Metarhodopsin II Research Articles

Reduced G Protein-coupled Signaling Efficiency in Retinal Rod Outer Segments in Response to n-3 Fatty Acid Deficiency

The fatty acid (FA) docosahexaenoic acid (DHA, 22: 6n-3) is highly enriched in membrane phospholipids of the central nervous system and retina. Loss of DHA because of n-3 FA deficiency leads to suboptimal function in learning, memory, olfactory-based discrimination, spatial learning, and visual acuity. G protein-coupled receptor (GPCR) signal transduction is a common signaling motif in these neuronal pathways. Here we investigated the effect of n-3 FA deficiency on GPCR signaling in retinal rod outer segment (ROS) membranes isolated from rats raised on n-3-adequate or -deficient diets. ROS membranes of second generation n-3 FA-deficient rats had approximately 80% less DHA than n-3-adequate rats. DHA was replaced by docosapentaenoic acid (22:5n-6), an n-6 FA. This replacement correlated with desensitization of visual signaling in n-3 FA-deficient ROS, as evidenced by reduced rhodopsin activation, rhodopsin-transducin (G(t)) coupling, cGMP phosphodiesterase activity, and slower formation of metarhodopsin II (MII) and the MII-G(t) complex relative to n-3 FA-adequate ROS. ROS membranes from n-3 FA-deficient rats exhibited a higher degree of phospholipid acyl chain order relative to n-3 FA-adequate rats. These findings reported here provide an explanation for the reduced amplitude and delayed response of the electroretinogram a-wave observed in n-3 FA deficiency in rodents and nonhuman primates. Because members of the GPCR family are widespread in signaling pathways in the nervous system, the effect of reduced GPCR signaling due to the loss of membrane DHA may serve as an explanation for the suboptimal neural signaling observed in n-3 FA deficiency.

Manipulation of Cholesterol Levels in Rod Disk Membranes by Methyl-β-cyclodextrin: EFFECTS ON RECEPTOR ACTIVATION

The effect of cholesterol on rod outer segment disk membrane structure and rhodopsin activation was investigated. Disk membranes with varying cholesterol concentrations were prepared using methyl-beta-cyclodextrin as a cholesterol donor or acceptor. Cholesterol exchange followed a simple equilibrium partitioning model with a partition coefficient of 5.2 +/- 0.8 in favor of the disk membrane. Reduced cholesterol in disk membranes resulted in a higher proportion of photolyzed rhodopsin being converted to the G protein-activating metarhodopsin II (MII) conformation, whereas enrichment of cholesterol reduced the extent of MII formation. Time-resolved fluorescence anisotropy measurements using 1,6-diphenyl-1,3,5-hexatriene showed that increasing cholesterol reduced membrane acyl chain packing free volume as characterized by the parameter f(v). The level of MII formed showed a positive linear correlation with f(v) over the range of 4 to 38 mol % cholesterol. In addition, the thermal stability of rhodopsin increased with mol % of cholesterol in disk membranes. No evidence was observed for the direct interaction of cholesterol with rhodopsin in either its agonist- or antagonist-bound form. These results indicate that cholesterol mediates the function of the G protein-coupled receptor, rhodopsin, by influencing membrane lipid properties, i.e. reducing acyl chain packing free volume, rather than interacting specifically with rhodopsin.

Anions stabilize a metarhodopsin II-like photoproduct with a protonated Schiff base.

In rhodopsin, the retinal chromophore is covalently bound to the apoprotein by a protonated Schiff base, which is stabilized by the negatively charged counterion Glu113, conferring upon it a pK(a) of presumably >16. Upon photoexcitation and conformational relaxation of the initial photoproducts, the Schiff base proton neutralizes the counterion, a step that is considered a prerequisite for formation of the active state of the receptor, metarhodopsin II (MII). We show that the pK(a) of the Schiff base drops below 2.5 in MII. In the presence of solute anions, however, it may be increased considerably, thereby leading to the formation of a MII photoproduct with a protonated Schiff base (PSB) absorbing at 480 nm. This PSB is not stabilized by Glu113, which is shown to be neutral, but by stoichiometric binding of an anion near the Schiff base. Protonation of the Schiff base in MII changes neither coupling to G protein, as assessed by binding to a transducin-derived peptide, nor the conformation of the protein, as judged by FTIR and UV spectroscopy. A PSB and an active state conformation are therefore compatible, as suggested previously by mutants of rhodopsin. The anion specificity of the stabilization of the PSB follows the series thiocyanate > iodide > nitrate > bromide > chloride > sulfate in order of increasing efficiency. This specificity correlates inversely with the strength of hydration of the respective anion species in solution and seems therefore to be determined mainly by its partitioning into the considerably less polar protein interior.

