchangeable with those of other G(o)-coupled receptors. The mutants bearing the second loop of other receptors, however, had little ability for G protein activation, suggesting that the second loop of rhodopsin contains a specific region essential for rhodopsin to be a G protein-activating form. Systematic chimeric and point mutational studies indicate that three amino acids (Glu(134), Val(138), and Cys(140)) in the N-terminal region of the second loop of rhodopsin are crucial for efficient G protein activation. These results suggest that the second and third cytoplasmic loops of bovine rhodopsin have distinct roles in G protein activation and subtype specificity.

Movement of retinal along the visual transduction path.

Movement of the ligand/receptor complex in rhodopsin (Rh) has been traced. Bleaching of diazoketo rhodopsin (DK-Rh) containing 11-cis-3-diazo-4-oxo-retinal yields batho-, lumi-, meta-I-, and meta-II-Rh intermediates corresponding to those of native Rh but at lower temperatures. Photoaffinity labeling of DK-Rh and these bleaching intermediates shows that the ionone ring cross-links to tryptophan-265 on helix F in DK-Rh and batho-Rh, and to alanine-169 on helix D in lumi-, meta-I-, and meta-II-Rh intermediates. It is likely that these movements involving a flip-over of the chromophoric ring trigger changes in cytoplasmic membrane loops resulting in heterotrimeric guanine nucleotide-binding protein (G protein) activation.

Local and distant protein structural changes on photoisomerization of the retinal in bacteriorhodopsin.

The photoisomerization of the retinal in bacteriorhodopsin is selective and efficient and yields perturbation of the protein structure within femtoseconds. The stored light energy in the primary intermediate is then used for the net translocation of a proton across the membrane in the microsecond to millisecond regime. This study is aimed at identifying how the protein changes on photoisomerization by using the O-H groups of threonines as internal probes. Polarized Fourier-transform IR spectroscopy of [3-(18)O]threonine-labeled and unlabeled bacteriorhodopsin indicates that 3 of the threonines (of a total of 18) change their hydrogen bonding. One is exchangeable in D(2)O, but two are not. A comprehensive mutation study indicates that the residues involved are Thr-89, Thr-17, and Thr-121 (or Thr-90). The perturbation of only three threonine side chains suggests that the structural alteration at this stage of the photocycle is local and specific. Furthermore, the structural change of Thr-17, which is located >11 A from the retinal chromophore, implicates a specific perturbation channel in the protein that accompanies the retinal motion.

Amino acid residues controlling the properties and functions of rod and cone visual pigments.

The visual transduction processes in rod and cone photoreceptor cells are initiated by photon absorption by the different types of visual pigments. In relation to the functional difference between these cells, cone visual pigments in chicken retinas exhibit faster regeneration from 11-cis-retinal and opsin and faster decay of physiologically active intermediate (Meta II) than rod visual pigment, rhodopsin. Replacement of the amino acid residue at position 122 of chicken rhodopsin by the residues present in the respective cone pigments dramatically changes both the decay rate of Meta II and the rate of regeneration into those of the cone pigment-type, indicating that the residue at this position is a major determinant controlling these properties. Thus, the single replacement of amino acid residue at this position would be one of the key steps of the divergence into twilight and daylight vision.

Chimeric nature of pinopsin between rod and cone visual pigments.

