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1.
Cross-linking of dark-adapted frog photoreceptor disk membranes. Evidence for monomeric rhodopsin. 总被引:1,自引:0,他引:1
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N W Downer 《Biophysical journal》1985,47(3):285-293
A model for random cross-linking of identical monomers diffusing in a membrane was formulated to test whether rhodopsin's cross-linking behavior was quantitatively consistent with a monomeric structure. Cross-linking was performed on rhodopsin both in intact retinas and in isolated rod outer segment (ROS) membranes using the reagent glutaraldehyde. The distribution of covalent oligomers formed was analyzed by SDS-polyacrylamide gel electrophoresis and compared to predictions for the random model. A similar analysis was made for ROS membranes cross-linked by diisocyanatohexane and retinas cross-linked by cupric ion complexed with o-phenanthroline. Patterns of cross-linking produced by these three reagents are reasonably consistent with the monomer model. Glutaraldehyde was also used to cross-link the tetrameric protein aldolase in order to verify that cross-linking of a stable oligomer, under conditions comparable to those used for ROS, yielded the pattern predicted for a tetrameric protein having D2 symmetry. This pattern is markedly different from the one for a random-collision model. Moreover, a comparison of rates showed that aldolase cross-linking with glutaraldehyde is significantly faster than cross-linking of membrane-bound rhodopsin. It is concluded that rhodopsin is monomeric in dark-adapted photoreceptor membranes and that the observed cross-linking results from collisions between diffusing rhodopsin molecules. 相似文献
2.
Absorbance difference spectra were recorded at 20 degrees C with a dense sequence of delay times from 1 to 128 micros after photolysis of lauryl maltoside suspensions of rhodopsin prepared from hypotonically washed bovine rod outer segments. Data were best fit by two-exponential components with a small, fast component (tau = 12 micros) occurring during the period that lumirhodopsin has been presumed to be stable. The shape of the spectral change corresponds to an approximately 2 nm red shift of the lumirhodopsin spectrum. Measurements with linearly polarized light verified that no absorbance changes associated with rotational diffusion were present in these preparations on this time scale, and experiments designed to enhance isorhodopsin production during photolysis showed no effect on the relative amplitude of the fast process. A similar process was previously observed in membrane suspensions of rhodopsin, but there the similarity of the change to rotational diffusion artifacts made conclusive identification of a second lumirhodopsin difficult. However, reexamination of polarized light measurements on rhodopsin in membrane supports the fact that the fast process seen here in detergent also takes place there. The new absorbance process occurs when time-resolved resonance Raman experiments have shown that the protonated Schiff base is moving from one hydrogen bond acceptor to another. The results are discussed in the context of possibly related processes on the same time scale that have been observed recently in artificial visual pigments with synthetic retinylidene chromophores and in a related rhodopsin mutant. The details of lumirhodopsin behavior are important because it is the last protonated Schiff base intermediate that occurs under physiological conditions. 相似文献
3.
Spin-label saturation-transfer electron spin resonance detection of transient association of rhodopsin in reconstituted membranes 总被引:2,自引:0,他引:2
Rotational diffusion of rhodopsin in reconstituted membranes of phosphatidylcholines of various alkyl chain lengths has been measured by using saturation-transfer electron spin resonance spectroscopy as a function of temperature and lipid/rhodopsin mole ratio. For dipalmitoyl-phosphatidylcholine, the rotational correlation time is 20 microseconds at physiological concentration, the same as in rod outer segment (ros) membranes. Dilution reduces the time to 10 microseconds, a value that is ascribed to well-dispersed monomeric rhodopsin. Use of phospholipids with longer or shorter chains results in sharply increased rotational correlation times. It is concluded that rhodopsin molecules are transiently associated in both reconstituted and ros membranes and that the nature of the association is determined by lipid type and composition. 相似文献
4.
