Metarhodopsin I Research Articles

Influence of soft matter on rhodopsin activation.

Rhodopsin is a prototypical member of G protein-coupled receptors (GPCRs) whose activation and conformational changes can be studied using UV-visible and FTIR spectroscopy. Our hypothesis is that the photoreceptor activation is influenced by the soft matter (water and lipids) and proteins in a membrane environment. Hydration primarily influences metarhodopsin-II (MII) formation rather than the deformation of lipid bilayer [1,2]. Our goal was to develop an accurate and simple experiment using UV-visible spectroscopy to investigate the influence of the soft matter and G-protein-derived peptide on the activation equilibrium of metarhodopsin-I (MI) and MII. Understanding the influence of thermodynamic parameters upon the rhodopsin activation is an important objective. Surprisingly, we observed that the light-dark difference UV-visible spectra were significantly time-dependent close to ambient temperature, which required monitoring of the spectral changes due to deprotonation of the retinal Schiff base in the active MII state. The time-dependent changes were fitted to an exponential decay to obtain the initial value and as well as the change in rate constant. The MII protonated Schiff base (PSB) fraction was calculated and plotted as a function of pH at various time points. Titration curves of initial values and partial decay of the MII fraction displayed similar tendencies consistent with our previous observations [1]. Furthermore, acid denaturation assays were conducted to address the question of whether the changes are due to the hydrolysis of the Schiff base linkage. Alternatively, the observed time-dependent changes can also be due to generation of the storage form metarhodopsin-III (MIII), which is yet to be understood. The effects of the lipid to protein ratio and the G-protein derived peptides upon rhodopsin activation are currently under investigation.

Physiological changes in bilayer thickness induced by cholesterol control GPCR rhodopsin function

We monitored the effect on function of the G-protein-coupled receptor (GPCR) rhodopsin from small, stepwise changes in bilayer thickness induced by cholesterol. Over a range of phosphatidylcholine bilayers with hydrophobic thickness from ≈21 Å to 38 Å, the metarhodopsin-I (MI)/metarhodopsin-II (MII) equilibrium was monitored with UV-visible spectroscopy while ordering of hydrocarbon chains was probed by 2H-NMR. Addition of cholesterol shifted equilibrium toward MII for bilayers thinner than the average length of hydrophobic transmembrane helices (27 Å) and to MI for thicker bilayers, while small bilayer thickness changes within the range of the protein hydrophobic thickness drastically up- or downregulated MII formation. The cholesterol-induced shifts toward MII for thinner membranes correlated with the cholesterol-induced increase of bilayer hydrophobic thickness measured by NMR, consistent with continuum elastic modeling. The energetic penalty of adding cholesterol to thick bilayers caused rhodopsin oligomerization and a shift toward MI. In membranes of physiological thickness, changes in bilayer mechanical properties induced by cholesterol potentiated the interplay between bilayer and protein thickness resulting in large swings of the MI-MII equilibrium. In membrane containing cholesterol, elastic deformations near the protein are a dominant energetic contribution to the functional equilibrium of the model GPCR rhodopsin.