Optimization of Receptor-G Protein Coupling by Bilayer Lipid Composition I

The role of membrane composition in modulating the rate of G protein-receptor complex formation was examined using rhodopsin and transducin (G(t)) as a model system. Metarhodopsin II (MII) and MII-G(t) complex formation rates were measured, in the absence of GTP, via flash photolysis for rhodopsin reconstituted in 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0,18:1PC) and 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0,22:6PC) bilayers, with and without 30 mol% cholesterol. Variation in bilayer lipid composition altered the lifetime of MII-G(t) formation to a greater extent than the lifetime of MII. MII-G(t) formation was fastest in 18:0,22:6PC and slowest in 18:0,18:1PC/30 mol% cholesterol. At 37 degrees C and a G(t) to photolyzed rhodopsin ratio of 1:1 in 18:0,22:6PC bilayers, MII-G(t) formed with a lifetime of 0.6 +/- 0.06 ms, which was not significantly different from the lifetime for MII formation. Incorporation of 30 mol% cholesterol slowed the rate of MII-G(t) complex formation by about 400% in 18:0,18:1PC, but by less than 25% in 18:0,22:6PC bilayers. In 18:0,22:6PC, with or without cholesterol, MII-G(t) formed rapidly after MII formed. In contrast, cholesterol in 18:0,18:1PC induced a considerable lag time in MII-G(t) formation after MII formed. These results demonstrate that membrane composition is a critical factor in determining the temporal response of a G protein-coupled signaling system.

Optimization of receptor-G protein coupling by bilayer lipid composition II: formation of metarhodopsin II-transducin complex.

The visual transduction system was used as a model to investigate the effects of membrane lipid composition on receptor-G protein coupling. Rhodopsin was reconstituted into large, unilamellar phospholipid vesicles with varying acyl chain unsaturation, with and without cholesterol. The association constant (K(a)) for metarhodopsin II (MII) and transducin (G(t)) binding was determined by monitoring MII-G(t) complex formation spectrophotometrically. At 20 degrees C, in pH 7.5 isotonic buffer, the strongest MII-G(t) binding was observed in 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0,22:6PC), whereas the weakest binding was in 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0,18:1PC) with 30 mol% cholesterol. Increasing acyl chain unsaturation from 18:0,18:1PC to 18:0,22:6PC resulted in a 3-fold increase in K(a). The inclusion of 30 mol% cholesterol in the membrane reduced K(a) in both 18:0,22:6PC and 18:0,18:1PC. These findings demonstrate that membrane compositions can alter the signaling cascade by changing protein-protein interactions occurring predominantly in the hydrophilic region of the proteins, external to the lipid bilayer. These findings, if extended to other members of the superfamily of G protein-coupled receptors, suggest that a loss in efficiency of receptor-G protein binding is a contributing factor to the loss of cognitive skills, odor and spatial discrimination, and visual function associated with n-3 fatty acid deficiency.

The role of docosahexaenoic acid containing phospholipids in modulating G protein-coupled signaling pathways: visual transduction.

In order to understand the role of the high levels of docosahexaenoic acid (DHA) in neuronal and retinal tissue, a study of the effect of membrane lipid composition on the visual pathway, a G protein-coupled system, was undertaken. The level of metarhodopsin II (MII) formation was determined to be a function of phospholipid acyl-chain unsaturation, with the highest levels seen in DHA-containing bilayers. Similarly, the rate of coupling of MII to the retinal G protein, Gt, to form a MII-Gt complex, was enhanced in DHA bilayers relative to less unsaturated phospholipids. Complex formation initiates the first stage of amplification in the visual pathway. The activation of the cGMP phosphodiesterase (PDE), the effector enzyme, represents the integrated pathway function. DHA-containing bilayers were found to support PDE levels comparable to those of the rod outer segment (ROS) disk membranes. Inclusion of 30 mol% cholesterol in the reconstituted bilayers had an inhibitory effect on each step in the visual pathway studied. Inclusion of cholesterol reduced MII formation and PDE activity and increased the lag time between the appearance of MII and the formation of the MII-Gt complex. However, signaling in DHA bilayers was far less affected by the addition of cholesterol than in bilayers containing less unsaturated phospholipids. These studies point up the importance of DHA acyl chains in promoting optimal function in G protein-coupled signaling pathways. The results reported here suggest that visual and cognitive deficits observed in n-3 deficiency may result from decreased efficiency in related neurotransmitter and visual signaling pathways in the absence of DHA.

