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Membrane proteins, encoded by ~20% of genes in almost all organisms, including humans, are critical for cellular communication, electrical and ion balances, structural integrity of the cells and their adhesions, and other functions. Atomic-resolution structures of these proteins furnish important information for understanding their molecular organization and constitute major breakthroughs in our understanding of how they participate in physiological processes. However, obtaining structural information about these proteins has progressed slowly (1, 2), mostly because of technical difficulties in the purification and handling of integral membrane proteins. Instability of the proteins in environments lacking phospholipids, the tendency for them to aggregate and precipitate, and/or difficulties with highly heterogeneous preparations of these proteins isolated from heterologous expression systems have hindered application of standard structure determination techniques to these molecules. Among membrane proteins, G-protein-coupled receptors (GPCRs)1 are of special importance because they form one of the largest and most diverse groups of receptor proteins. More than 400 nonsensory receptors identified in the human genome are involved in the regulation of virtually all physiological processes. Drug addiction, mood control, and memory (via 5-HT6 or neuropeptide receptors) are just a short list of processes in which GPCRs are critically implicated. Another even larger group of GPCRs consist of sensory receptors involved in the fundamental process of translation of light energy (rhodopsin and cone pigments), the detection of chemoattractant molecules, or the detection of compounds stimulating the taste buds (3, 4). The activity of GPCRs comes about when binding of diffusable extracellular ligands causes them to switch from quiescent forms to an active conformation capable of interaction with hundreds of G-proteins. Their roles as extracellular ligand-binding proteins make them attractive targets for drug design. GPCRs account for ~40% of all therapeutic intervention, and major GPCR research projects are found throughout the pharmaceutical industry (5, 6). A paucity of structural data is available for GPCRs. The crystal structure of a member of the largest subgroup (I) of GPCRs, rhodopsin (7), and a ligand-binding domain of the metabotropic glutamate receptor with and without the ligand (8) have been determined recently. The data allow models, firmly based on the atomic-resolution structural information, to be further tested as to the conformational changes that these receptors undergo in going from the quiescent to the signaling state. In this article, we describe the further refinement of rhodopsin (7) and provide some clues about how the receptor could be activated by light.

Through low-temperature absorption and circular dichroism (CD) spectroscopies, and G-protein (transducin) binding experiments, we have investigated molecular properties of the meta II intermediate of iodopsin, a cone visual pigment present in chicken red-sensitive cones. The meta II intermediate of iodopsin (metaiodopsin II, lambda max = 390 nm) displayed a positive CD band at about 390 nm and a large negative CD band below 300 nm. It dissociated into all-trans-retinal and the protein moiety. A long-lived intermediate corresponding to the meta III intermediate of rhodopsin was not observed in iodopsin, under our experimental conditions. Decay of metaiodopsin II was significantly suppressed in the presence of transducin, but not in the presence of both transducin and GTP, indicating that metaiodopsin II can interact with transducin and activate it. Both metaiodopsin II and metarhodopsin II displayed a large negative CD band below 300 nm. This fact suggested that during the formation of both meta II intermediates, some aromatic amino acid residues and/or a disulfide bond are rearranged, which may be important for expression of catalytic activity for exchange of GDP to GTP on transducin. On the other hand, metaiodopsin II decayed more than 10 times faster than metarhodopsin II. This fact may be one of the reasons why cones are less photosensitive than rods.

The photoactivation phenomena of the photosensitive enzyme nitrile hydratase (NHase) was studied by various spectroscopic methods. We have already shown that the photoactivation of NHase accompanies oxidation of an iron atom in the NHase [Honda et al. (1992) FEBS Lett. 301, 177-180]. From the results obtained in the present study by absorption, action, and fluorescence spectra, we show that the chromophore responsible for the photoactivation process is the iron complex, and the tryptophan residues in NHase induce the oxidation of the iron atom via an energy-transfer process. The nanosecond flash photolysis experiment revealed that this photoactivation process is completed within 50 ns, which suggests that the changes observable in the absorption spectra originate from an intramolecular electron transfer occurring from an electronically excited state. Also the role of a stabilizing reagent, namely, n-butyric acid (BA), was investigated using the above methods, which revealed that BA, besides its stabilizing effect, contributes to the increase in apparent photoactivation rate.