Lakowicz JR 《Biophysical chemistry》1984,19(1):13-23
It is generally assumed that the rotational diffusion coefficients of fluorophores are independent of time subsequent to excitation, and that the rotational diffusion coefficients of the ground and the excited states are the same. We now describe a linkage between the extent of solvent relaxation and the rate of fluorescence depolarization. Specifically, if a fluorophore displays time-dependent solvent relaxation it may also show a time-dependent decrease in its rotational rate. A decreased rate of rotation could result from the increased interaction with polar solvent molecules which occurs as a result of solvent relaxation. The decays of anisotropy predicted from our model closely mimic those often observed for fluorophores which are bound to macromolecules. For example, the decays are more complex than a single exponential, and the time-resolved anisotropy can display a limiting value which does not decay to zero. The effect of solvent relaxation upon the rates of rotational diffusion is expected to be most dramatic for solvent-sensitive fluorophores in a viscous environment. These conditions are frequently encountered for fluorophore-macromolecule complexes. Consideration of the linkage between solvent relaxation and rotational diffusion leads to two unusual predictions. First even spherical fluorophores in an isotropic environment could display multi- or nonexponential decays of fluorescence anisotropy. Secondly, for the special case in which the fluorophore dipole moment decreases upon excitation, the theory predicts that the anisotropy decay rate may increase with time subsequent to pulsed excitation. The predictions of this theory are consistent with published data on the effects of red-edge excitation upon the apparent rotational rates of fluorophores in polar solvents. 相似文献
5.
Bovine rhodopsin has been reconstituted in seven different saturated diacylphosphatidylcholine species of odd and even chain lengths from C-12 to C-18 at a lipid/protein ratio (60:1 mol/mol) comparable to that in the native rod outer segment disk membrane. All recombinants were found to be photochemically active, in that optical bleaching produced a temperature- and lipid chain-length-dependent mixture of species absorbing at 480 and 380 nm. Both the rotational diffusion of rhodopsin and lipid-protein interactions in the various recombinants were studied by saturation transfer and conventional electron spin resonance spectroscopy of spin-labeled rhodopsin and of spin-labeled phosphatidylcholine, respectively. In the fluid lipid phase, the rotational diffusion rate of rhodopsin was found to be dependent on the lipid chain length of the different recombinants in a nonmonotonic manner. The diffusion rate in dilauroylphosphatidylcholine was found to be very slow, indicating extensive protein aggregation, whereas that in dipentadecanoylphosphatidylcholine was rapid (effective correlation time ca. 7 microseconds), consistent with the presence of monomeric protein. For recombinants with longer lipid chain lengths, the rotational diffusion rate again decreased, indicating the presence of di- or oligomeric protein. The fraction of lipid motionally restricted at temperatures in the fluid phase was also dependent on the chain length of the phosphatidylcholine used in the reconstitution.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
6.
Understanding the mechanism of G-protein coupled receptors action is of major interest for drug design. The visual rhodopsin is the prototype structure for the family A of G-protein coupled receptors. Upon photoisomerization of the covalently bound retinal chromophore, visual rhodopsins undergo a large-scale conformational change that prepares the receptor for a productive interaction with the G-protein. The mechanism by which the local perturbation of the retinal cis-trans isomerization is transmitted throughout the protein is not well understood. The crystal structure of the visual rhodopsin from squid solved recently suggests that a chain of water molecules extending from the retinal toward the cytoplasmic side of the protein may play a role in the signal transduction from the all-trans retinal geometry to the activated receptor. As a first step toward understanding the role of water in rhodopsin function, we performed a molecular dynamics simulation of squid rhodopsin embedded in a hydrated bilayer of polyunsaturated lipid molecules. The simulation indicates that the water molecules present in the crystal structure participate in favorable interactions with side chains in the interhelical region and form a persistent hydrogen-bond network in connecting Y315 to W274 via D80. 相似文献
7.
8.
In fluid membranes, mobile molecules are thought to collide at high frequencies. Concern has been expressed as to whether these colliding molecules are cross-linked during the chemical cross-linking of membrane molecules, thereby creating problems in interpreting such experiments. Hemoglobin was used as a model to test this possibility. Oligomers larger than the tetramer could be cross-linked depending on factors such as hemoglobin concentration, duration of the cross-linking reaction and the type of reagent. Under certain conditions, however, such as a hemoglobin concentration less than 150 μM or a duration of cross-linking shorter than 15 min, larger oligomers were not detectable. Analysis of these data suggests that the probability of random collisional cross-links under normal conditions is insignificant. 相似文献
9.