Extent of Internal Hydration influence the Activation of GPCR Rhodopsin

Rhodopsin is an archetypical GPCR responsible for dim‐light vision, which adopts a dynamic equilibrium between two metastable intermediates, metarhodopsin‐I (MI) and metarhodopsin‐II (MII), upon absorption of light. MI is an inactive conformational state of this GPCR, while MII is a fully active conformational state. We hypothesized that the MI to MII transition following isomerization of the retinal ligand involves a flood of bulk water into the protein core. To observe this change in the hydration level of the receptor during activation that is typically invisible to structural techniques such as X‐ray crystallography or cryo‐EM, we carried out a set of experiments using an osmotic stress approach. In this approach, the hydration level of rhodopsin was changed by placing the protein in native disk membranes in solutions with varying levels of osmotic pressure. Inert hydrophilic polyethylene glycol (PEG) solutes with different molecular weights and concentrations were used as the osmolytes. The effect of osmotic pressure on conformational dynamics was then quantified by measuring the reversible shifting of the metarhodopsin equilibrium using UV‐Visible spectroscopy. Results from these experiments show an influx of 80–100 water molecules into the rhodopsin interior, forming a partially disordered, solvent‐swollen active MII conformational state during photoactivation, a result supported by atomistic molecular dynamics simulations work [1, 2]. Additionally, we observed that the size of the polymer osmolyte played an important role in the modulation of rhodopsin’s activation: large osmolytes (PEG > 600) back shift the equilibrium to inactive MI state, while small osmolytes (PEG < 600) forward‐shift the equilibrium to MII state. We attribute this size dependence to the degree of osmolyte penetration into the rhodopsin core. Large polymers behave similarly to ideal osmolytes and dehydrate rhodopsin, while smaller polymers wriggle into the rhodopsin interior and stabilize the open MII conformation. These results necessitate a new understanding of GPCR activation in which the hydration level of protein is paramount in governing conformational energy landscapes. [1] U. Chawla et al. (2021) Angew. Chem. Int. Ed. 60, 2288–2295. [2] N. Leioatts et al. (2014) Biochemistry 53, 376−385.

Effects of Soft Matter on G-Protein-Coupled Receptor Activation

G protein-coupled receptors (GPCRs) constitute both the largest class of medicinal targets and the largest family of human membrane proteins. Current three-dimensional structures have shown the existence of many water molecules immersed inside GPCRs. However, the functional role of these water molecules has remained uncertain. Our previous work has shown that water modulates the activation of visual rhodopsin, a prototypical GPCR [1]. Here we expanded our approach to further investigate how the conformational energetics of GPCR activation are modulated by water using a series of osmolytes that included polyethylene glycols (PEGs), dextrans, polyvinylpyrrolidones (PVPs), and their monomers. We hypothesized that osmotic pressure affects the thermodynamic stability of conformational substates of photoactivated rhodopsin. Accordingly, we changed the osmotic stress on rhodopsin using different concentrations of various polymer solutes. Shifting of the metarhodopsin equilibrium due to the osmotic pressure was probed using UV-visible spectroscopy. The larger osmolytes (e.g., PEG 1500-6000) promote the inactive metarhodopsin-I (MI) state, while the small organic osmolytes such as smaller molar mass PEGs, their monomers, and small sugars increase the active metarhodopsin-II (MII) fraction. These size effects are a result of the degree of penetration of osmolytes into the protein core of rhodopsin. Small polymers penetrate the protein core and favor the open MII state of rhodopsin, while the large polymers behave like ideal osmolytes and dehydrate rhodopsin. Most interestingly, PEGs and dextrans with extremely large molar masses deviated in their trends from other large polymers (PEGs), which we attribute to a combined result of crowding and penetration effects. The results provide us with new insights into the interplay between osmotic stress, penetration, and crowding effects. Our findings further the understanding of the vital role of water in the GPCR activation mechanisms. [1] U. Chawla et al. (2020) Angew.Chem.Int.Ed. doi:10.1002/anie.202003342.

Modulation of GPCR Function by Membrane Lipids and Water

G‐protein‐coupled receptors (GPCRs) are integral membrane proteins that regulate a wide range of normal and disease‐related physiological processes. Our understanding of the role of soft matter such as membrane lipids and cellular water on activation and regulation of the GPCR function remains incomplete. In the standard biochemical model, GPCRs behave as primarily agonist dependent bimodal switches, with little influence of the surrounding medium. However, using the visual receptor rhodopsin as a model GPCR, we show that water drives rhodopsin to a partially disordered, solvent‐swollen conformational ensemble upon light absorption, rendering the standard model obsolete [1], [2]. We placed rhodopsin under varying degrees of osmotic stress using polyethylene glycol solutes and investigated the activation equilibrium response using UV‐Visible spectroscopy. Our results show a flood of ~ 80 water molecules into the rhodopsin interior during photoactivation, a result supported by atomistic molecular dynamics simulations [3]. Furthermore, the osmolyte effects on rhodopsin activation are size dependent: large osmolytes back shift the equilibrium to inactive metarhodopsin‐I (MI), while small osmolytes forward‐shift the equilibrium to active metarhodopsin‐II (MII). We attribute these size effects to varying degrees of osmolyte penetration into the rhodopsin core. Large polymers behave similarly to ideal osmolytes and dehydrate rhodopsin, while smaller polymers wriggle into the rhodopsin interior and stabilize the open MII conformation of rhodopsin. Besides osmotic pressure, the application of hydrostatic pressure also back shifts the metarhodopsin equilibrium but for fundamentally different reasons. Integrating the two force‐based methods together with neutron scattering experiments indicates that the active state of rhodopsin is more hydrated yet simultaneously more structurally collapsed [4]. At the same time, the active GPCR undergoes dynamic volume fluctuations and solvent coupling, which give rise to greater thermal volume. Our results necessitate a new understanding of GPCR activation in which the surrounding soft matter is paramount in governing conformational energy landscapes.Support or Funding InformationNSF (MCB 11817862 and CHE 1904125) and US NIH (EY012049 and EY02604)

