Dynamics of the Internal Water Molecules in Squid Rhodopsin

Understanding the mechanism of G-protein coupled receptors action is of major interest for drug design. The visual rhodopsin is the prototype structure for the family A of G-protein coupled receptors. Upon photoisomerization of the covalently bound retinal chromophore, visual rhodopsins undergo a large-scale conformational change that prepares the receptor for a productive interaction with the G-protein. The mechanism by which the local perturbation of the retinal cis-trans isomerization is transmitted throughout the protein is not well understood. The crystal structure of the visual rhodopsin from squid solved recently suggests that a chain of water molecules extending from the retinal toward the cytoplasmic side of the protein may play a role in the signal transduction from the all-trans retinal geometry to the activated receptor. As a first step toward understanding the role of water in rhodopsin function, we performed a molecular dynamics simulation of squid rhodopsin embedded in a hydrated bilayer of polyunsaturated lipid molecules. The simulation indicates that the water molecules present in the crystal structure participate in favorable interactions with side chains in the interhelical region and form a persistent hydrogen-bond network in connecting Y315 to W274 via D80.     

Role of membrane integrity on G protein-coupled receptors: Rhodopsin stability and function

Rhodopsin is a prototypical G protein-coupled receptor (GPCR) - a member of the superfamily that shares a similar structural architecture consisting of seven-transmembrane helices and propagates various signals across biological membranes. Rhodopsin is embedded in the lipid bilayer of specialized disk membranes in the outer segments of retinal rod photoreceptor cells where it transmits a light-stimulated signal. Photoactivated rhodopsin then activates a visual signaling cascade through its cognate G protein, transducin or Gt, that results in a neuronal response in the brain. Interestingly, the lipid composition of ROS membranes not only differs from that of the photoreceptor plasma membrane but is critical for visual transduction. Specifically, lipids can modulate structural changes in rhodopsin that occur after photoactivation and influence binding of transducin. Thus, altering the lipid organization of ROS membranes can result in visual dysfunction and blindness.     

How a small change in retinal leads to G-protein activation: initial events suggested by molecular dynamics calculations

Rhodopsin is the prototypical G-protein coupled receptor, coupling light activation with high efficiency to signaling molecules. The dark-state X-ray structures of the protein provide a starting point for consideration of the relaxation from initial light activation to conformational changes that may lead to signaling. In this study we create an energetically unstable retinal in the light activated state and then use molecular dynamics simulations to examine the types of compensation, relaxation, and conformational changes that occur following the cis-trans light activation. The results suggest that changes occur throughout the protein, with changes in the orientation of Helices 5 and 6, a closer interaction between Ala 169 on Helix 4 and retinal, and a shift in the Schiff base counterion that also reflects changes in sidechain interactions with the retinal. Taken together, the simulation is suggestive of the types of changes that lead from local conformational change to light-activated signaling in this prototypical system.     

Molecular simulations and solid-state NMR investigate dynamical structure in rhodopsin activation

Rhodopsin has served as the primary model for studying G protein-coupled receptors (GPCRs)-the largest group in the human genome, and consequently a primary target for pharmaceutical development. Understanding the functions and activation mechanisms of GPCRs has proven to be extraordinarily difficult, as they are part of a complex signaling cascade and reside within the cell membrane. Although X-ray crystallography has recently solved several GPCR structures that may resemble the activated conformation, the dynamics and mechanism of rhodopsin activation continue to remain elusive. Notably solid-state ((2))H NMR spectroscopy provides key information pertinent to how local dynamics of the retinal ligand change during rhodopsin activation. When combined with molecular mechanics simulations of proteolipid membranes, a new paradigm for the rhodopsin activation process emerges. Experiment and simulation both suggest that retinal isomerization initiates the rhodopsin photocascade to yield not a single activated structure, but rather an ensemble of activated conformational states. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Circular dichroism,optical rotatory dispersion,and absorption studies on the conformation of bovine rhodopsin in situ and solubilized with detergent