Interactions of Metarhodopsin II: ARRESTIN PEPTIDES COMPETE WITH ARRESTIN AND TRANSDUCIN

Arrestin blocks the interaction of rhodopsin with the G protein transducin (G(t)). To characterize the sites of arrestin that interact with rhodopsin, we have utilized a spectrophotometric peptide competition assay. It is based on the stabilization of the active intermediates metarhodopsin II (MII) and phosphorylated MII by G(t) and arrestin, respectively (extra MII monitor). The protocol involves native disc membranes and three sets of peptides 10-30 amino acids in length spanning the arrestin sequence. In the absence of arrestin, not one of the peptides by itself had an effect on the amount of MII formed. However, inhibition of arrestin-dependent extra MII was found for the peptides at residues 11-30 and 51-70 (IC(50) < 100 microm) and residues 231-260 (IC(50) < 200 microm). A similar pattern of inhibition by arrestin peptides was seen when arrestin was replaced by G(t) or the farnesylated G(t)gamma C-terminal peptide. Only arrestin-(11-30) inhibited MII.G(t) less (IC(50) = 300 microm) than phosphorylated MII.arrestin. We interpreted the data by competition of the arrestin peptides for interaction sites at rhodopsin, exposed in the MII conformation and specific for both arrestin and G(t). The arrestin sites are located in both the C- and N-terminal domains of the arrestin structure.

Transducin-Dependent Protonation of Glutamic Acid 134 in Rhodopsin

A highly conserved carboxylic acid residue in rhodopsin, Glu(134), modulates transducin (G(t)) interaction. It has been postulated that Glu(134) becomes protonated upon receptor activation. We studied the interaction between rhodopsin and G(t) using Fourier transform infrared (FTIR) difference spectroscopy combined with attenuated total reflection (ATR). Formation of the complex between G(t) and photoactivated rhodopsin reconstituted into phosphatidylcholine vesicles caused prominent infrared absorption increases at 1641, 1550, and 1517 cm(-)(1). The rhodopsin mutant E134Q was also studied. When measured in the presence of G(t), replacement of Glu(134) by glutamine abolished the low-frequency part of a broad absorption band at 1735 cm(-)(1) that is normally superimposed on the light-induced absorption changes of Asp(83) and Glu(122) of rhodopsin. In addition, a negative absorption band at 1400 cm(-)(1) that is evoked by interaction of native metarhodopsin II (MII) with G(t) was not observed in the difference spectrum of the E134Q mutant. Thus, Glu(134) is ionized in the dark and exhibits a symmetrical COO(-) stretching vibration at 1400 cm(-)(1). Glu(134) becomes protonated in the G(t)-MII complex and displays a C=O stretching mode near 1730 cm(-)(1). The E134Q mutation also affects absorption changes attributable to lipids, suggesting that the protonation of Glu(134) is linked to transfer of the carboxylic acid side chain from a polar to a nonpolar environment by becoming exposed to the lipid phase when G(t) binds. These results show directly that Glu(134) becomes protonated in MII upon G(t) binding and suggest that changes in receptor conformation affect lipid-protein interactions.

The molecular origin of the inhibition of transducin activation in rhodopsin lacking the 9-methyl group of the retinal chromophore: a UV-Vis and FTIR spectroscopic study.

