Rhodopsin In Solution Research Articles

X-ray Footprinting Studies on Photoactivation of Bovine Rhodopsin

Rhodopsin, the visual G-protein coupled receptor (GPCR) in the rod cells of the vertebrate retina, is fundamental to vision. The light-activated intermediate of rhodopsin, Meta-II, initiates a signalling cascade that culminates in an electrical impulse in the visual cortex of the brain. The molecular details of agonist-induced structural change that are likely to be conserved among the members of the GPCR super family are not fully understood. We used X-ray Footprinting to study the conformational change in rhodopsin in solution upon photoactivation. Purified rhodopsin is exposed in tens of milliseconds with high flux focused X-rays. The hydroxyl radicals that are generated by photolysis of water react with the solvent accessible side chains and form stable modification products. The peptic fragments are analyzed by mass spectrometry to quantify the extent and identify the sites of oxidation. Monitoring the changes in the radiolytic modification as function of the exposure time provides information that is directly correlated with the solvent accessibilities of individual peptide or side chain residues within the protein. The difference in solvent accessibilities between dark state and light activated Meta-II state shows conformational changes near the retinal binding site, but not a large structural change as predicted by some models of GPCR activation. Labeling was also observed in the trans-membrane helical regions, this was also unexpected. We introduce a novel O18 labeling method to determine if transmembrane labeling arises from exchange with bulk water or is mediated by activation of bound, conserved water molecules in the GPCR structure. This is a novel approach that can probe the details of bound water structure and dynamics that function in a number of ion channels and receptors.

Monomeric G protein-coupled receptor rhodopsin in solution activates its G protein transducin at the diffusion limit.

G protein-coupled receptors mediate biological signals by stimulating nucleotide exchange in heterotrimeric G proteins (Galphabetagamma). Receptor dimers have been proposed as the functional unit responsible for catalytic interaction with Galphabetagamma. To investigate whether a G protein-coupled receptor monomer can activate Galphabetagamma, we used the retinal photoreceptor rhodopsin and its cognate G protein transducin (G(t)) to determine the stoichiometry of rhodopsin/G(t) binding and the rate of catalyzed nucleotide exchange in G(t). Purified rhodopsin was prepared in dodecyl maltoside detergent solution. Rhodopsin was monomeric as concluded from fluorescence resonance energy transfer, copurification studies with fluorescent labeled and unlabeled rhodopsin, size exclusion chromatography, and multiangle laser light scattering. A 1:1 complex between light-activated rhodopsin and G(t) was found in the elution profiles, and one molecule of GDP was released upon complex formation. Analysis of the speed of catalytic rhodopsin/G(t) interaction yielded a maximum of approximately 50 G(t) molecules per second and molecule of activated rhodopsin. The bimolecular rate constant is close to the diffusion limit in the diluted system. The results show that the interaction of G(t) with an activated rhodopsin monomer is sufficient for fully functional G(t) activation. Although the activation rate in solution is at the physically possible limit, the rate in the native membrane is still 10-fold higher. This is likely attributable to the precise orientation of the G protein to the membrane surface, which enables a fast docking process preceding the actual activation step. Whether docking in membranes involves the formation of rhodopsin dimers or oligomers remains to be elucidated.

Designer short peptide surfactants stabilize G protein-coupled receptor bovine rhodopsin.

Membrane proteins play vital roles in every aspect of cellular activities. To study diverse membrane proteins, it is crucial to select the right surfactants to stabilize them for analysis. Despite much effort, little progress has been made in elucidating their structure and function, largely because of a lack of suitable surfactants. Here we report the stabilization of a G protein-coupled receptor bovine rhodopsin in solution, using a new class of designer short and simple peptide surfactants. These surfactants consist of seven amino acids with a hydrophilic head, aspartic acid or lysine, and a hydrophobic tail with six consecutive alanines. These peptide surfactants not only enhance the stability of bovine rhodopsin in the presence of lipids and the common surfactants n-dodecyl-beta-D-maltoside and octyl-D-glucoside, but they also significantly stabilize rhodopsin under thermal denaturation conditions, even after lipids are removed. These peptide surfactants are simple, versatile, effective, and affordable. They represent a designer molecular nanomaterial for use in studies of diverse elusive membrane proteins.