In the visual process, one photoexcited rhodopsin (R*) catalyzes the activation of hundreds of G-proteins. It remains to be determined whether G-protein and R* find one another by membrane surface diffusion of these components (diffusion model) or by diffusion of G-protein through the aqueous phase (hopping model). A monolayer of each main rod outer segment (ROS) phospholipid interacting with a subphase containing G-protein, has been used to simulate the interaction of G-protein with the cytoplasmic surface of discal membranes. The possible diffusion of G-protein through the aqueous phase was then measured by observing its adsorption-desorption in the monolayer of each main ROS phospholipid. From examination of surface pressure and ellipsometric isotherms at the nitrogen-water interface, we have determined that once incorporated into the monolayer, the G-protein remains associated, independent of surface pressure, thus providing evidence against the hopping model. 相似文献
10.
All-trans retinol in rod photoreceptor outer segments moves unrestrictedly by passive diffusion
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The visual pigment protein of vertebrate rod photoreceptors, rhodopsin, contains an 11-cis retinyl moiety that is isomerized to all-trans upon light absorption. Subsequently, all-trans retinal is released from the protein and reduced to all-trans retinol, the first step in the recycling of rhodopsin's chromophore group through the series of reactions that constitute the visual cycle. The concentration of all-trans retinol in photoreceptor outer segments can be monitored from its fluorescence. We have used two-photon excitation (720 nm) of retinol fluorescence and fluorescence recovery after photobleaching to characterize the mobility of all-trans retinol in frog photoreceptor outer segments. Retinol produced after rhodopsin bleaching moved laterally in the disk membrane bilayer with an apparent diffusion coefficient of 2.5 +/- 0.3 micro m(2) s(-1). The diffusion coefficient of exogenously added retinol was 3.2 +/- 0.5 micro m(2) s(-1). These diffusion coefficients are in close agreement with those reported for lipids, suggesting that retinol is not tightly bound to protein sites that would be diffusing much more slowly in the plane of the membrane. In agreement with this interpretation, a fluorescent-labeled C-16 fatty acid diffused laterally with a similar diffusion coefficient, 2.2 +/- 0.2 micro m(2) s(-1). Retinol also moved along the length of the rod outer segment, with an apparent diffusion coefficient of 0.07 +/- 0.01 micro m(2) s(-1), again suggesting that it is not tightly bound to proteins that would confine it to the disks. The axial diffusion coefficient of exogenously added retinol was 0.05 +/- 0.01 micro m(2) s(-1). In agreement with passive diffusion, the rate of axial movement was inversely proportional to the square of the length of the rod outer segment. Diffusion of retinol on the plasma membrane of the outer segment can readily account for the measured value of the axial diffusion coefficient, as the plasma membrane comprises approximately 1% of the total outer-segment membrane. The values of both the lateral and axial diffusion coefficients are consistent with most of the all-trans retinol in the outer segments moving unrestricted and not being bound to carrier proteins. Therefore, and in contrast to other steps of the visual cycle, there does not appear to be any specialized processing for all-trans retinol within the rod outer segment. 相似文献
11.
The effect of illumination on the electrical conductance of rhodopsin solutions 总被引:1,自引:1,他引:1
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HARA T 《The Journal of general physiology》1958,41(5):857-877
An apparatus was constructed in order to record continuously and simultaneously changes in extinction and electrical conductance of rhodopsin solutions. With this apparatus, changes in electrical conductance on exposing rhodopsin to light were investigated. On illumination solutions of rhodopsin revealed a conductance change so long as they preserved their photosensitivity. The conductance change begins almost immediately upon illumination and is almost proportional to the amount of rhodopsin decomposed, continuing until rhodopsin is converted to indicator yellow. Near pH 7 the conductance is apt to increase slightly, while it decreases considerably outside the range of pH 6–9, being accompanied by a pH change towards neutrality. The conductance change is regarded as an essential property of rhodopsin, because it occurs in aqueous suspension as well as in digitonin solution; it may be caused by hydrogen or hydroxyl ions and some other conductive substances. It is also noteworthy that the petroleum ether-soluble component of the rod outer segments—presumably the lipide—tends to increase the conductance change. In suspensions of rod outer segments and retinal homogenates, the conductance increases on illumination irrespective of pH: this may be due to secondary reactions following the photic reaction of rhodopsin. We shall discuss the significance of the conductance change in relation to the initiation of visual excitation. 相似文献
12.
