Light-induced protein conformational changes in the photolysis of octopus rhodopsin.

Light-induced protein conformational changes in the photolysis of octopus rhodopsin were measured with a highly sensitive time-resolved transient UV absorption spectrophotometer with nanosecond time resolution. A negative band around 280 nm in the lumirhodopsin minus rhodopsin spectra suggests that alteration of the environment of some of the tryptophan residues has taken place before the formation of lumirhodopsin. A small recovery of the absorbance at 280 nm was observed in the transformation of lumirhodopsin to mesorhodopsin. Kinetic parameters suggest that major conformational changes have taken place in the transformation of mesorhodopsin to acid metarhodopsin. In this transformation, drastic changes of amplitude and a shift of a difference absorption band around 280 nm take place, which suggest that some of the tryptophan residues of rhodopsin become exposed to a hydrophilic environment.     

Discussion to II. Light-induced conformational changes of the rhodopsin molecule

European Biophysics Journal -     

Localization of light-induced conformational changes in bovine rhodopsin

Conformational changes in the extradiscal regions of rhodopsin induced by illumination were investigated by modifying the visual pigment by mild treatment with cyanogen bromide prior to and after light exposure. Light induced an increased yield of cleavage of the Met bond 253-254 and a new cleavage at the Met bond 155-156 of the rhodopsin polypeptide chain. These residues, located at the beginnings of the membrane-buried helices 6 and 4, respectively, were concluded to become extradiscally exposed upon illumination.     

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.     

Agonist-induced conformational changes in bovine rhodopsin: insight into activation of G-protein-coupled receptors

Activation of G-protein-coupled receptors (GPCRs) is initiated by conformational changes in the transmembrane (TM) helices and the intra- and extracellular loops induced by ligand binding. Understanding the conformational changes in GPCRs leading to activation is imperative in deciphering the role of these receptors in the pathology of diseases. Since the crystal structures of activated GPCRs are not yet available, computational methods and biophysical techniques have been used to predict the structures of GPCR active states. We have recently applied the computational method LITiCon to understand the ligand-induced conformational changes in β₂-adrenergic receptor by ligands of varied efficacies. Here we report a study of the conformational changes associated with the activation of bovine rhodopsin for which the crystal structure of the inactive state is known. Starting from the inactive (dark) state, we have predicted the TM conformational changes that are induced by the isomerization of 11-cis retinal to all-trans retinal leading to the fully activated state, metarhodopsin II. The predicted active state of rhodopsin satisfies all of the 30 known experimental distance constraints. The predicted model also correlates well with the experimentally observed conformational switches in rhodopsin and other class A GPCRs, namely, the breaking of the ionic lock between R135^3.50 at the intracellular end of TM3 (part of the DRY motif) and E247^6.30 on TM6, and the rotamer toggle switch on W265^6.48 on TM6. We observe that the toggling of the W265^6.48 rotamer modulates the bend angle of TM6 around the conserved proline. The rotamer toggling is facilitated by the formation of a water wire connecting S298^7.45, W265^6.48 and H211^5.46. As a result, the intracellular ends of TMs 5 and 6 move outward from the protein core, causing large conformational changes at the cytoplasmic interface. The predicted outward movements of TM5 and TM6 are in agreement with the recently published crystal structure of opsin, which is proposed to be close to the active-state structure. In the predicted active state, several residues in the intracellular loops, such as R69, V139^3.54, T229, Q237, Q239, S240, T243 and V250^6.33, become more water exposed compared to the inactive state. These residues may be involved in mediating the conformational signal from the receptor to the G protein. From mutagenesis studies, some of these residues, such as V139^3.54, T229 and V250^6.33, are already implicated in G-protein activation. The predicted active state also leads to the formation of new stabilizing interhelical hydrogen-bond contacts, such as those between W265^6.48 and H211^5.46 and E122^3.37 and C167^4.56. These hydrogen-bond contacts serve as potential conformational switches offering new opportunities for future experimental investigations. The calculated retinal binding energy surface shows that binding of an agonist makes the receptor dynamic and flexible and accessible to many conformations, while binding of an inverse agonist traps the receptor in the inactive state and makes the other conformations inaccessible.     