The formation of the active rhodopsin state metarhodopsin II (MII) is believed to be partially governed by specific steric constraints imposed onto the protein by the 9-methyl group of the retinal chromophore. We studied the properties of the synthetic pigment 9-demethyl rhodopsin (9dm-Rho), consisting of the rhodopsin apoprotein regenerated with synthetic retinal lacking the 9-methyl group, by UV-vis and Fourier transform infrared difference spectroscopy. Low activation rates of the visual G-protein transducin by the modified pigment reported in previous studies are shown to not be caused by the reduced activity of its MII state, but to be due to a dramatic equilibrium shift from MII to its immediate precursor, MI. The MII state of 9dm-Rho displays only a partial deprotonation of the retinal Schiff base, leading to the formation of two MII subspecies absorbing at 380 and 470 nm, both of which seem to be involved in transducin activation. The rate of MII formation is slowed by 2 orders of magnitude compared to rhodopsin. The dark state and the MI state of 9dm-Rho are distinctly different from their respective states in the native pigment, pointing to a more relaxed fit of the retinal chromophore in its binding pocket. The shifted equilibrium between MI and MII is therefore discussed in terms of an increased entropy of the 9dm-Rho MI state due to changed steric interactions.

FTIR spectroscopy of complexes formed between metarhodopsin II and C-terminal peptides from the G-protein α- and γ-subunits

Metarhodopsin II (MII) provides the active conformation of rhodopsin for interaction with the G-protein, Gt. Fourier transform infrared spectra from samples prepared by centrifugation reflect the pH dependent equilibrium between MII and inactive metarhodopsin I. C-terminal synthetic peptides (Gtα(340–350) and Gtγ(60–71)farnesyl) stabilize MII. We find that both peptides cause similar spectral changes not seen with control peptides (Gtα (K341R, L349A) and non-farnesylated Gtγ). The spectra reflect all the protonation dependent bands normally observed when MII is formed at acidic pH. Beside the protonation dependent bands, additional features, similar with both peptides, appear in the amide I and II regions.

Effect of Ethanol and Osmotic Stress on Receptor Conformation: REDUCED WATER ACTIVITY AMPLIFIES THE EFFECT OF ETHANOL ON METARHODOPSIN II FORMATION

The combined effects of ethanol and osmolytes on both the extent of formation of metarhodopsin II (MII), which binds and activates transducin, and on acyl chain packing were examined in rod outer segment disc membranes. The ethanol-induced increase in MII formation was amplified by the addition of neutral osmolytes. This enhancement was linear with osmolality. At 360 milliosmolal, the osmolality of human plasma, 50 mM ethanol was 2.7 times more potent than at 0 osmolality, demonstrating the importance of water activity in in vitro experiments dealing with ethanol potency. Ethanol disordered acyl chain packing, and increasing osmolality enhanced this acyl chain disordering. Prior osmotic stress data showed a release of 35 +/- 2 water molecules upon MII formation. Ethanol increases this number to 49 water molecules, suggesting that ethanol replaces 15 additional water molecules upon MII formation. Amplification of ethanol effects on MII formation and acyl chain packing by osmolytes suggests that ethanol increases the equilibrium concentration of MII both by disordering acyl chain packing and by disrupting rhodopsin-water hydrogen bonds, demonstrating a direct effect of ethanol on rhodopsin. At physiologically relevant levels of osmolality and ethanol, about 90% of ethanol's effect is due to disordered acyl chain packing.

32] Intrinsic biophysical monitors of transducin activation: Fluorescence, UV-visible spectroscopy, light scattering, and evanescent field techniques

Information on the temporal order, the kinetics, and the energetics of each of the reaction intermediates is required in order to fully elucidate the mechanism of catalytic interaction of G proteins with their receptors. Intrinsic monitors, such as light scattering, absorption, and fluorescence, exploit endogenous properties of proteins, and therefore offer the advantage of leaving the system under investigation undisturbed. Such assays currently available for G proteins include the well-known intrinsic fluorescence changes of a tryptophan residue located near the G protein's active center. In addn., the visual system offers UV-visible spectrophotometric assays in which the retinal chromophore itself serves as an intrinsic reporter group. These assays include monitoring the transducin sensitive formation of the active metarhodopsin-II (MII) conformation, as well as the photoregeneration of dark state rhodopsin from MII. A third technique is kinetic light scattering, which makes use of the unique activation-dependent soly. of the rod G protein. While these monitors are highly specialized, more generally applicable monitors have been developed in recent years and successfully tested with the visual system. This family of techniques is based on the evanescent field in the vicinity of a reflecting optical surface, and includes surface plasmon resonance and resonant mirror spectroscopy. Applications and limitations of these techniques are described. (c) 2000 Academic Press. [on SciFinder (R)]

Effect of protein hydration on receptor conformation: decreased levels of bound water promote metarhodopsin II formation.

