Light-induced Conformational Changes In Rhodopsin Research Articles

Conformational activation of visual rhodopsin in native disc membranes.

Rhodopsin is the G protein-coupled receptor (GPCR) that serves as a dim-light receptor for vision in vertebrates. We probed light-induced conformational changes in rhodopsin in its native membrane environment at room temperature using time-resolved wide-angle x-ray scattering. We observed a rapid conformational transition that is consistent with an outward tilt of the cytoplasmic portion of transmembrane helix 6 concomitant with an inward movement of the cytoplasmic portion of transmembrane helix 5. These movements were considerably larger than those reported from the basis of crystal structures of activated rhodopsin, implying that light activation of rhodopsin involves a more extended conformational change than was previously suggested.

Mapping of the local environmental changes in proteins by cysteine scanning.

Protein conformational changes, which regulate the activity of proteins, are induced by the alternation of intramolecular interactions. Therefore, the detection of the local environmental changes around the key amino acid residues is essential to understand the activation mechanisms of functional proteins. Here we developed the methods to scan the local environmental changes using the vibrational band of cysteine S-H group. We validated the sensitivity of this method using bathorhodopsin, a photoproduct of rhodopsin trapped at liquid nitrogen temperature, which undergoes little conformational changes from the dark state as shown by the X-ray crystallography. The cysteine residues were individually introduced into 15 positions of Helix III, which contains several key amino acid residues for the light-induced conformational changes of rhodopsin. The shifts of S-H stretching modes of these cysteine residues and native cysteine residues upon the formation of bathorhodopsin were measured by Fourier transform infrared spectroscopy. While most of cysteine residues demonstrated no shift of S-H stretching mode, cysteine residues introduced at positions 117, 118, and 122, which are in the vicinity of the chromophore, demonstrated the significant changes. The current results are consistent with the crystal structure of bathorhodopsin, implying that the cysteine scanning is sensitive enough to detect the tiny conformational changes.

Constraints on the conformation of the cytoplasmic face of dark-adapted and light-excited rhodopsin inferred from antirhodopsin antibody imprints.

Rhodopsin is the best-understood member of the large G protein-coupled receptor (GPCR) superfamily. The G-protein amplification cascade is triggered by poorly understood light-induced conformational changes in rhodopsin that are homologous to changes caused by agonists in other GPCRs. We have applied the "antibody imprint" method to light-activated rhodopsin in native membranes by using nine monoclonal antibodies (mAbs) against aqueous faces of rhodopsin. Epitopes recognized by these mAbs were found by selection from random peptide libraries displayed on phage. A new computer algorithm, FINDMAP, was used to map the epitopes to discontinuous segments of rhodopsin that are distant in the primary sequence but are in close spatial proximity in the structure. The proximity of a segment of the N-terminal and the loop between helices VI and VIII found by FINDMAP is consistent with the X-ray structure of the dark-adapted rhodopsin. Epitopes to the cytoplasmic face segregated into two classes with different predicted spatial proximities of protein segments that correlate with different preferences of the antibodies for stabilizing the metarhodopsin I or metarhodopsin II conformations of light-excited rhodopsin. Epitopes of antibodies that stabilize metarhodopsin II indicate conformational changes from dark-adapted rhodopsin, including rearrangements of the C-terminal tail and altered exposure of the cytoplasmic end of helix VI, a portion of the C-3 loop, and helix VIII. As additional antibodies are subjected to antibody imprinting, this approach should provide increasingly detailed information on the conformation of light-excited rhodopsin and be applicable to structural studies of other challenging protein targets.

Light-induced conformational changes of rhodopsin probed by fluorescent alexa594 immobilized on the cytoplasmic surface.