Circular dichroism, optical rotatory dispersion and absorption of rhodopsin, the visual pigment of bovine rod outer segment membranes, were studied in situ and in membranes solubilized with various detergents. The -helical content of the membrane protein is approximately 30%. The membrane protein possesses little -structure. Solubilization of the membrane by the detergents, Emulphogene BC-720 and cetyltrimethylammonium salts, results in loss of protein helical structure and perturbation of aromatic residues. These effects are not observed on digitonin solubilization.In regard to the structural stability of the membrane during bleaching, the following conclusions were reached: (1) Delocalized conformational changes of rhodopsin in situ involving secondary and/or tertiary structure are very unlikely. (2) Localized conformational changes of rhodopsin in situ involving secondary structure must be limited to the involvement of no more than three amino acid residues and localized conformational changes involving tertiary structure must be limited to very short segments of the protein chain containing, at the most, only a few aromatic residues. (3) Large changes in the interaction of lipid and protein moieties of the membrane are unlikely. (4) The detergents, Emulphogene, cetyltrimethylammonium salts, and digitonin, significantly decrease the conformational stability of rhodopsin as compared to the in situ conditions. The effect is smaller with digitonin.Evidence is presented against a proposed mechanism by which optical activity of the prosthetic group, retinal, is induced by resonance coupling of the transition dipoles of retinal and the lowest energy transitions of the aromatic groups of the apoprotein, opsin. A mechanism in which atropisomers of retinal are preferentially bound by opsin is consistent with the present results. The optical activity of the prosthetic group is markedly changed upon solubilization of the membrane by detergent. This change in optical activity is probably coupled to changes in conformation of the protein moiety induced by solubilization.This work is based in part upon a Ph.D. dissertation submitted by C.N.R. to The Ohio State University (1974). A preliminary report of this work was presented at the sixteenth annual meeting of the Biophysical Society, Toronto, Canada, February, 1972, Abstracts SaPM-H8 and SaPM-H9     

Docking of human rhodopsin mutant (Gly90→Asp) with beta-arrestin and cyanidin 3-rutinoside to cure night blindness

MOTIVATION: Rhodopsin is a visual pigment present in rod cells of retina. It belongs to GPCR family and involves photoisomerization of 11-cis-retinal to all-trans-retinal isomers, conformational changes in rhodopsin and signal transduction cascade to generate a nerve impulse. This signaling pathway has been targeted to eliminate the effect of a mutation (Gly90→Asp) responsible for abnormal activation of G-protein without retinal conformations in the absence of light leading to congenital night blindness. A theoretical model of rhodopsin with induced mutation has been deliberated in order to find potential ligands which can offset this mutational effect. The binding interactions between the target mutated rhodopsin model and potential ligands have been predicted with the help of molecular docking. The results indicated strong functional benefits of ligands as an inhibitor and an agonist for mutated rhodopsin model. Therefore, we propose a new visual cascade model which can initiate the normal signaling of rhodopsin mutant with the help of proposed ligands and can provide a hope for vision in future.     

Human Blue Cone Opsin Regeneration Involves Secondary Retinal Binding with Analog Specificity

Human color vision is mediated by the red, green, and blue cone visual pigments. Cone opsins are G-protein-coupled receptors consisting of an opsin apoprotein covalently linked to the 11-cis-retinal chromophore. All visual pigments share a common evolutionary origin, and red and green cone opsins exhibit a higher homology, whereas blue cone opsin shows more resemblance to the dim light receptor rhodopsin. Here we show that chromophore regeneration in photoactivated blue cone opsin exhibits intermediate transient conformations and a secondary retinoid binding event with slower binding kinetics. We also detected a fine-tuning of the conformational change in the photoactivated blue cone opsin binding site that alters the retinal isomer binding specificity. Furthermore, the molecular models of active and inactive blue cone opsins show specific molecular interactions in the retinal binding site that are not present in other opsins. These findings highlight the differential conformational versatility of human cone opsin pigments in the chromophore regeneration process, particularly compared to rhodopsin, and point to relevant functional, unexpected roles other than spectral tuning for the cone visual pigments.     