Neutral solutes were used to investigate the effects of osmotic stress both on the ability of rhodopsin to undergo its activating conformation change and on acyl chain packing in the rod outer segment (ROS) disk membrane. The equilibrium concentration of metarhodopsin II (MII), the conformation of photoactivated rhodopsin, which binds and activates transducin, was increased by glycerol, sucrose, and stachyose in a manner which was linear with osmolality. Analysis of this shift in equilibrium in terms of the dependence of ln(Keq) on osmolality revealed that 20 +/- 1 water molecules are released during the MI-to-MII transition at 20 degrees C, and at 35 degrees C 13 +/- 1 waters are released. At 35 degrees C the average time constant for MII formation was increased from 1.20 +/- 0.09 ms to 1.63 +/- 0.09 ms by addition of 1 osmolal sucrose or glycerol. The effect of the neutral solutes on acyl chain packing in the ROS disk membrane was assessed via measurements of the fluorescence lifetime and anisotropy decay of 1,6-diphenyl-1,3,5-hexatriene (DPH). Analysis of the anisotropy decay of DPH in terms of the rotational diffusion model showed that the angular width of the equilibrium orientational distribution of DPH about the membrane normal was progressively narrowed by increased osmolality. The parameter fv, which is proportional to the overlap between the DPH orientational probability distribution and a random orientational distribution, was reduced by the osmolytes in a manner which was linear with osmolality. This study highlights the potentially opposing interplay between the effect of membrane surface hydration on both the lipid bilayer and integral membrane protein structure. Our results further demonstrate that the binding and release of water molecules play an important role in modulating functional conformational changes for integral membrane proteins, as well as for soluble globular proteins.

Signal transfer from rhodopsin to the G-protein: evidence for a two-site sequential fit mechanism.

Photoactivation of the retinal photoreceptor rhodopsin proceeds through a cascade of intermediates, resulting in protein-protein interactions catalyzing the activation of the G-protein transducin (Gt). Using stabilization and photoregeneration of the receptor's signaling state and Gt activation assays, we provide evidence for a two-site sequential fit mechanism of Gt activation. We show that the C-terminal peptide from the Gt gamma-subunit, Gtgamma(50-71)farnesyl, can replace the holoprotein in stabilizing rhodopsin's active intermediate metarhodopsin II (MII). However, the peptide cannot replace the Gtbeta gamma complex in direct activation assays. Competition by Gtgamma(50-71)farnesyl with Gt for the active receptor suggests a pivotal role for Gtbeta gamma in signal transfer from MII to Gt. MII stabilization and competition is also found for the C-terminal peptide from the Gt alpha-subunit, Gtalpha(340-350), but the capacity of this peptide to interfere in MII-Gt interactions is paradoxically low compared with its activity to stabilize MII. Besides this disparity, the pH profiles of competition with Gt are characteristically different for the two peptides. We propose a two-site sequential fit model for signal transfer from the activated receptor, R*, to the G-protein. In the center of the model is specific recognition of conformationally distinct sites of R* by Gtalpha(340-350) and Gtgamma(50-71)farnesyl. One matching pair of domains on the proteins would, on binding, lead to a conformational change in the G-protein and/or receptor, with subsequent binding of the second pair of domains. This process could be the structural basis for GDP release and the formation of a stable empty site complex that is ready to receive the activating cofactor, GTP.

A comparison of the efficiency of G protein activation by ligand-free and light-activated forms of rhodopsin

Activation of the photoreceptor G protein transducin (Gt) by opsin, the ligand-free form of rhodopsin, was measured using rod outer segment membranes with densities of opsin and Gt similar to those found in rod cells. When GTPgammaS was used as the activating nucleotide, opsin catalyzed transducin activation with an exponential time course with a rate constant k(act) on the order of 2 x 10(-3)s(-1). Comparison under these conditions to activation by flash-generated metarhodopsin II (MII) revealed that opsin- and R*-catalyzed activation showed similar kinetics when MII was present at a surface density approximately 10(-6) lower than that of opsin. Thus, in contrast to some previous reports, we find that the catalytic potency of opsin is only approximately 10(-6) that of MII. In the presence of residual retinaldehyde-derived species present in membranes treated with hydroxylamine after bleaching, the apparent k(act) observed was much higher than that for opsin, suggesting a possible explanation for previous reports of more efficient activation by opsin. These results are important for considering the possible role of opsin in the diverse phenomena in which it has been suggested to play a key role, such as bleaching desensitization and retinal degeneration induced by continuous light or vitamin A deprivation.