The supramolecular structure of the GPCR rhodopsin in solution and native disc membranes

Rhodopsin, the prototypical G-protein-coupled receptor, which is densely packed in the disc membranes of rod outer segments, was proposed to function as a monomer. However, a growing body of evidence indicates dimerization and oligomerization of numerous G-protein-coupled receptors, and atomic force microscopy images revealed rows of rhodopsin dimers in murine disc membranes. In this work we demonstrate by electron microscopy of negatively stained samples, blue native- and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, chemical crosslinking, and by proteolysis that native bovine rhodopsin exists mainly as dimers and higher oligomers. These results corroborate the recent findings from atomic force microscopy and molecular modeling on the supramolecular structure and packing arrangement of murine rhodopsin dimers.

Absolute quantification of the G protein-coupled receptor rhodopsin by LC/MS/MS using proteolysis product peptides and synthetic peptide standards.

Methods for the absolute quantification of a membrane protein are described using isotopically labeled or unlabeled synthetic peptides as standards. Synthetic peptides are designed to mimic peptides that are cleaved from target analyte proteins by proteolytic or chemical digestion, and the peptides selected serve as standards for quantification by LC/MS/MS on a triple quadrupole mass spectrometer. The technique is complementary to relative quantification techniques in widespread use by providing absolute quantitation of selected targets with greater sensitivity, dynamic range, and precision. Proteins that are found to be of interest by global proteome searches can be selected as targets for quantitation by the present method. This method has a much shorter analytical cycle time (minutes versus hours for the global proteome experiments), making it well suited for high-throughput environments. The present approach using synthetic peptides as standards, in conjunction with proteolytic or chemical cleavage of target proteins, allows mass spectrometry to be used as a highly selective detector for providing absolute quantification of proteins for which no standards are available. We demonstrate that quantification is simple and reliable for the integral membrane protein rhodopsin with reasonable recoveries for replicate experiments using low-micromolar solutions of rhodopsin from rod outer segments.

On the electrical conductivity of rhodopsin solutions

Measurements were made on the conductivity of digitonin extracts of frog rhodopsin with and without previous light exposure. The light-dark difference in conductivity is observed at low concentrations of rhodopsin and detergent.

Chromophore of a long-lived photoproduct formed with metarhodopsin III in the isolated frog retina.

Abstract Long‐lived photoproducts of frog rhodopsin in isolated retina and digitonin solution have been investigated by spectrophotometry and their chromophores have been analyzed by high‐pressure liquid chromatography (HPLC). By irradiation (> 560 nm) at 3°C and pH 8.6, a product analogous to metarhodopsin III (MIII) is formed, whose absorption maximum is at about 450 nm. This product decays more slowly than MIII does. The results of HPLC analysis indicate that the chromophore of this photoproduct is 7‐cis retinal and that of MIII is all‐trans retinal. The product possessing 7‐cis retinal is called 7‐cis photoproduct. The amount of 7‐cis isomer in rhodopsin solution irradiated at various temperatures between 15°C and –82°C, has been determined. The results suggest that the 7‐cis photoproduct can be formed by the photoconversion of lumirhodopsin and metarhodopsin I.

22] Purification of rhodopsin on hydroxyapatite columns, detergent exchange, and recombination with phospholipids

The purpose of this chapter is to describe the details of the purification of rhodopsin on hydroxyapatite columns and the preparation of recombinant rhodopsin—lipid vesicles. In this procedure, all buffers are at pH 6.6, and all manipulations involving rhodopsin are carried out under dim red light or in total darkness at 4° unless otherwise specified. Dodecyltrimethylammonium bromide (DTAB) is synthesized by refluxing excess anbydrous trimethylamine with dodecylbromide in methanol. The purification procedure for rhodopsin outlined is highly reproducible and satisfactory with DTAB but less so for other detergents, which may, however, be preferred for investigations subsequent to the purification. The DTAB solution of crude rhodopsin is immediately loaded on the hydroxyapatite column at 4° and eluted with a 300-ml linear gradient of 0–0.5 M sodium chloride. During the elution, the concentrations of phosphate, DTAB, and dithiothreitol are constant at the values used in the equilibration buffer. The purification procedure for rhodopsin outlined is highly reproducible and satisfactory with DTAB but less so for other detergents, which may, however, be preferred for investigations subsequent to the purification.

Principal absorption axes of rhodopsin and prelumirhodopsin

The experimental data on the absorption of plane polarized light by a solution of cattle rhodopsin at −196‡ C have been theoretically analysed to model the directional absorption properties of rhodopsin and prelumirhodopsin. It has been found that rhodopsin and prelumirhodopsin are planar absorbers having ratios of about 100∶7 and 100∶4, respectively, between the extinction coefficients along the long axis and perpendicular to it. These results support that the chromophore in prelumirhodopsin is more linear than the chromophore in rhodopsin.