Conformational energetics of rhodopsin modulated by nonlamellar-forming lipids

Rhodopsin is an important example of a G protein-coupled receptor (GPCR) in which 11-cis-retinal is the ligand and acts as an inverse agonist. Photolysis of rhodopsin leads to formation of the activated meta II state from its precursor meta I. Various mechanisms have been proposed to explain how the membrane composition affects the meta I-meta II conformational equilibrium in the visual process. For rod disk membranes and recombinant membranes containing rhodopsin, the lipid properties have been discussed in terms of elastic deformation of the bilayer. Here we have investigated the relation of nonlamellar-forming lipids, such as dioleoylphosphatidylethanolamine (DOPE), together with dioleoylphosphatidylcholine (DOPC), to the photochemistry of membrane-bound rhodopsin. We conducted flash photolysis experiments for bovine rhodopsin recombined with DOPE/DOPC mixtures (0:100 to 75:25) as a function of pH to explore the dependence of the photochemical activity on the monolayer curvature free energy of the membrane. It is well-known that DOPC forms bilayers, whereas DOPE has a propensity to adopt the nonlamellar, reverse hexagonal (H(II)) phase. In the case of neutral DOPE/DOPC recombinants, calculations of the membrane surface pH confirmed that an increase in DOPE favored the meta II state. Moreover, doubling the PE headgroup content versus the native rod membranes substituted for the polyunsaturated, docosahexaenoic acyl chains (22:6 omega 3), suggesting rhodopsin function is associated with a balance of forces within the bilayer. The data are interpreted by applying a flexible surface model, in which the meta II state is stabilized by lipids tending to form the H(II) phase, with a negative spontaneous curvature. A simple theory, based on principles of surface chemistry, for coupling the energetics of membrane proteins to material properties of the bilayer lipids is described. For rhodopsin, the free energy balance of the receptor and the lipids is altered by photoisomerization of retinal and involves curvature stress/strain of the membrane (frustration). A new biophysical principle is introduced: matching of the spontaneous curvature of the lipid bilayer to the mean curvature of the lipid/water interface adjacent to the protein, which balances the lipid/protein solvation energy. In this manner, the thermodynamic driving force for the meta I-meta II conformational change of rhodopsin is tightly controlled by mixtures of nonlamellar-forming lipids having distinctive material properties.     

Mechanism and specificity of rhodopsin phosphorylation.

Partial separation of protein kinase activity from rhodopsin in isolated bovine retinal photoreceptor outer segments was accomplished by mild ultrasonic treatment followed by ultracentrifugation. Residual kinase activity in the rhodopsin-rich sediment was destroyed by chemical denaturation which did not affect the spectral properties of the rhodopsin. The retinal outer segment kinase was found to be specific for rhodopsin, since in these preparations it alone of several bovine protein kinases was capable of phosphorylating rhodopsin in the light. The phosphorylation reaction apparently requires a specific conformation of the rhodopsin molecule since it is abolished by heat denaturation of rhodopsin, and it is greatly reduced or abolished by treatment of the visual pigment protein with potassium alum after the rhodopsin has been "bleached" by light. When kinase and rhodopsin or opsin fractions were prepared from dark-adapted and bleached outer segments and the resultant fractions were mixed in various combinations of bleached and unbleached preparations, the observed pattern of light-activated phosphorylation was consistent only with the interpretation that a conformational change in the rhodopsin molecule in the light exposes a site on the visual pigment protein to the kinase and ATP. These results rule out the possibility of a direct or indirect (rhodopsin-mediated) light activation of the kinase. Finally, phosphorylation of retinal outer segment protein in monochromatic lights of various wavelengths followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that both rhodopsin and the higher molecular weight visual pigment protein reported by several laboratories have the same action spectrum for phosphorylation. This result is consistent with the suggestion that the higher molecular weight species is a rhodopsin dimer.     