G-Protein-Coupled Receptors are Solvent-Swollen in the Functionally Active State

G-protein-coupled receptors (GPCRs) are transmembrane receptors that play critical roles in a number of physiological signaling processes. In the standard biochemical model, GPCRs behave as primarily agonist-dependent bimodal switches, with little influence of the surrounding medium. However, using the visual receptor rhodopsin as a model GPCR, we show that water drives rhodopsin to a partially disordered, solvent-swollen conformational ensemble upon light absorption, rendering the standard model obsolete. We placed rhodopsin under varying degrees of osmotic stress using polyethylene glycol solutes and investigated the activation equilibrium response using UV-visible spectroscopy. Our results show a flood of ∼80 water molecules into the rhodopsin interior during photoactivation, a result supported by atomistic MD simulations [1]. Furthermore, the osmolyte effects on rhodopsin activation are size-dependent: large osmolytes back shift the equilibrium to inactive metarhodopsin-I (MI), while small osmolytes forward-shift the equilibrium to active metarhodopsin-II (MII). We attribute these size effects to varying degrees of osmolyte penetration into the rhodopsin core. Large polymers behave similarly to ideal osmolytes and dehydrate rhodopsin, while smaller polymers wriggle into the rhodopsin interior and stabilize the open MII conformation of rhodopsin. Besides osmotic pressure, the application of hydrostatic pressure also back shifts the metarhodopsin equilibrium but for fundamentally different reasons. Integrating the two force-based methods together with neutron scattering experiments [2] indicates that the active state of rhodopsin is more hydrated yet more locally collapsed. At the same time, the active GPCR undergoes volume fluctuations and solvent coupling, which give rise to greater thermal volume. The combined force-based results necessitate a new understanding of GPCR activation in which the surrounding soft matter is paramount in governing conformational energy landscapes. [1] N. Leioatts et al. (2014) Biochemistry 53, 376−385. [2] S.M.D.C. Perera et al. (2018) J. Phys. Chem. Lett.9, 7064−7071.

Controlling GPCR Rhodopsin Function by Small, Physiologically Relevant Changes in Bilayer Hydrophobic Thickness

It was established previously that the G protein-coupled membrane receptor rhodopsin has transmembrane helices which match a hydrophobic bilayer thickness of 27±1 A. Here we demonstrate that small changes of bilayer thickness of ±2 A about that match point translate in the considerable changes of rhodopsin activation measured as the metarhodopsin I (MI)/metarhodopsin II (MII) equilibrium. We observed a biphasic behavior of the MI/MII equilibrium, with a sharp decline towards MI from 25-27 A followed by a rapid increase of MII from 27-29 A. Results are qualitatively identical for thickness changes induced by mixing of 16:0-16:1 PC and 18:0-18:1 PC, or 16:1-16:1 PC and 18:1-18:1 PC, or addition of 0-30 mol% cholesterol to 16:0-16:1 PC. The biphasic behavior was observed regardless of lipids used to alter bilayer hydrophobic thickness suggesting a relationship between small changes in hydrophobic thickness and rhodopsin function. It strongly favors an explanation based on a change of elastic stresses in lipid bilayers upon the transition from negative curvature in lipid monolayers near the protein below 27 A hydrophobic thickness to positive monolayer curvature above the match point. A continuum elastic model of the membrane, including the effect of lipid monolayer curvature near the protein, predicts membrane mediated clustering of rhodopsin and stabilization of the MI photointermediate at the matching point. Small, physiologically relevant changes in cholesterol content of bilayers with a thickness in the physiologically relevant range do drastically down- or up regulate the amount of MII which is the state that activates G protein.