A novel fluorescence method has been developed for detecting the light-induced conformational changes of rhodopsin and for monitoring the interaction between photolyzed rhodopsin and G-protein or arrestin. Rhodopsin in native membranes was selectively modified with fluorescent Alexa594-maleimide at the Cys(316) position, with a large excess of the reagent Cys(140) that was also derivatized. Modification with Alexa594 allowed the monitoring of fluorescence changes at a red excitation light wavelength of 605 nm, thus avoiding significant rhodopsin bleaching. Upon absorption of a photon by rhodopsin, the fluorescence intensity increased as much as 20% at acidic pH with an apparent pK(a) of approximately 6.8 at 4 degrees C, and was sensitive to the presence of hydroxylamine. These findings indicated that the increase in fluorescence is specific for metarhodopsin II. In the presence of transducin, a significant increase in fluorescence was observed. This increase of fluorescence emission intensity was reduced by addition of GTP, in agreement with the fact that transducin enhances the formation of metarhodopsin II. Under conditions that favored the formation of a metarhodopsin II-Alexa594 complex, transducin slightly decreased the fluorescence. In the presence of arrestin, under conditions that favored the formation of metarhodopsin I or II, a phosphorylated, photolyzed rhodopsin-Alexa594 complex only slightly decreased the fluorescence intensity, suggesting that the cytoplasmic surface structure of metarhodopsin II is different in the complex with arrestin and transducin. These results demonstrate the application of Alexa594-modified rhodopsin (Alexa594-rhodopsin) to continuously monitor the conformational changes in rhodopsin during light-induced transformations and its interactions with other proteins.

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.

Probing intramolecular orientations in rhodopsin and metarhodopsin II by polarized infrared difference spectroscopy.

The light-induced conformational changes of rhodopsin, which lead to the formation of the G-protein activating metarhodopsin II intermediate, are studied by polarized attenuated total reflectance infrared difference spectroscopy. Orientations of protein groups as well as the retinylidene chromophore were calculated from the linear dichroism of infrared difference bands. These bands correspond to changes in the vibrational modes of individual molecular groups that are structurally active during receptor activation, i.e., during the rhodopsin to metarhodopsin II transition. The orientation of the transition dipole moments of bands previously assigned to the carboxyl (C=O) groups of Asp83 and Glu113 has been determined. The orientation of specific groups in the retinylidene chromophore has been inferred from the dichroism of the bands associated with the polyene C-C, C=C, and hydrogen-out-of-plane vibrations. Interestingly, the use of polarized infrared light reveals several difference bands in the rhodopsin to metarhodopsin II difference spectrum which were previously undetected, e.g., at 1736 and 939 cm(-1). The latter is tentatively assigned to the hydrogen-out-of-plane mode of the HC(11)=C(12)H segment of the chromophore. Our data suggest a significant change in orientation of this group in the late phase of rhodopsin activation. On the basis of available site-directed mutagenesis data, bands at 1406, 1583, and 1736 cm(-1) are tentatively assigned to Glu134. The main features in the amide regions in the dichroic difference spectrum are discussed in terms of a slight reorientation of helical segments upon receptor activation.

Photoactivation of Rhodopsin Causes an Increased Hydrogen-Deuterium Exchange of Buried Peptide Groups

A key step in visual transduction is the light-induced conformational changes of rhodopsin that lead to binding and activation of the G-protein transducin. In order to explore the nature of these conformational changes, time-resolved Fourier transform infrared spectroscopy was used to measure the kinetics of hydrogen/deuterium exchange in rhodopsin upon photoexcitation. The extent of hydrogen/deuterium exchange of backbone peptide groups can be monitored by measuring the integrated intensity of the amide II and amide II′ bands. When rhodopsin films are exposed to D 2O in the dark for long periods, the amide II band retains at least 60% of its integrated intensity, reflecting a core of backbone peptide groups that are resistant to H/D exchange. Upon photoactivation, rhodopsin in the presence of D 2O exhibits a new phase of H/D exchange which at 10°C consists of fast (time constant ∼30 min) and slow (∼11 h) components. These results indicate that photoactivation causes buried portions of the rhodopsin backbone structure to become more accessible.