Effect of packing density on rhodopsin stability and function in polyunsaturated membranes

Rod outer segment disk membranes are densely packed with rhodopsin. The recent notion of raft or microdomain structures in disk membranes suggests that the local density of rhodopsin in disk membranes could be much higher than the average density corresponding to the lipid/protein ratio. Little is known about the effect of high packing density of rhodopsin on the structure and function of rhodopsin and lipid membranes. Here we examined the role of rhodopsin packing density on membrane dynamic properties, membrane acyl chain packing, and the structural stability and function of rhodopsin using a combination of biophysical and biochemical techniques. We reconstituted rhodopsin into large unilamellar vesicles consisting of polyunsaturated 18:0,22:6n3PC, which approximates the polyunsaturated nature of phospholipids in disk membranes, with rhodopsin/lipid ratios ranging from 1:422 to 1:40. Our results showed that increased rhodopsin packing density led to reduced membrane dynamics revealed by the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene, increased phospholipid acyl chain packing, and reduced rhodopsin activation, yet it had minimal impact on the structural stability of rhodopsin. These observations imply that densely packed rhodopsin may impede the diffusion and conformational changes of rhodopsin, which could reduce the speed of visual transduction.     

Conformations of the active and inactive states of opsin

The signaling state metarhodopsin II of the visual pigment rhodopsin decays to the apoprotein opsin and all-trans retinal, which are then regenerated to rhodopsin by the visual cycle. Opsin is known to have at neutral pH only a small residual constitutive activity toward its G protein transducin, which is thought to play a considerable role in light adaptation (bleaching desensitization). In this study we show with Fourier-transform infrared spectroscopy that after metarhodopsin II decay, opsin exists in two conformational states that are in a pH-dependent equilibrium at 30 degrees C with a pK of 4.1 in the presence of hydroxylamine scavenging the endogenous all-trans retinal. Despite the lack of the native agonist in its binding pocket, the low pH opsin conformation is very similar to that of metarhodopsin II and is likewise stabilized by peptides derived from rhodopsin's cognate G protein, transducin. The high pH form, on the other hand, has some conformational similarity to the inactive metarhodopsin I state. We therefore conclude that the opsin apoprotein displays intrinsic conformational states that are merely modulated by bound all-trans retinal.     

Time-resolved rapid-scan Fourier transform infrared difference spectroscopy on a noncyclic photosystem: rhodopsin photointermediates from Lumi to Meta II

The visual pigment rhodopsin has been extensively studied for the kinetics of its photointermediates by various spectroscopic methods. Unlike such archaeal retinal proteins as bacteriorhodopsin, visual rhodopsin does not thermally recover its dark state after photoexcitation, which precludes repeated excitation of a single sample and thereby complicates time-resolved experiments. Kinetic data on the late rhodopsin photointermediates have so far been available mainly from time-resolved ultraviolet (UV)-visible spectroscopy, but not from Fourier transform infrared (FTIR) spectroscopy. The latter has the advantage of being informative of structural changes of both chromophore and protein, but does not allow the highly reproducible, automated sample exchange procedures available to UV-visible spectroscopy. Using rapid-scan FTIR difference spectroscopy, we obtained time-resolved data sets that were analyzed by a maximum entropy inverse Laplace-transform. Covering the time range from 8 ms to 15 s at temperatures of 0 and -7 degrees C, the transitions from the Lumi to the Meta I and from the Meta I to the Meta II photoproduct states could be resolved. In the transition from Meta I to Meta II, our data reveal a partial deprotonation of the retinal Schiff base preceding the conformational change of the receptor protein to Meta II. The technique and the results are discussed in regard to its advantages as well as its limitations.     