Contribution of glutamic acid in the conserved E/DRY triad to the functional properties of rhodopsin.

Rhodopsin is a G protein-coupled receptor specialized for photoreception and contains a light-absorbing chromophore retinal that binds to the lysine residue of opsin through a protonated Schiff base linkage. Light converts rhodopsin to an equilibrium mixture of the active state metarhodopsin II (MII) and its precursor, metarhodopsin I (MI), which have deprotonated and protonated Schiff base chromophores, respectively. This equilibrium was thought to depend on the pKa of not the Schiff base chromophore but glutamic acid E134 in the highly conserved E/DRY triad in helix III. We performed mutational analyses of E134 and nearby residues to examine whether the equilibrium is really dependent on the pKa of E134 and to obtain clues about the contribution of E134 to the G protein activation characteristics of rhodopsin. All the single mutants at position 134 except for E134D lost the characteristic pH-dependent equilibrium, indicating that the carboxyl group of E134 is responsible for the equilibrium. Interestingly, mutation at position 134 caused little change in the MI or MII spectra or G protein activation efficiency of MII, while it caused a shift of the MI-MII equilibrium. The mutants containing hydrophobic or amide-containing residues at position 134 formed an equilibrium in favor of MII, resulting in an increase in light-induced G protein activation efficiency. On the other hand, the wild type exhibited an opsin activity lower than those of the mutants, which exhibited reasonable light-dependent activities. These results strongly suggest that the evolutionary significance of E134 is not an increase in G protein activity but rather suppression of the opsin activity.

Rhodopsin Crowding in Model Lipid Bilayers - Functional Implications

In the rod outer segment disks of the retina, rhodopsin is densely packed in phospholipid bilayers with a high content of polyunsaturated acyl chains. It has been hotly debated if oligomerizationof rhodopsin is a critical step for efficient activation of G-protein. Here, we investigated the effect of rhodopsin density in synthetic membranes on the interaction with its cognate G-protein transducin (Gt). Experiments were conducted at rhodopsin:lipid ratios ranging from 1:4000 to 1:70 with sn1-stearoyl sn2-oleoyl phosphatidylcholine (18:0-18:1 PC) model bilayers at ambient temperature.The amount of metarhodopsin-II (MII) formed after photoactivation was determined by UV-visible spectroscopy. Guanine nucleotide exchange rate measured using labeled GTPγS was used to monitor Gt affinity for activated rhodopsin, the rate of rhodopsin catalyzed Gt activation, and the decay rate of the active photointermediate.At low rhodopsin density (1:1000 and below), MII concentration was the highest and independent of rhodopsin concentration. Increasing rhodopsin packing density correlated with a shift in the metarhodopsin-I (MI)/MII equilibrium towards MI. After photoactivation, rhodopsin decayed at two different rates (t1/2 ∼ 3 min and > 60 min) and the proportion of the fast decaying photointermediate decreased with increasing rhodopsin density. Finally, MII-catalyzed GDP-GTPγS nucleotide exchange rates for Gt were crucially affected by rhodopsin density. The enzymatic power of rhodopsin (Vmax/Km for Gt) was higher by 2 orders of magnitude at low rhodopsin density as compared to high rhodopsin density.Our results suggest that, in model membranes, rhodopsin exists as an equilibrium between at least two populations: monomeric at low rhodopsin density with rapid decay and high catalytic efficiency and oligomeric at high rhodopsin density with slow decay and inefficient catalysis of nucleotide exchange.