Calcium regulates the rate of rhodopsin disactivation and the primary amplification step in visual transduction 总被引:3,自引:0,他引:3
The kinetics of the light-induced activation of transducin as well as the subsequent disactivation process can be monitored by means of a specific light scattering transient PA. In this communication it is demonstrated that the rate of transducin disactivation is calcium dependent, increasing when the calcium concentration is decreased. As a consequence of the accelerated recovery in low calcium, the time to the peak of the transducin activation process is shortened and the gain of the primary amplification step, i.e. the number of transducin molecules activated per bleached rhodopsin, is reduced. Experiments using hydroxylamine as an artificial quencher of rhodopsin activity suggest that calcium acts upon rhodopsin kinase and not upon the rate of the GTPase. This would indicate that calcium may control visual adaptation not only by regulating guanine cyclase activity, but also by affecting the primary step in the transduction cascade, the rhodopsin-transducin coupling. 相似文献
13.
Changes in cGMP concentration correlate with some, but not all, aspects of the light-regulated conductance of frog rod photoreceptors 总被引:2,自引:0,他引:2
R H Cote G D Nicol S A Burke M D Bownds 《The Journal of biological chemistry》1986,261(28):12965-12975
Cyclic GMP has been implicated in controlling the light-regulated conductance of rod photoreceptors of the vertebrate retina. However, there is little direct evidence correlating changes in cGMP concentration with the light-regulated permeability mechanism in living cells. A preparation of intact frog rod outer segments suspended in a Ringer's medium containing low Ca2+ has been used to demonstrate that initial changes in total cellular cGMP concentration parallel changes in the light-regulated membrane current over a wide range of light intensities. At light intensities bleaching from 160 to 5.6 X 10(6) rhodopsin molecules/rod/s, decreases in the response latency for the cGMP kinetics parallel decreases in the latent period of the electrical response. Further, changes in the rate of the cGMP decrease parallel the rate of membrane current suppression as the light intensity is varied. Up to 10(5) cGMP molecules are hydrolyzed per photolyzed rhodopsin, consistent with in vitro studies showing that each bleached rhodopsin can activate over 100 phosphodiesterase molecules. Addition of the Ca2+ ionophore, A23187, does not affect the initial kinetics of the cGMP decrease or of the electrical response, excluding a direct role for Ca2+ in the initial events of phototransduction. These results are consistent with cGMP being the intracellular messenger that links rhodopsin isomerization with changes in membrane permeability upon illumination. It is unlikely, however, that light-induced changes in total cGMP concentration are the sole regulators of membrane current. This is suggested by several observations: at bright light intensities, the subsecond light-induced cGMP decrease is essentially complete prior to complete suppression of membrane current; maximal light-induced decreases in cGMP concentration occur at all light intensities tested, whereas the extent of membrane current suppression varies over the same range of light intensities; changing the external Ca2+ concentration from 1 mM to 10 nM in the dark causes an increase in membrane current that is significantly more rapid than corresponding changes in cGMP concentration. Thus, light-induced changes in total cellular cGMP concentration correlate with some, but not all, aspects of the visual excitation process in vertebrate photoreceptors. 相似文献
14.