视紫红质及RPE65蛋白在光致视网膜变性疾病中的作用机制

视紫红质是感光细胞中的一种视色素,在光线的接收和视觉电位的产生方面具有重要的生理作用,由视紫红质介导的过度光信号传导是光性视网膜变性的主要原因。近年的研究表明,视网膜色素上皮细胞中的RPE65蛋白作为影响视紫红质再生的关键因素,与视网膜光损伤的易感性密切相关。就视紫红质和RPE65蛋白在光致视网膜变性中的作用机理作一探讨。     

Deuterium NMR structure of retinal in the ground state of rhodopsin

The conformation of retinal bound to the G protein-coupled receptor rhodopsin is intimately linked to its photochemistry, which initiates the visual process. Site-directed deuterium ((2)H) NMR spectroscopy was used to investigate the structure of retinal within the binding pocket of bovine rhodopsin. Aligned recombinant membranes were studied containing rhodopsin that was regenerated with retinal (2)H-labeled at the C(5), C(9), or C(13) methyl groups by total synthesis. Studies were conducted at temperatures below the gel to liquid-crystalline phase transition of the membrane lipid bilayer, where rotational and translational diffusion of rhodopsin is effectively quenched. The experimental tilt series of (2)H NMR spectra were fit to a theoretical line shape analysis [Nevzorov, A. A., Moltke, S., Heyn, M. P., and Brown, M. F. (1999) J. Am. Chem. Soc. 121, 7636-7643] giving the retinylidene bond orientations with respect to the membrane normal in the dark state. Moreover, the relative orientations of pairs of methyl groups were used to calculate effective torsional angles between different planes of unsaturation of the retinal chromophore. Our results are consistent with significant conformational distortion of retinal, and they have important implications for quantum mechanical calculations of its electronic spectral properties. In particular, we find that the beta-ionone ring has a twisted 6-s-cis conformation, whereas the polyene chain is twisted 12-s-trans. The conformational strain of retinal as revealed by solid-state (2)H NMR is significant for explaining the quantum yields and mechanism of its ultrafast photoisomerization in visual pigments. This work provides a consensus view of the retinal conformation in rhodopsin as seen by X-ray diffraction, solid-state NMR spectroscopy, and quantum chemical calculations.     

The effect of illumination on the electrical conductance of rhodopsin solutions

An apparatus was constructed in order to record continuously and simultaneously changes in extinction and electrical conductance of rhodopsin solutions. With this apparatus, changes in electrical conductance on exposing rhodopsin to light were investigated. On illumination solutions of rhodopsin revealed a conductance change so long as they preserved their photosensitivity. The conductance change begins almost immediately upon illumination and is almost proportional to the amount of rhodopsin decomposed, continuing until rhodopsin is converted to indicator yellow. Near pH 7 the conductance is apt to increase slightly, while it decreases considerably outside the range of pH 6–9, being accompanied by a pH change towards neutrality. The conductance change is regarded as an essential property of rhodopsin, because it occurs in aqueous suspension as well as in digitonin solution; it may be caused by hydrogen or hydroxyl ions and some other conductive substances. It is also noteworthy that the petroleum ether-soluble component of the rod outer segments—presumably the lipide—tends to increase the conductance change. In suspensions of rod outer segments and retinal homogenates, the conductance increases on illumination irrespective of pH: this may be due to secondary reactions following the photic reaction of rhodopsin. We shall discuss the significance of the conductance change in relation to the initiation of visual excitation.     