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.Presented at the EMBO-Workshop on Transduction Mechanism of Photoreceptors, Jülich, Germany, October 4–8, 1976     

Electron spin resonance study of light-induced conformational changes in nitroxide labelled bovine rhodopsin

Bovine rhodopsin was isolated in the unbleached form as a retinal disc membrane suspension and spin-labelled with 4-maleimido-2,2,6,6-tetramethylpiperidine-N-oxyl. Both conventional and saturation transfer electron spin resonance methods were used to investigate the sensitivity of the spin-label to conformational changes of rhodopsin induced by both transient and long-term exposure to light. The results indicate that the ESR methods do display sensitivity to such changes. An exponential decay curve with a time constant of 10 s was obtained by following the height of a single peak in the saturation transfer electron spin resonance spectrum in response to a single light flash.     

Light-induced conformational changes in full-length Arabidopsis thaliana cryptochrome

Cryptochromes (CRYs) are widespread flavoproteins with homology to photolyases (PHRs), a class of blue-light-activated DNA repair enzymes. Unlike PHRs, both plant and animal CRYs have a C-terminal domain. This cryptochrome C-terminal (CCT) domain mediates interactions with other proteins, while the PHR-like domain converts light energy into a signal via reduction and radical formation of the flavin adenine dinucleotide cofactor. However, the mechanism by which the PHR-like domain regulates the CCT domain is not known. Here, we applied the pulsed-laser-induced transient grating method to detect conformational changes induced by blue-light excitation of full-length Arabidopsis thaliana cryptochrome 1 (AtCRY1). A significant reduction in the diffusion coefficient of AtCRY1 was observed upon photoexcitation, indicating that a large conformational change occurs in this monomeric protein. AtCRY1 containing a single mutation (W324F) that abolishes an intra-protein electron transfer cascade did not exhibit this conformational change. Moreover, the conformational change was much reduced in protein lacking the CCT domain. Thus, we conclude that the observed large conformational changes triggered by light excitation of the PHR-like domain result from C-terminal domain rearrangement. This inter-domain modulation would be critical for CRYs' ability to transduce a blue-light signal into altered protein-protein interactions for biological activity. Lastly, we demonstrate that the transient grating technique provides a powerful method for the direct observation and understanding of photoreceptor dynamics.     

Phosphorylated rhodopsin and heparin induce similar conformational changes in arrestin

Photoactivated rhodopsin is quenched upon its phosphorylation in the reaction catalyzed by rhodopsin kinase and the subsequent binding of a regulatory protein, arrestin. We have found that heparin and other polyanions compete with photoactivated, phosphorylated rhodopsin to bind arrestin (48-kDa protein, S-antigen). This is shown (a) by the suppression of stabilized metarhodopsin II; (b) by changes in the digestion of arrestin in the presence of heparin; and (c) by the restoration of arrestin-quenched phosphodiesterase activity. When bound to arrestin, heparin also mimics phosphorylated rhodopsin by similarly exposing arrestin to limited proteolysis. We conclude that heparin and rhodopsin have similar means of binding to arrestin, and we propose a cationic region of arrestin (beginning with Lys163 of the bovine sequence) as the interaction site. In agreement with previous kinetic data we interpret the results in terms of a binding conformation of arrestin which is stabilized by rhodopsin or heparin and is open to proteolytic attack.     

Light-induced changes in the structure and accessibility of the cytoplasmic loops of rhodopsin in the activated MII state