Membrane Curvature Elastic Stress Strongly Modulates Metarhodopsin II Formation

We studied the influence of curvature elastic stress in lipid monolayers induced by hydrophobic mismatch between lipid bilayer thickness and the hydrophobic length of rhodopsin transmembrane helices on the metarhodopsin I (MI)/metarhodopsin II (MII) equilibrium. Experiments were conducted at the low rhodopsin/lipid ratio of 1/1,000 to suppress rhodopsin oligomerization. Elastic stress was generated by reconstituting dark-adapted rhodopsin into a series of phosphatidylcholine (PC) bilayers with acyl chains of 14-20 carbons in length and cholesterol content varied from 0 to 30 mol %. 2H NMR was used to monitor the adjustment of the length of hydrocarbon chains to the protein and to characterize monolayer curvature at the protein-lipid interface. We found that the average length of hydrophobic regions on rhodopsin transmembrane helices is 2.6 ± 0.1 nm. Thinner bilayers stretch and bend with negative monolayer curvature to match the length of hydrophobic helices, while thicker bilayers get thinner with positive monolayer curvature near the protein. Shifts in the MI/MII equilibrium depended on the sign of monolayer curvature: negative curvature favored MI while positive curvature favored MII. The results suggest that MII formation generates negative curvature in monolayers near the protein, thus raising curvature stress for thin bilayers but releasing stress for thicker bilayers. Addition of cholesterol increases bilayer hydrophobic thickness, but otherwise showed identical behavior: bilayers with a hydrophobic thickness less than 2.6 nm favored MI while thicker bilayers favored MII. In case of severe hydrophobic mismatch between rhodopsin and bilayers, the behavior was complicated by rhodopsin oligomerization that seems to favor MI. The sensitivity of the MI/MII equilibrium to negative curvature elastic stress was observed earlier in experiments with phosphatidylethanolamines by us and the Brown laboratory. Negative curvature stress upon MII formation is critical for understanding shifts in the MI/MII equilibrium.

Quantifying the differential effects of DHA and DPA on the early events in visual signal transduction

A range of evidence from animal, clinical and epidemiological studies indicates that highly polyunsaturated acyl chains play important roles in development, cognition, vision and other aspects of neurological function. In a number of these studies n3 polyunsaturated fatty acids (PUFAs) appear to be more efficacious than n6 PUFAs. In a previous study of retinal rod outer segments obtained from rats raised on either an n3 adequate or deficient diet, we demonstrated that the replacement of 22:6n3 by 22:5n6 in the n3 deficient rats led to functional deficits in each step in the visual signaling process (Niu et al., 2004). In this study, we examined rhodopsin and phosphodiesterase function and acyl chain packing properties in membranes consisting of phosphatidylcholines with sn-1=18:0, and sn-2=22:6n3, 22:5n6, or 22:5n3 in order to determine if differences in function are due to the loss of one double bond or due to differences in double bond location. At 37°C the n6 lipid shifted the equilibrium between the active metarhodopsin II (MII) state and inactive metarhodopsin I (MI) state towards MI. In addition, 22:5n6 reduced the rates of MII formation and MII-transducin complex formation by 2- and 6-fold, respectively. At a physiologically relevant level of rhodopsin light stimulation, the activity of phosphodiesterase was reduced by 50% in the 22:5n6 membrane, relative to either of the n3 membranes. Activity levels in the two n3 membranes were essentially identical. Ensemble acyl chain order was assessed with time-resolved fluorescence measurements of the membrane probe diphenylhexatriene (DPH). Analysis in terms of the orientational distribution of DPH showed that acyl chain packing in the two n3 membranes is quite similar, while in the 22:5n6 membrane there was considerably less packing disorder in the bilayer midplane. These results demonstrate that the n3 bond configuration uniquely optimizes the early steps in signaling via a mechanism which may involve acyl chain packing deep in the bilayer.