The photobleaching process of iodopsin (a chicken red-sensitive cone visual pigment) purified in a detergent system containing CHAPS and phosphatidylcholine was investigated by means of nanosecond laser photolysis at room temperature. Excitation of iodopsin with a nanosecond laser pulse (wavelength, 560 nm; pulse width, 17 ns) resulted in the formation of at least four intermediates on the nanosecond to millisecond time scale. The earliest intermediate detected had an absorption maximum at 571 nm, which was very close to that of original iodopsin (lambda max = 567 nm), and remarkably blue-shifted as compared with that of bathoiodopsin [lambda max = 625 nm; Kandori et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8908-8912]. The intermediate, named BL-iodopsin, converted to the next intermediate, lumiiodopsin (lambda max = 535 nm), with a time constant of 130 ns. The BL intermediate had an absorption maximum just between batho- and lumiiodopsins, and an extinction coefficient comparable with these intermediates. These properties are different from those of the corresponding intermediate of rhodopsin [BL(BSI)-rhodopsin], suggesting that the binding of chloride to iodopsin, but not to rhodopsin, has an influence upon changes of the chromophore-opsin interaction in the early stage of photobleaching of iodopsin. Lumiiodopsin converted to metaiodopsin I (lambda max < 500 nm) with a time constant of 230 microseconds, and then to metaiodopsin II (lambda max = 390 nm) with a time constant of 6 ms. A thermal equilibrium between metaiodopsin I and II was established, but unlike meta intermediates of rhodopsin, they showed little temperature dependence.

Photochemical and subsequent thermal reactions of rhodopsin containing 9-cis-retinal [Rh(9)] or one of four analogues with 9-cis geometries formed from ring-modified retinals, alpha-retinal [alpha Rh(9)], acyclic retinal [AcRh(9)], acyclic alpha-retinal [Ac alpha Rh(9)], and 5-isopropyl-alpha-retinal [P alpha Rh(9)] were investigated by low-temperature spectrophotometry and nanosecond laser photolysis. Irradiation of each pigment at -180 degrees C produced a photosteady-state mixture containing the original 9-cis pigment, its 11-cis pigment, and a photoproduct, indicating that the primary process of each pigment is a photoisomerization of its chromophore. The photoproduct produced by the irradiation of AcRh(9) had an absorption spectrum red shifted from the original AcRh(9) and was identified as the batho intermediate of AcRh(9). It was converted to the lumi intermediate through a metastable species, the BL intermediate, which has never been detected in Rh(9) at low temperature and whose absorption maximum was at shorter wavelengths than that of the batho intermediate. In contrast, the absorption maxima of the photoproducts produced from the other analogue pigments were at shorter wavelengths than those of the original pigments. They were identified as BL intermediates on the basis of their absorption maxima and thermal stabilities. The formation time constant of the lumi intermediate at room temperature was found to be dependent on the extent of modification of the ring portion of the chromophore, decreasing with the complete truncation of the cyclohexenyl ring [Ac alpha Rh(9)] and increasing with the attachment of the isopropyl group to the ring [P alpha Rh(9)].(ABSTRACT TRUNCATED AT 250 WORDS)

To identify how many rhodopsin intermediates interact with retinal G-protein transducin, the photobleaching process of chicken rhodopsin has been investigated in the presence or absence of transducin by means of time-resolved low-temperature spectroscopy. Singular value decomposition (SVD) analysis of the spectral data showed that a new intermediate called meta Ib is present between formally identified metarhodopsin I (now referred to as meta Ia) and metarhodopsin II (meta II). Since the absorption maximum of meta Ib (460 nm) is similar to that of meta Ia (480 nm), but considerably different from that of meta II (380 nm), meta Ib should have a protonated retinylidene Schiff base as its chromophore. Whereas transducin showed no effect on the conversion process between lumirhodopsin (lumi) and meta Ia, it affected the process between meta Ia and meta Ib and that between meta Ib and meta II. These results suggest that at least two intermediates (meta Ib and meta II) interact with transducin. The addition of GTPgammaS had no effect on the meta Ib-transducin interaction, while it abolished the ability of transducin to interact with meta II. Thus, meta Ib only binds to transducin, while meta II catalyzes a GDP-GTP exchange in transducin. These results suggest that deprotonation of the Schiff base chromophore is not necessary for the binding to transducin, while changes in protein structure including Schiff base deprotonation are needed to induce the GDP-GTP exchange in transducin.