Bovine rhodopsin was specifically labeled on the cytoplasmic surface at cysteine 140 (the first residue of the loop connecting helices III and IV) or at cysteine 316 (in the loop connecting helix VII and the palmitoylation sites) with the fluorescent labels fluorescein and Texas Red. These loops are involved in activation and signal transduction. The time-resolved fluorescence depolarization was measured in the dark state and in the M(II) state, with labeled samples consisting of rhodopsin-octylglucoside micelles or rod outer segment (ROS) membranes. In this way the diffusional dynamics of the flexible loops of rhodopsin were measured for the first time directly on the nanosecond time scale. Control experiments showed that the large number of weak excitation pulses required in these single photon counting experiments leads to <5% bleaching of the sample. Rhodopsin was trapped in the activated M(II) state for the duration of the fluorescence experiments ( approximately 20 min) after illumination at pH 6 and 5 degrees C. For both types of samples and at both labeled positions the dynamics of the label and loop motion as monitored by the time constants of the depolarization were not significantly different in the two states of the receptor. The end-anisotropy increased, however, from 0.09 in the dark to 0.16 in the M(II) state for ROS samples labeled at C140. The corresponding numbers for the C316 position are 0.06 and 0.12. Light-induced activation in M(II) is thus associated with a large increase in the loop steric hindrance due to a changed loop domain structure on the cytoplasmic surface. These results are supported by fluorescence quenching experiments with I(-), which indicate a significant decrease in the collisional quenching constant k(q) and in accessibility in the M(II) state at both positions. The rotational correlation time of the rhodopsin micelles increased from 48 ns in the dark state to 60 ns in M(II). This increase is caused by a change in volume and/or shape and is consistent with a structural change. These results demonstrate that time-resolved fluorescence depolarization is a powerful tool to study the changes in conformation and dynamics of the cytoplasmic loops that accompany the activation of rhodopsin and other G-protein coupled receptors.     

Structural changes of water molecules during the photoactivation processes in bovine rhodopsin

Internal water molecules of rhodopsins play an important role in stabilizing the crucial ion pair comprised by the protonated retinal Schiff base and its counterion. Previous low-temperature FTIR spectroscopy of archaeal rhodopsins observed water O-D stretching vibrations at 2400-2100 cm(-1) in D(2)O, corresponding to strong hydrogen bonds. Since a water molecule bridges the protonated Schiff base and an aspartate in archaeal rhodopsins, the observed water molecules presumably hydrate the negative charges in the Schiff base region. In contrast, the FTIR spectroscopy data of bovine rhodopsin presented here revealed that there are no spectral changes of water molecules under strongly hydrogen-bonding conditions (in the range <2400 cm(-1) for O-D stretch) during the photoactivation processes. The only observed water bands were located in the >2500 cm(-1) region that corresponds to weak hydrogen bonding. These results imply that the ion pair state in vertebrate visual rhodopsins is stabilized in a manner different from that in archaeal rhodopsins. In addition, the internal water molecules that hydrate the negative charges do not play important role in the photoactivation processes of rhodopsin that involve proton transfer from the Schiff base to Glu113 upon formation of Meta II. Structural changes of the H-D exchangeable peptide amide of a beta-sheet are observed upon formation of metarhodopsin II, suggesting that motion of a beta-sheet is coupled to the proton transfer reaction from the Schiff base to its counterion.     

Light-induced conformational changes in photosynthetic reaction centers: redox-regulated proton pathway near the dimer

The influence of the hydrogen bonds on the light-induced structural changes were studied in the wild type and 11 mutants with different hydrogen bonding patterns of the primary electron donor of reaction centers from Rhodobacter sphaeroides. Previously, using the same set of mutants at pH 8, a marked light-induced change of the local dielectric constant in the vicinity of the dimer was reported in wild type and in mutants retaining Leu L131 that correlated with the recovery kinetics of the charge-separated state [ Deshmukh et al. (2011) Biochemistry, 50, 340-348]. In this work after prolonged illumination the recovery of the oxidized dimer was found to be multiphasic in all mutants. The fraction of the slowest phase, assigned to a recovery from a conformationally altered state, was strongly pH dependent and found to be extremely long at room temperature, at pH 6, with rate constants of ～10(-3) s(-1). In wild type and in mutants with Leu at L131 the very long recovery kinetics was coupled to a large proton release at pH 6 and a decrease of up to 79 mV of the oxidation potential of the dimer. In contrast, in the mutants carrying the Leu to His mutation at the L131 position, only a negligible fraction of the dimer exhibited lowered potential, the large proton release was not observed, the oxidized dimer recovered 1 or 2 orders of magnitude faster depending on the pH, and the very long-lived state was not or barely detectable. These results are modeled as arising from the loss of a proton pathway from the bacteriochlorophyll dimer to the solvent when His is present at the L131 position.     