K Palczewski J P Van Hooser G G Garwin J Chen G I Liou J C Saari 《Biochemistry》1999,38(37):12012-12019
Photoisomerization of 11-cis-retinal to all-trans-retinal and reduction to all-trans-retinol occur in photoreceptor outer segments whereas enzymatic esterification of all-trans-retinol, isomerization to 11-cis-retinol, and oxidation to 11-cis-retinal occur in adjacent cells. The processes are linked into a visual cycle by intercellular diffusion of retinoids. Knowledge of the mechanistic aspects of the visual cycle is very limited. In this study, we utilize chemical analysis of visual cycle retinoids to assess physiological roles for components inferred from in vitro experiments and to understand why excised mouse eyes fail to regenerate their bleached visual pigment. Flash illumination of excised mouse eyes or eyecups, in which regeneration of rhodopsin does not occur, produced a block in the visual cycle after all-trans-retinal formation; constant illumination of eyecups produced a block in the cycle after all-trans-retinol formation; and constant illumination of whole excised eyes resulted in a block of the cycle after formation of all-trans-retinyl ester. These blocks emphasize the role of cellular metabolism in the visual cycle. Interphotoreceptor retinoid-binding protein (IRBP) has been postulated to play a role in intercellular retinoid transfer in the retina; however, the rates of recovery of 11-cis-retinal and of regeneration of rhodopsin in the dark in IRBP-/- mice were very similar to those found with wild-type (wt) mice. Thus, IRBP is necessary for photoreceptor survival but is not essential for a normal rate of visual pigment turnover. Arrestin forms a complex with activated rhodopsin, quenches its activity, and affects the release of all-trans-retinal in vitro. The rate of recovery of 11-cis-retinal in arrestin-/- mice was modestly delayed relative to wt, and the rate of rhodopsin recovery was approximately 80% of that observed with wt mice. Thus, the absence of arrestin appeared to have a minor effect on the kinetics of the visual cycle. 相似文献
15.
The condensation of retinene1 with opsin to form rhodopsin is optimal at pH about 6, a pH which favors the condensation of retinene1 with sulfhydryl rather than with amino groups. The synthesis of rhodopsin, though unaffected by the less powerful sulfhydryl reagents, monoiodoacetic acid and its amide, is inhibited completely by p-chloromercuribenzoate (PCMB). This inhibition is reversed in part by the addition of glutathione. PCMB does not attack rhodopsin itself, nor does it react with retinene1. Its action in this system is confined to the —SH groups of opsin. Under some conditions the synthesis of rhodopsin is aided by the presence of such a sulfhydryl compound as glutathione, which helps to keep the —SH groups of opsin free and reduced. By means of the amperometric silver titration of Kolthoff and Harris, it is shown that sulfhydryl groups are liberated in the bleaching of rhodopsin, two such groups for each retinene1 molecule that appears. This is true equally of rhodopsin from the retinas of cattle, frogs) and squid. The exposure of new sulfhydryl groups adds an important element to the growing evidence that relates the bleaching of rhodopsin to protein denaturation. The place of sulfhydryl groups in the structure of rhodopsin is still uncertain. They may be concerned directly in binding the chromophore to opsin; or alternatively they may furnish hydrogen atoms for some reductive change by which the chromophore is formed from retinene1. In the amperometric silver titration, the bleaching of rhodopsin yields directly an electrical variation. This phenomenon may have some fundamental connection with the role of rhodopsin in visual excitation, and may provide a model of the excitation process in general. 相似文献
16.
Protein structural change at the cytoplasmic surface as the cause of cooperativity in the bacteriorhodopsin photocycle. 总被引:3,自引:1,他引:2
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The effects of excitation light intensity on the kinetics of the bacteriorhodopsin photocycle were investigated. The earlier reported intensity-dependent changes at 410 and 570 nm are explained by parallel increases in two of the rate constants, for proton transfers to D96 from the Schiff base and from the cytoplasmic surface, without changes in the others, as the photoexcited fraction is increased. Thus, it appears that the pKa of D96 is raised by a cooperative effect within the purple membrane. This interpretation of the wild-type kinetics was confirmed by results with several mutant proteins, where the rates are well separated in time and a model-dependent analysis is unnecessary. Based on earlier results that demonstrated a structural change of the protein after deprotonation of the Schiff base that increases the area of the cytoplasmic surface, and the effects of high hydrostatic pressure and lowered water activity on the photocycle steps in question, we suggest that the pKa of D96 is raised by a lateral pressure that develops when other bacteriorhodopsin molecules are photoexcited within the two-dimensional lattice of the purple membrane. Expulsion of no more than a few water molecules bound near D96 by this pressure would account for the calculated increase of 0.6 units in the pKa. 相似文献
17.