Flash photolysis of cattle rhodopsin in solution: determination of kinetic activation parameters

Flash photolysis of cattle rhodopsin in solution has been analysed theoretically and the results are found to be in good agreement with the experimental results of Williams (1970). The values of various kinetic activation parameters ( i.e. activation energy, free energy of activation, enthalpy of activation, entropy of activation and the pre-exponential factor of Arrhenius equation) for the reactions metarhodopsin I → metarhodopsin I′, metarhodopsin I′ → metarhodopsin I, metarhodopsin I′ → metarhodopsin II and metarhodopsin II → metarhodopsin I′ have been determined. Also the quantum efficiency of the conversion of metarhodopsin I into rhodopsin has been determined.

The directional absorption properties of rhodopsin and its photoproducts.

The experimental data on the absorption of a plane polarised light by a solution of cattle rhodopsin at -196 degrees C have been theoretically analysed to model the directional absorption properties of rhodopsin and its photoproducts. It is seen that these molecules behave like planar absorbers having a ratio of about 100 : 7 between the extinction coefficients along the long axis and perpendicular to it. Using this result and the experimental observations on absorption and dichroism in the retina in situ, a model for the configuration of chromophores in the disc membranes has been derived. In this model the plane of the chromophore is perpendicular to that of the disc and the long axis of the chromophore makes an angle of 6.6 degrees with the plane of the disc. The solution of the problem depends on the assumption that the absorption axes are the same for the rhodopsin, prelumirhodopsin and isorhodopsin.

Responses of retinal rods to single photons.

1. A suction electrode was used to record the membrane current of single rod outer segments in pieces of toad retina. During dim illumination the membrane current showed pronounced fluctuations. 2. Amplitude histograms of responses to dim flashes of fixed intensity exhibited two discrete peaks, one at 0 pA and one near 1 pA, suggesting that the response was quantized. By setting a criterion amplitude level, flash responses could be classed as 'failures' (no response) or as 'successes' (at least one quantal event). 3. The variation of fraction of successes with flash intensity was consistent with the hypothesis that each quantal electrical event resulted from a single photoisomerization. 4. The quantal event had a mean amplitude of about 1 pA (5% of the standing dark current) and a standard deviation of 0.2 pA. Dispersion in the event amplitude prevented identification of histogram peaks corresponding to two or more photoisomerizations. 5. Individual quantal responses exhibited a smooth shape very similar to that of the average quantal response. This suggests that a single photoisomerization releases many particles of transmitter and that radial diffusion of internal transmitter is not a major source of delay in the light response. 6. The 'quantum efficiency' with which an absorbed photon generated an electrical event was measured as 0.5 +/- 0.1 (S.E. of mean, n = 4). This is slightly lower than the quantum efficiency of photoisomerization obtained previously for rhodopsin in solution. 7. At wavelengths between 420 and 700 nm the quantal event was invariant in size, although the cell's sensitivity varied over a range of 10(5). 8. The power spectrum of the fluctuations in dim steady light was predicted by assuming that a random series of quantal events occurred independently. 9. In brighter light the fluctuations were faster, and the response to an incremental flash was reduced in size and duration. The power spectrum could be predicted by assuming random superposition of events with the shape of the incremental flash response.

Flash photolysis of cattle rhodopsin in solution: determination of kinetic activation parameters

Flash photolysis of cattle rhodopsin in solution has been analysed theoretically and the results are found to be in good agreement with the experimental results of Williams (1970). The values of various kinetic activation parameters ( i.e. activation energy, free energy of activation, enthalpy of activation, entropy of activation and the pre-exponential factor of Arrhenius equation) for the reactions metarhodopsin I → metarhodopsin I′, metarhodopsin I′ → metarhodopsin I, metarhodopsin I′ → metarhodopsin II and metarhodopsin II → metarhodopsin I′ have been determined. Also the quantum efficiency of the conversion of metarhodopsin I into rhodopsin has been determined.

Biochemical aspects of the visual process. XXXIII. A convenient purification procedure of rhodopsin by means of affinity chromatography

An improved technique is described for the purification of the visual pigment rhodopsin by means of affinity chromatography over a concanavalin A-sepharose 4B column in the presence of the detergent dodecyltrimethylammoniumbromide. All phospholipids and all proteins other than rhodopsin appear to be separated from the rhodopsin during this purification procedure. The resulting concentrated rhodopsin solutions show excellent spectral properties. They are very suitable for the preparation of lipid- and detergent-free rhodopsin and for the assembly of lipid-rhodopsin vesicles, since the detergent can be removed by dialysis.