Light-induced conformational changes in photosynthetic reaction centers: dielectric relaxation in the vicinity of the dimer

Conformational changes near the bacteriochlorophyll dimer induced by continuous illumination were identified in the wild type and 11 different mutants of reaction centers from Rhodobacter sphaeroides. The properties of the bacteriochlorophyll dimer, which has a different hydrogen bonding pattern with the surrounding protein in each mutant, were characterized by steady-state and transient optical spectroscopy. After illumination for 1 min, in the absence of the secondary quinone, the recovery of the charge-separated states was nearly 1 order of magnitude slower in one group of mutants including the wild type than in the mutants carrying the Leu to His mutation at the L131 position. The slower recovery was accompanied by a substantial decrease in the electrochromic absorption changes associated with the Q(y) bands of the nearby monomers during the illumination. The other set of mutants containing the Leu L131 to His substitution exhibited slightly altered electrochromic changes that decreased only half as much during the illumination as in the other family of mutants. The correlation between the recovery of the charge-separated states in the light-induced conformation and the electrochromic absorption changes suggests a dielectric relaxation of the protein that stabilizes the charge on the dimer.     

Acid pH-induced conformational changes in bovine liver rhodanese.

The enzyme rhodanese is greatly stabilized in the range pH 4-6, and samples at pH 5 are fully active after several days at 23 degrees C. This is very different from results at pH greater than 7, where there is significant loss of activity within 1 h. A pH-dependent conformational change occurs below pH 4 in a transition centered around pH 3.25 that leads slowly to inactive rhodanese at pH 3 (t 1/2 = 22 min at pH3). The inactive rhodanese can be reactivated by incubation under conditions required for detergent-assisted refolding of denatured rhodanese. The inactive enzyme at pH 3 has the maximum of its intrinsic fluorescence spectrum shifted to 345 nm from 335 nm, which is characteristic of native rhodanese at pH greater than 4. At pH 3, rhodanese shows increased exposure of organized hydrophobic surfaces as measured by 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid binding. The secondary structure is maintained over the entire pH range studied (pH 2-7). Fluorescence anisotropy measurements of the intrinsic fluorescence provide evidence suggesting that the pH transition produces a state that does not display greatly increased average flexibility at tryptophan residues. Pepsin digestibility of rhodanese follows the pH dependence of conformational changes reported by activity and physical methods. Rhodanese is resistant to proteolysis above pH 4 but becomes increasingly susceptible as the pH is lowered. The form of the enzyme at pH 3 is cleaved at discrete sites to produce a few large fragments. It appears that pepsin initially cleaves close to one end of the protein and then clips at additional sites to produce species of a size expected for the individual domains into which rhodanese is folded. Overall, it appears that in the pH range between pH 3 and 4, titration of groups on rhodanese leads to opening of the structure to produce a conformation resembling, but more rigid than, the molten globule state that is observed as an intermediate during reversible unfolding of rhodanese.     

Light-induced conformational change in rhodopsin detected by modification of G-protein binding, GTP gamma S binding and cGMP phosphodiesterase activation

CNBr treatment of rod outer segments was performed in dark and in light conditions. With the subsequent modified rhodopsin and opsin the cGMP phosphodiesterase activation system was reconstituted. The recombination systems exhibited greatly reduced G-protein binding, GTP gamma S binding and cGMP phosphodiesterase activation. The reduction in activity of these three steps of the PDE activation cascade is most significant with modified opsin and is shown to be due to its inability to bind the G alpha subunit. The correlation between the localization of CNBr cleavage in dark and light conditions and these results is strongly indicative that a light-induced conformational change occurs in two extradiscal regions of rhodopsin.     