Protein-bound water molecules play crucial roles in the structure and function of proteins. The functional role of water molecules has been discussed for rhodopsin, the light sensor for twilight vision, on the basis of X-ray crystallography, Fourier transform infrared (FTIR) spectroscopy, and a radiolytic labeling method, but nothing is known about the protein-bound waters in our color visual pigments. Here we apply low-temperature FTIR spectroscopy to monkey red (MR)- and green (MG)-sensitive color pigments at 77 K and successfully identify water vibrations using D(2)O and D(2)(18)O in the whole midinfrared region. The observed water vibrations are 6-8 for MR and MG, indicating that several water molecules are present near the retinal chromophore and change their hydrogen bonds upon retinal photoisomerization. In this sense, color visual pigments possess protein-bound water molecules essentially similar to those of rhodopsin. The absence of strongly hydrogen-bonded water molecules (O-D stretch at <2400 cm(-1)) is common between rhodopsin and color pigments, which greatly contrasts with the case of proton-pumping microbial rhodopsins. On the other hand, two important differences are observed in water signal between rhodopsin and color pigments. First, the water vibrations are identical between the 11-cis and 9-cis forms of rhodopsin, but different vibrational bands are observed at >2550 cm(-1) for both MR and MG. Second, strongly hydrogen-bonded water molecules (2303 cm(-1) for MR and 2308 cm(-1) for MG) are observed for the all-trans form after retinal photoisomerization, which is not the case for rhodopsin. These specific features of MR and MG can be explained by the presence of water molecules in the Cl(-)-biding site, which are located near positions C11 and C9 of the retinal chromophore. The averaged frequencies of the observed water O-D stretching vibrations for MR and MG are lower as the λ(max) is red-shifted, suggesting that water molecules are involved in the color tuning of our vision. 相似文献
18.
1. The proposed models of two independent groups, which relate the different states of the visual pigment to the excitation of the membrane in invertebrate photoreceptors (with particular reference to the prolonged depolarising afterpotential, the PDA) are compared and evaluated. 2. The validity of the late receptor potential (the "normal" receptor response) as an index of photoreceptor sensitivity, i.e., an index of the number of rhodopsin to metarhodopsin transitions, is verified by concurrent spectrophotometry. 3. Electrophysiological observations alone allow the calculation of 1.3 x 10(8) photopigment molecules in the rhabdom of an R1-6 photoreceptor of a vitamin A-bred Calliphora. 4. The PDA is shown to be quantifiable in terms of the number of rhodopsin to metarhodopsin conversions by the absorption of single light quanta. 5. The comparison of discrete membrane fluctuations (quantum bumps) during the PDA and during exposure to sustained light stimuli that mimic the PDA suggest that, the PDA, similar to the late receptor potential, may be due to the summation of quantum bumps. 相似文献
19.
Two types of experiments indicate that light adaptation and excitation are initiated by the same, rather than different, populations of visual pigment. (a) The criterion action spectra of light adaptation and excitation are the same. (b) Increment-threshold curves were measured with a voltage-clamp technique under conditions of high and low concentration of plasma membrane rhodopsin (Rhpm). SD, the dark-adapted sensitivity, and 1/I2, the inverse of the background irradiance that desensitized by 0.3 log units, underwent the same fractional change when the rhodopsin concentration was changed. Both quantities appear to be linearly related to Rhpm. Reversible reductions in Rhpm were achieved by orange irradiation during a brief increase of extracellular pH from 7.8 to 10. This procedure would be unlikely to produce similar concentration changes in a hypothetical intracellular pigment because the concurrent change in intracellular pH, measured using the dye, phenol red, was only 0.45 pH units. It is thus unlikely that an intracellular pigment initiates light adaptation. On the assumption that light adaptation is mediated by a light-induced release of Ca++ from an intracellular store. the results reported here imply that an intracellular transmitter is needed to couple Rhpm to the intracellular store. 相似文献
20.
G protein-coupled receptors form dimers and higher-order oligomers in membranes, but the precise mode of receptor-receptor interaction remains unknown. To probe the intradimeric proximity of helix 8 (H8), we conducted chemical cross-linking of endogenous cysteines in rhodopsin in disk membranes. We identified a Cys316-Cys316 cross-link using partial proteolysis and liquid chromatography with mass spectrometry. These results show that a symmetric dimer interface mediated by H1 and H8 contacts is present in native membranes. 相似文献