Cyclic GMP and photoreceptor function

A single photon can be detected by a rod photoreceptor cell. The absorption of light by rhodopsin triggers a cascade of reactions that amplifies the photon signal and results in ion channel closure with hyperpolarization of the rod photoreceptor cell. Light-induced conformational changes in rhodopsin facilitate the binding of a guanosine nucleotide-binding protein, transducin, which then undergoes a GTP-GDP exchange reaction and dissociation of the transducin complex. A subunit of transducin then activates a phosphodiesterase complex that hydrolyzes cyclic GMP. In darkness, cyclic GMP binds to cation channels of the photoreceptor plasma membrane, maintaining them in an open configuration. The light-induced reduction in cyclic GMP concentration dissociates the bound cyclic GMP, resulting in channel closure and hyperpolarization. Down-regulation of the cascade involves other proteins that block the interaction of transducin with rhodopsin and another protein that may interfere with transducin recycling. Cone photoreceptors possess a light-activated cascade that follows the rod format, but it is composed of proteins that are homologous to those of rod photoreceptors. Phototransduction in invertebrate photoreceptors uses rhodopsin to activate a cascade that uses phosphoinositides and calcium ion to regulate membrane polarization.

Effects of lipid environment on the light-induced conformational changes of rhodopsin. 2. Roles of lipid chain length, unsaturation, and phase state.

When rhodopsin is incorporated into the saturated short-chain phospholipid dimyristoylphosphatidylcholine, photolysis of the protein results in an abnormal sequence of spectral transitions, and the dominant product of metarhodopsin I decay is free retinal plus opsin [Baldwin, P. A., & Hubbell, W. L. (1985) Biochemistry (preceding paper in this issue)]. By incorporation of rhodopsin into a series of phosphatidylcholines of defined composition, we have determined the properties of the lipid environment that are responsible for the altered spectral behavior. Metarhodopsin II is not found in appreciable amounts in bilayers containing acyl chains that are too short (14 or fewer carbon atoms in length), in the presence of only n-alkyl chains, or below the characteristic phase-transition temperature of recombinant membranes. Double bonds are not required for the formation of the metarhodopsin II intermediate, as it is observed in diphytanoylphosphatidylcholine recombinants.

Effects of lipid environment on the light-induced conformational changes of rhodopsin. 1. Absence of metarhodopsin II production in dimyristoylphosphatidylcholine recombinant membranes.

Photolysis of bovine rhodopsin in dimyristoylphosphatidylcholine recombinant membranes results in the production of a relatively stable metarhodopsin I like photointermediate that decays slowly to a species with a broad absorbance maximum centered at about 380 nm [O'Brien, D. F., Costa, L. F., & Ott, R. A. (1977) Biochemistry 16, 1295-1303]. On the basis of the results of a variety of chemical and spectroscopic tests, we show that this process corresponds to the production of free retinal plus opsin and not to the slow production of metarhodopsin II. Electron spin resonance studies using a novel disulfide spin-label that is covalently linked to rhodopsin indicate that the apparent arrest of the protein at the metarhodopsin I stage is not due to simple aggregation of the protein in this short-chain, saturated lipid bilayer but must be understood in terms of the effect of the lipid host on the conformational energies of individual protein molecules. Limited production of metarhodopsin II is observed under acidic conditions. Thus, the rhodopsin-dimyristoylphosphatidylcholine recombinants offer a unique system for the study of the effect of the phospholipid bilayer environment on the conformation of an intrinsic membrane protein.

Transient light-induced conformational changes in rhodopsin.

Arguments are presented which support the possibility that the unfolding of the rhodopsin molecule during photolysis up to the stage of metarhodopsin II is followed by a spontaneous refolding of the protein, once the isomerized retinaldehyde has left its original binding site. Such a transient conformational change might imply a very similar conformation for rhodopsin and opsin, apart from the presence of the chromophore.