Light-induced conformational changes of cyanobacterial phytochrome Cph1 probed by limited proteolysis and autophosphorylation

Photoreceptor chromoproteins undergo light-induced conformational changes that result in a modulation of protein interaction and enzymatic activity. Bacterial phytochromes such as Cph1 from the cyanobacterium Synechocystis PCC 6803 are light-regulated histidine kinases in which the light signal is transferred from the N-terminal chromophore module to the C-terminal kinase module. In this study, purified recombinant Cph1 was subjected to limited proteolysis using trypsin and endoproteinase Glu-C (V8). Cleavage sites of chromopeptide fragments were determined by MALDI-TOF and micro-HPLC on-line with tandem mass spectrometry in an ion trap mass spectrometer. Trypsin produced three major chromopeptides, termed F1 (S56 to R520), F2 (T64 to R472), and F3 (L81 to R472). F1 was produced only in the far-red absorbing form Pfr within 15 min and remained stable up to >1 h; F2 and F3 were obtained in the red-light absorbing form Pr within ca. 5-10 min. When F1 was photoconverted to Pr in the presence of trypsin, this fragment degraded to F2 and F3 within 1-2 min. On size exclusion chromatography, F1 eluted as a dimer in the Pfr and as a monomer in the Pr form, whereas F2 and F3 behaved always as monomers, irrespective of the light conditions. These and other results are discussed in the context of light-dependent subunit interactions, in which amino acids 473-520 within the PHY domain are required for chromophore-module subunit interaction within the homodimer. V8 proteolysis yielded five major chromopeptides, F4 (T17 to N449), F5 (T17 to E335), F6 (T17 to E323), F7 (unknown sequence), and F8 (tentatively L121 to E323). F6 and F8 were formed in the Pr form, whereas F4, F5, and F7 were preferentially formed in the Pfr form. Three amino acids next to specific cleavage sites, R520, R472, and E323, were altered by site-directed mutagenesis. The mutants were analyzed by UV-vis spectroscopy, size exclusion chromatography, and autophosphorylation. Histidine kinase activity was low in R472A, R520P, and R520A; in all mutants, the ratio of phosphorylation intensity between Pr and Pfr was reduced. Thus, light regulation of autophosphorylation is negatively affected in all mutants. In R472P, E323P, and E323D, the phosphorylation intensity of the Pfr form exceeded that of the wild-type control. This result shows that the histidine kinase activity of Cph1 is actively inhibited by photoconversion into Pfr.     

Monitoring the conformational changes of photoactivated rhodopsin from microseconds to seconds by transient fluorescence spectroscopy

The transient changes of the tryptophan fluorescence of bovine rhodopsin in ROS membranes were followed in time from 1 micros to 10 s after flash excitation of the photoreceptor. Up to about 100 micros the fluorescence did not change, suggesting that the tryptophan lifetimes in rhodopsin and the M(I) intermediate are similar. The fluorescence then decreases on the millisecond time scale with kinetics that match the rise of the M(II) state as measured on the same sample by the transient absorption increase at 360 nm. Both the sign and kinetics of the fluorescence change strongly suggest that it is due to an increase in energy transfer to the retinylidene chromophore caused by the increased spectral overlap in M(II). Calculation of the Forster radius of each tryptophan from the high-resolution crystal structure suggests that W265 and W126 are already completely quenched in the dark, whereas W161, W175, and W35 are located at distances from the retinal chromophore that are comparable to their Forster radii. The fluorescence from these residues is thus sensitive to an increase in energy transfer in M(II). Similar results were obtained at other temperatures and with monomeric rhodopsin in dodecyl maltoside micelles. A large light-induced transient fluorescence increase was observed with ROS membranes that were selectively labeled with Alexa594 at cysteine 316 in helix 8. Using transient absorption spectroscopy the kinetics of this structural change at the cytoplasmic surface was compared to the formation of the signaling state M(II) (360 nm) and to the kinetics of proton uptake as measured with the pH indicator dye bromocresol purple (605 nm). The fluorescence kinetics lags behind the deprotonation of the Schiff base. The proton uptake is even further delayed. These observations show that in ROS membranes (at pH 6) the sequence of events is Schiff base deprotonation, structural change, and proton uptake. From the temperature dependence of the kinetics we conclude that the Schiff base deprotonation and the transient fluorescence have comparable activation energies, whereas that of proton uptake is much smaller.