Cholesterol Enhances or Reduces Metarhodopsin II Formation Depending on Bilayer Thickness

Cholesterol is one of the most efficient modulators of G Protein-Coupled Receptor (GPCR) function. We monitored the effect of cholesterol on rhodopsin function for a set of bilayers with different hydrophobic thickness. Surprisingly, cholesterol shifts the Metarhodopsin-I (MI)/Metarhodopsin-II (MII) equilibrium toward MII for bilayers thinner than the average length of hydrophobic transmembrane helices (2.7 nm), and to MI for thicker bilayers. In previous work conducted on rod outer segment disks and model membranes, increasing cholesterol concentration always shifted the equilibrium towards MI. It was proposed that the cholesterol effect is primarily related to a tighter packing of lipid hydrocarbon chains which generates a less permissive environment for the formation of MII. To gain deeper insights into mechanisms, we followed changes in lipid-rhodopsin interaction by 2H NMR using deuterated lipids. It was reported by us and the Brown laboratory that an increase of bilayer hydrophobic thickness in the absence of cholesterol favors MII with a turnover to MI for bilayers that are very thick. Indeed, the cholesterol-induced shifts towards MII for thinner membranes correlated nicely with the cholesterol-induced increase of bilayer hydrophobic thickness measured by NMR suggesting that the increase in bilayer thickness by cholesterol plays a major role in controlling the energetics of the MI-MII equilibrium. Furthermore, changes in average lipid order parameters due to the presence of rhodopsin were much larger in cholesterol-containing membranes than in cholesterol-free membranes suggesting strongly that the perturbation in the lipid matrix from protein insertion reaches much further away from the protein. This is expected for membranes that are stiffer due to the presence of cholesterol. The consequences of these findings for lipid mediated shifts in rhodopsin function and rhodopsin-rhodopsin interactions will be discussed.

Rhodopsin - Rhodopsin Oligomerization in Model Lipid Bilayers - Functional Implications

We studied rhodopsin oligomerization as a function of rhodopsin concentration and lipid composition and related oligomerization to shifts in rhodopsin function. In the rod outer segment disks of the retina, rhodopsin is densely packed in phospholipid bilayers with a high content of polyunsaturated acyl chains. In model membranes, increasing rhodopsin packing density was linked to a shift in the metarhodopsin-I (MI)/metarhodopsin-II (MII) equilibrium towards MI as well as to lower rates of MII formation. We reconstituted rhodopsin into various phosphatidylcholine bilayers at rhodopsin/lipid ratios ranging from 1:1,000 to 1:70 and followed rhodopsin oligomerization by cross linking of rhodopsin and changes in lipid-rhodopsin interactions by 2H NMR. The amount of MII formed after photoactivation was determined by UV/vis spectroscopy and the rate of transducin activation studied with a GTPγS-assay. At low rhodopsin concentrations (1/300 and lower) rhodopsin appears to be predominantly monomeric. At rhodopsin/lipid ratios higher than 1/300, the level of oligomerization increases in a highly cooperative fashion with concentration such that at physiological concentrations rhodopsin is mostly oligomeric. Protein function correlated tightly with rhodopsin oligomerization. Data on the influence of bilayer properties on the monomer - oligomer transition of rhodopsin and the rate of transducin activation will be presented.

Contribution of Membrane Elastic Energy to Rhodopsin Function

We considered the issue of whether shifts in the metarhodopsin I (MI)-metarhodopsin II (MII) equilibrium from lipid composition are fully explicable by differences in bilayer curvature elastic stress. A series of six lipids with known spontaneous radii of monolayer curvature and bending elastic moduli were added at increasing concentrations to the matrix lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and the MI-MII equilibrium measured by flash photolysis followed by recording UV-vis spectra. The average area-per-lipid molecule and the membrane hydrophobic thickness were derived from measurements of the 2H NMR order parameter profile of the palmitic acid chain in POPC. For the series of ethanolamines with different levels of headgroup methylation, shifts in the MI-MII equilibrium correlated with changes in membrane elastic properties as expressed by the product of spontaneous radius of monolayer curvature, bending elastic modulus, and lateral area per molecule. However, for the entire series of lipids, elastic energy explained the shifts only partially. Additional contributions correlated with the capability of the ethanolamine headgroups to engage in hydrogen bonding with the protein, independent of the state of ethanolamine methylation, with introduction of polyunsaturated sn-2 hydrocarbon chains, and with replacement of the palmitic acid sn-1 chains by oleic acid. The experiments point to the importance of interactions of rhodopsin with particular lipid species in the first layer of lipids surrounding the protein as well as to membrane elastic stress in the lipid-protein domain.