Properties of early photolysis intermediates of rhodopsin are affected by glycine 121 and phenylalanine 261.

Glycine 121 in transmembrane (TM) helix 3 and phenylalanine 261 in TM helix 6 of bovine rhodopsin have been shown to be critical residues for creating an appropriate chromophore binding pocket for 11-cis-retinal [Han, M., Lin, S. W., Smith, S. O., and Sakmar, T. P. (1996) J. Biol. Chem. 271, 32330-32336; Han, M., Lin, S. W., Minkova, M., Smith, S. O., and Sakmar, T. P. (1996) J. Biol. Chem. 271, 32337-32342]. To further explore structure-function relationships in the vicinity of receptor helices 3 and 6, time-resolved absorption difference spectra of rhodopsin mutants G121A, G121V, and G121L/F261A were obtained at 20 degrees C. Data were collected from 30 ns to 690 ms after laser photolysis with 7 ns pulses (lambdamax = 477 nm) and analyzed using a global exponential fitting procedure after singular value decomposition (SVD). For each mutant, the decay of its bathorhodopsin photoproduct (batho) into an equilibrium with its blue-shifted intermediate (bsi) was too fast to resolve (<20 ns). The reaction scheme found for the mutants G121A and G121L/F261A was batho/bsi --> lumirhodopsin (lumi) --> metarhodopsin I (MI) --> metarhodopsin II (MII). For G121V, an additional early 380 nm absorber, with a back-reaction to lumi, had to be included in the above scheme. For the three Gly121 mutants, the main pathway to reach the active MII state is via lumi and MI. This is in contrast to rhodopsin where the main pathway in detergent samples is via lumi and an early 380 nm absorber, MI380. From the accelerated batho decay present in all three mutants, we conclude that Gly121 is likely to participate in the earliest chromophore-protein interactions. In addition, bsi decay is further accelerated in mutant G121L/F261A, suggesting that Phe261 is an essential determinant of the protein processes involved in bsi decay.

Time-resolved spectroscopy of the early photolysis intermediates of rhodopsin Schiff base counterion mutants.

Time-resolved absorption difference spectra of COS-cell expressed rhodopsin and rhodopsin mutants (E113D, E113A/A117E, and G90D), solubilized in detergent, were collected from 20 ns to 510 ms after laser photolysis with 7 ns pulses (lambda(max) = 477 nm). The data were analyzed using a global exponential fitting procedure following singular value decomposition (SVD). Over the entire time range excellent agreement was achieved between results for COS-cell and rod outer segment rhodopsin both in kinetics and in the lambda(max) values of the intermediates. The Schiff base counterion mutant E113D showed strong similarities to rhodopsin up to lumi, following the established scheme: batho <==> bsi --> lumi. Including late delay times (past 1 micros), the mutant E113D lumi decayed to metarhodopsin II (MII), showing that the detergent strongly favors MII over metarhodopsin I (MI). However, a back-reaction from MII to lumi was observed that was not seen for rhodopsin. The kinetic schemes for the mutants E113A/A117E and G90D were significantly different from that of rhodopsin. In both mutants batho decay into an equilibrium with bsi was too fast to resolve (<20 ns). The batho/bsi mixtures decayed with the following reaction scheme: batho/bsi <==> lumi <==> MI-like <==> MII-like. However, the back-reaction from MI-like to lumi was not seen in G90D. MI-like spectral intermediates absorbing around 460 nm appeared in both mutants. They have been shown to be the transducin-activating species (R*). These data, interpreted in the context of previous NMR, FTIR, and Raman data, are consistent with a picture in which the kinetics of batho decay is dependent on a protein-induced perturbation near C12-C13 of the retinal chromophore. The lambda(max) values of the bsi and lumi intermediates in the mutant pigments are interpreted in terms of movement of the Schiff base relative to its counterion.

Modulation of the metarhodopsin I/metarhodopsin II equilibrium of bovine rhodopsin by ionic strength--evidence for a surface-charge effect.