Chicken pineal pinopsin is the first example of extra-retinal opsins, but little is known about its molecular properties as compared with retinal rod and cone opsins. For characterization of extra-retinal photon signaling, we have developed an overexpression system providing a sufficient amount of purified pinopsin. The recombinant pinopsin, together with similarly prepared chicken rhodopsin and green-sensitive cone pigment, was subjected to photochemical and biochemical analyses by using low-temperature spectroscopy and the transducin activation assay. At liquid nitrogen temperature (-196 degrees C), we detected two kinds of photoproducts, bathopinopsin and isopinopsin, having their absorption maxima (lambda(max)) at 527 and approximately 440 nm, respectively, and we observed complete photoreversibility among pinopsin, bathopinopsin, and isopinopsin. A close parallel of the photoreversibility to the rhodopsin system strongly suggests that light absorbed by pinopsin triggers the initial event of cis-trans isomerization of the 11-cis-retinylidene chromophore. Upon warming, bathopinopsin decayed through a series of photobleaching intermediates: lumipinopsin (lambda(max) 461 nm), metapinopsin I (460 nm), metapinopsin II (385 nm), and metapinopsin III (460 nm). Biochemical and kinetic analyses showed that metapinopsin II is a physiologically important photoproduct activating transducin. Detailed kinetic analyses revealed that the formation of metapinopsin II is as fast as that of a chicken cone pigment, green, but that the decay process of metapinopsin II is as slow as that of the rod pigment, rhodopsin. These results indicate that pinopsin is a new type of pigment with a chimeric nature between rod and cone visual pigments in terms of the thermal behaviors of the meta II intermediate. Such a long-lived active state of pinopsin may play a role in the pineal-specific phototransduction process.

Effect of anion binding on iodopsin studied by low-temperature fourier transform infrared spectroscopy.

The effect of anion binding on iodopsin, the chicken red-sensitive cone visual pigment, was studied by measurements of the Fourier transform infrared spectra of chloride- and nitrate-bound forms of iodopsin at 77 K. In addition to the blue shift of the absorption maximum upon substituting nitrate for chloride, the C=C stretching vibrations of iodopsin and its photoproducts were upshifted 5-6 cm(-)(1). The C=NH and C=ND stretching vibrations were the same in wavenumber between the chloride- and nitrate-bound forms, indicating that the binding of either chloride or nitrate has no effect on the interaction between the protonated Schiff base and the counterion. The vibrational bands of iodopsin in the fingerprint and the hydrogen out-of-plane wagging regions were insensitive to anion substitution, suggesting that local chromophore interactions with the anions are not crucial for the absorption spectral shift. In contrast, bathoiodopsin in the chloride-bound form exhibited an intense C(14)H wagging mode, whose intensity was considerably weakened upon substitution of nitrate for chloride. These results suggest that binding of chloride changes the environment near the C(14) position of the chromophore, which could be one of the factors in the thermal reverse reaction of bathoiodopsin to iodopsin in the chloride-bound form.

Structural change of threonine 89 upon photoisomerization in bacteriorhodopsin as revealed by polarized FTIR spectroscopy.

The all-trans to 13-cis photoisomerization of the retinal chromophore of bacteriorhodopsin occurs selectively, efficiently, and on an ultrafast time scale. The reaction is facilitated by the surrounding protein matrix which undergoes further structural changes during the proton-transporting reaction cycle. Low-temperature polarized Fourier transform infrared difference spectra between bacteriorhodopsin and the K intermediate provide the possibility to investigate such structural changes, by probing O-H and N-H stretching vibrations [Kandori, Kinoshita, Shichida, and Maeda (1998) J. Phys. Chem. B 102, 7899-7905]. The measurements of [3-18O]threonine-labeled bacteriorhodopsin revealed that one of the D2O-sensitive bands (2506 cm(-1) in bacteriorhodopsin and 2466 cm(-1) in the K intermediate, in D2O exhibited 18(O)-induced isotope shift. The O-H stretching vibrations of the threonine side chain correspond to 3378 cm(-1) in bacteriorhodopsin and to 3317 cm(-1) in the K intermediate, indicating that hydrogen bonding becomes stronger after the photoisomerization. The O-H stretch frequency of neat secondary alcohol is 3340-3355 cm(-1). The O-H stretch bands are preserved in the T46V, T90V, T142N, T178N, and T205V mutant proteins, but diminished in T89A and T89C, and slightly shifted in T89S. Thus, the observed O-H stretching vibration originates from Thr89. This is consistent with the atomic structure of this region, and the change of the S-H stretching vibration of the T89C mutant in the K intermediate [Kandori, Kinoshita, Shichida, Maeda, Needleman, and Lanyi (1998) J. Am. Chem. Soc. 120, 5828-5829]. We conclude that all-trans to 13-cis isomerization causes shortening of the hydrogen bond between the OH group of Thr89 and a carboxyl oxygen atom of Asp85.