Direct Observation of the pH-Dependent Equilibrium between Metarhodopsins I and II and the pH-Independent Interaction of Metarhodopsin II with Transducin C-Terminal Peptide

Bovine rhodopsin contains 11-cis-retinal as a light-absorbing chromophore that binds to a lysine residue of the apoprotein opsin via a protonated Schiff base linkage. Light isomerizes 11-cis-retinal into the all-trans form, which eventually leads to the formation of an enzymatically active state, metarhodopsin II (MII). It is widely believed that MII forms a pH-dependent equilibrium with metarhodopsin I (MI), but direct evidence for this equilibrium has not been reported. Here, we confirmed this equilibrium by direct observation of the mutual conversions of MI and MII upon changing the pH of the MI/MII mixture. We also observed a reversible binding of the synthetic peptide constituting the C-terminal 11 amino acids of the transducin alpha-subunit to MII, which resulted in change of the amounts of MI and MII in the equilibrium. Interestingly, addition of the peptide did not induce a simple pK(a) shift but rather induced an increase of the MII fraction at high pH. These results indicate that in addition to the MII that is formed from MI in a pH-dependent manner there also exists another MII, which is in equilibrium with MI in a pH-independent manner and can bind to the peptide. Therefore, there is no need for proton uptake by the protein moiety of opsin for the binding to the peptide.

Salt Dependence of the Formation and Stability of the Signaling State in G Protein-Coupled Receptors: Evidence for the Involvement of the Hofmeister Effect

We studied the salt dependence of both the stability and the equilibrium of the late photoproducts metarhodopsin I (MI) and II (MII) of the artificial visual pigment 9-demethyl rhodopsin (9dm-Rho). In the photoproducts of 9dm-Rho, all-trans-9-demethyl retinal acts only as a partial agonist, enabling us to study the photoproduct equilibrium of the pigment both in membranes and in detergent micelles. Chloride, bromide, and phosphate salts shift this equilibrium from the inactive MI to the active MII receptor conformation both in native membranes and even more with purified pigment in detergent micelles. In the presence of these salts, the induced MII state seems to be structurally intact, as judged by Fourier transform infrared (FTIR) and UV-vis spectroscopy. In the long term, however, we observe an increased instability of the photoproducts and a change in the decay pathways. Both MII enhancement and destabilization are particularly pronounced with the strong chaotropic salts KI and KSCN. The results fit into the framework of the Hofmeister effect and are assigned to an increased solvation of the peptide moiety of the solvent-exposed domains, their resulting partial disordering favoring MII over MI. In this picture, increased solvation also affects helix-helix interactions, thereby leading to a structural instability of the protein in the long term. The reported influences of salts on conformation and stability of this membrane protein are likely to be general and may therefore also apply to other transmembrane proteins and particularly to other G protein-coupled receptors.

Spectroscopic evidence for interaction between transmembrane helices 3 and 5 in rhodopsin.

Recent molecular models of rhodopsin (Rho) propose a specific interaction between transmembrane (TM) helices 3 and 5, which appears to be mediated by amino acid residues Glu122 and His211 on TM helices 3 and 5, respectively. To test this proposed interaction, four single-site histidine replacement mutants (H100N, H152N, H211N, and H211F), two single-site glutamic acid replacement mutants (E122Q and E122A), and three double-site replacement mutants (E122Q/H211F, E122Q/H211N, and E122A/H211F) of Rho were prepared. The expressed mutant pigments reconstituted into membranes were studied by FTIR difference spectroscopy addressing especially the transition to metarhodopsin I (MI). It is shown that the lipid environment influences bands typical of the MI state. Spectra of mutants with substituted Glu122 allowed assignments of the C=O stretch of protonated Glu122 in the dark state and in MI of Rho. Mutation of His211, but not of other histidine residues, affects these vibrational modes assigned to Glu122. In addition, replacements of His211 affect protein modes that are proposed to arise from a third, hydroxyl-bearing group, which also interacts with Glu122. These modes are influenced as well when Glu122 is replaced by Ala in mutant E122A but not when it is replaced by Gln in mutant E122Q. These results provide direct experimental evidence for an interaction between TM helices 3 and 5 in Rho, which is mediated by Glu122 and His211.

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.