Molecular flow resonance Raman effect from retinal and rhodopsin.

We have performed resonance enhanced Raman measurements of retinal isomers in solution (all-trans, 11-cis, 9-cis, and 13-cis) and cetyltrimethylammonium bromide (CTAB) detergent extracts of bovine rhodopsin near physiological temperatures (17 degrees C). In order to measure these photolabile systems, we have developed a general technique which allows Raman measurements of any photosensitive material. This technique involves imposing a molecular velocity transverse to the Raman exciting laser beam sufficient to ensure that any given molecule moves through the beam so that it has little probability of absorbing a photon. We have also measured the resonance Raman spectra of crystals of the same retinal isomers. The data show that each isomer has a distinct and characteristic Raman spectra and that the spectrum of 11-cis-retinal is quite similar but not identical with that of rhodopsin and similarly for 9-cis-retinal compared with isorhodopsin. In agreement with previous work, the Raman data demonstrate that retinal and opsin are joined by a protonated Schiff base. Due to the fact that the Raman spectra of 11-cis-retinal (solution) and rhodopsin show bands near 998 and 1018 cm(-1), a spectral region previously assigned to C-Me stretching motions, it is suggested that 11-cis-retinal in solution is compased of a mixture of 12-s-trans and 12-s-cis, and that the conformation of rhodopsin is (perhaps distorted) 12-s-trans.

Effect of detergents on the conformation of spin-labeled rhodopsin

Rhodopsin was labeled with the spin probe 3 maleimido 2,2,5,5 tetramethyl-1-pyrrolidinyloxyl and studied with the electron spin resonance technique. Labeled rod outer segment membranes were solubilized by various detergents. The perturbations induced by detergent solubilization in the conformation of the spin labeled rhodopsin molecule can be classified as follows: digitonin, Triton X-100, emulphogene, cetyl trimethyl ammonium bromide, sodium dodecyl sulfate. Illumination of a rhodopsin solution produces larger changes in the electron spin resonance spectra than solubilization.

Upper limits to the bleaching of rhodopsin by high intensity flashes

Digitonin solutions of cattle rhodopsin are irradiated with intense flashes of various durations. Upper limits to bleaching are found which depend upon (1) the duration of the flash, (2) the temperature of the solution at the time of the flash, (3) the presence or absence of u.v. light in the flash. All flashes bleach no more than ca. 50 per cent at low temperatures and no more than ca. 88 per cent at high temperatures. Empirical equations are presented which describe the maximum bleach possible at various temperatures and it is proposed that these equations may prove helpful to those who wish to estimate the bleaching effectiveness of their flashes.

Dynamic aspects of metarhodopsin formation in photochemical cycle of extracted octopus rhodopsin.

Changes in the difference spectrum of the octopus rhodopsin solution with pH 10.3 were studied at about 2°C by applying the flash photolytic technique. The main peak (about 520nm) in the difference spectrum at 10 msec after a flash coincided with the peak of the calculated difference spectrum between rhodopsin and acid metarhodopsin. It was suggested that in the octopus rhodopsin cycle the acid metarhodopsin may be a precursor of the alkaline metarhodopsin even in alkaline solution.

Tunable laser resonance Raman spectroscopy of the visual process. I: The spectrum of rhodopsin

AbstractTunable laser resonance Raman spectra have been obained of a solution of bovine rhodopsin. The spectra were obtained without causing the cis‐trans isomerization of the retinylidene chromophore which is the primary photochemical act in vision. The spectra appear to be sensitive to the structure, the environment and the interactions of the retinylidene chromophore in its opsin matrix. The ease with which the spectra were obtained, the experimental flexibility of Raman spectroscopy and the apparent sensitivity of the spectra suggests a wide range of possible applications for resonance Raman spectroscopy in the elucidation of the principal molecular steps in vision.

Extraction, regeneration after bleaching, and ion-exchange chromatography of rhodopsin in Tween 80

Rhodopsin can be readily and somewhat, selectively extracted into Tween 80 solutions from the isolated photoreceptor particulate fraction of bovine retinal tissue. Approximately 80% of the rhodopsin is recovered from the particulate fraction with A 498 values of approximately 6 and spectral ratios ( A 278: A 498) of 1.8-1.9. The solutions are estimated to be approximately 97% pure based upon assay of protein and rhodopsin content and 98% pure based upon chromatography on DEAE-cellulose. The bulk of the rhodopsin can be regenerated after bleaching in Tween 80. Partial regenerability is retained when solutions of unbleached or bleached rhodopsin in Tween 80 are further purified by DEAE-cellulose chromatography.