The effects of ionic strength on formation and decay of metarhodopsin II (MII), the active photointermediate of bovine rhodopsin, were studied in the native membrane environment by means of ultraviolet/ visible and Fourier-transform infrared (FTIR) spectroscopy. By increasing the concentration of KCl in the range from hypotonic to 4 M, the apparent pKa of the metarhodopsin I(MI)/MII equilibrium is shifted by approximately pH three, in favor of the MII intermediate. In addition, the apparent rate of MII formation is enhanced by an increase in ionic strength (about twofold in the presence of 2 M KCl). MIII decay is independent of the salt concentration. Attenuated-total-reflectance/FTIR data show that the high-salt conditions have no effect on the rigidity of the membrane matrix and do not induce structural changes in the intermediates themselves. Different salts were tested for their ability to shift the MI/MII equilibrium; however, no clear ion dependence was observed. We interpret these results as an indication for direct involvement of the cytosolic surface charge in the regulation of the photochemical activity of bovine rhodopsin.

Surface plasmon resonance spectroscopy studies of membrane proteins: transducin binding and activation by rhodopsin monitored in thin membrane films

Surface plasmon resonance (SPR) spectroscopy can provide useful information regarding average structural properties of membrane films supported on planar solid substrates. Here we have used SPR spectroscopy for the first time to monitor the binding and activation of G-protein (transducin or Gt) by bovine rhodopsin incorporated into an egg phosphatidylcholine bilayer deposited on a silver film. Rhodopsin incorporation into the membrane, performed by dilution of a detergent solution of the protein, proceeds in a saturable manner. Before photolysis, the SPR data show that Gt binds tightly (Keq approximately equal to 60 nM) and with positive cooperativity to rhodopsin in the lipid layer to form a closely packed film. A simple multilayer model yields a calculated average thickness of about 57 A, in good agreement with the structure of Gt. The data also demonstrate that Gt binding saturates at a Gt/rhodopsin ratio of approximately 0.6. Moreover, upon visible light irradiation, characteristic changes occur in the SPR spectrum, which can be modeled by a 6 A increase in the average thickness of the lipid/protein film caused by formation of metarhodopsin II (MII). Upon subsequent addition of GTP, further SPR spectral changes are induced. These are interpreted as resulting from dissociation of the alpha-subunit of Gt, formation of new MII-Gt complexes, and possible conformational changes of Gt as a consequence of complex formation. The above results clearly demonstrate the ability of SPR spectroscopy to monitor interactions among the proteins associated with signal transduction in membrane-bound systems.

Specific tryptophan UV-absorbance changes are probes of the transition of rhodopsin to its active state.

The difference of rhodopsin and metarhodopsin II (MII) absorption spectra exhibits a characteristic pattern in the UV wavelength range, consisting of peaks at 278, 286, 294, 302 nm. These difference bands are thought to result from the perturbation of the environments of tryptophan and/or tyrosine residues. We used site-directed mutagenesis to investigate the contribution of tryptophan absorption to these spectral features. Each of the five tryptophan residues in bovine rhodopsin was replaced by either a phenylalanine or a tyrosine. The mutant pigments (W35F, W126F, W161F, W175F, W265F/Y) were prepared and studied by UV-visible photobleaching difference spectroscopy. The difference spectra of the W35F and W175F mutants were identical to that of rhodopsin, whereas in the W161F mutant, the magnitudes of the 294- and 302-nm bands were slightly lowered. The differential absorbance at 294 nm was reduced by over 50% in the W126F and W265F/Y mutants. The difference peak at 302 nm was reduced in the W265F/Y mutants, but was almost completely absent in the W126F mutant. These data indicate that the difference bands at 294 and 302 nm originate from the perturbations of Trp126 and Trp265 environments resulting from a general conformational change concomitant with MII formation and receptor activation. Model studies on tryptophan absorption indicate that the difference peak at 294 nm is due to the differential shift of the Lb absorption of the indole side chain as a result of decreased hydrophobicity or polarizability of the Trp126 and Trp265 environments. The resolution of the 302-nm band, assigned to the differential shift of the indole La absorption, is consistent with hydrogen-bonding interactions of the indole N-H groups of Trp126 and Trp265 becoming weaker in MII. These results suggest that the photoactivation of rhodopsin involves a change in the relative disposition of transmembrane helices 3 and 6, which contain Trp126 and Trp265 respectively, within the alpha-helical bundle of the receptor.