Rhodopsin and 9-demethyl-retinal analog: effect of a partial agonist on displacement of transmembrane helix 6 in class A G protein-coupled receptors

Rhodopsin is the visual pigment of rod cells and a prototypical G protein-coupled receptor. It is activated by cis-->trans photoisomerization of the covalently bound chromophore 11-cis-retinal, which acts in the cis configuration as an inverse agonist. Light-induced formation of the full agonist all-trans-retinal in situ triggers conformational changes in the protein moiety. Partial agonists of rhodopsin include a retinal analog lacking the methyl group at C-9, termed 9-demethyl-retinal (9-dm-retinal). Rhodopsin reconstituted with this retinal (9-dm-rhodopsin) activates G protein poorly. Here we investigated the molecular nature of the partial agonism in 9-dm-rhodopsin using site-directed spin labeling. Earlier site-directed spin labeling studies of rhodopsin identified a rigid-body tilt of the cytoplasmic segment of [corrected] transmembrane helix 6 (TM6) by approximately 6A as a central event in rhodopsin activation. Data presented here provide additional evidence for this mechanism. Only a small fraction of photoexcited 9-dm pigments reaches the TM6-tilted conformation. This fraction can be increased by increasing proton concentration or [corrected] by anticipation of the activating protonation step by the mutation E134Q in 9-dm-rhodopsin. These results on protein conformation are in complete accord with previous findings regarding the biological activity of the 9-dm pigments. When the proton concentration is further increased, a new state arises in 9-dm pigments that is linked to direct proton uptake at the retinal Schiff base. This state apparently has a conformation distinguishable from the active state.     

Crystal structure of squid rhodopsin with intracellularly extended cytoplasmic region

G-protein-coupled receptors play a key step in cellular signal transduction cascades by transducing various extracellular signals via G-proteins. Rhodopsin is a prototypical G-protein-coupled receptor involved in the retinal visual signaling cascade. We determined the structure of squid rhodopsin at 3.7A resolution, which transduces signals through the G(q) protein to the phosphoinositol cascade. The structure showed seven transmembrane helices and an amphipathic helix H8 has similar geometry to structures from bovine rhodopsin, coupling to G(t), and human beta(2)-adrenergic receptor, coupling to G(s). Notably, squid rhodopsin contains a well structured cytoplasmic region involved in the interaction with G-proteins, and this region is flexible or disordered in bovine rhodopsin and human beta(2)-adrenergic receptor. The transmembrane helices 5 and 6 are longer and extrude into the cytoplasm. The distal C-terminal tail contains a short hydrophilic alpha-helix CH after the palmitoylated cysteine residues. The residues in the distal C-terminal tail interact with the neighboring residues in the second cytoplasmic loop, the extruded transmembrane helices 5 and 6, and the short helix H8. Additionally, the Tyr-111, Asn-87, and Asn-185 residues are located within hydrogen-bonding distances from the nitrogen atom of the Schiff base.     

Leukemia inhibitory factor coordinates the down-regulation of the visual cycle in the retina and retinal-pigmented epithelium

Leukemia inhibitory factor (LIF), an interleukin-6 family neurocytokine, is up-regulated in response to different types of retinal stress and has neuroprotective activity through activation of the gp130 receptor/STAT3 pathway. We observed that LIF induces rapid, robust, and sustained activation of STAT3 in both the retina and retinal pigmented epithelium (RPE). Here, we tested whether LIF-induced STAT3 activation within the RPE can down-regulate RPE65, the central enzyme in the visual cycle that provides the 11-cis-retinal chromophore to photoreceptors in vivo. We generated conditional knock-out mice to specifically delete STAT3 or gp130 in RPE, retina, or both RPE and retina. After intravitreal injection of LIF, we analyzed the expression levels of visual cycle genes and proteins, isomerase activity of RPE65, levels of rhodopsin protein, and the rates of dark adaptation and rhodopsin regeneration. We found that RPE65 protein levels and isomerase activity were reduced and recovery of bleachable rhodopsin was delayed in LIF-injected eyes. In mice with functional gp130/STAT3 signaling in the retina, rhodopsin protein was also reduced by LIF. However, the LIF-induced down-regulation of RPE65 required a functional gp130/STAT3 cascade intrinsic to RPE. Our data demonstrate that a single cytokine, LIF, can simultaneously and independently affect both RPE and photoreceptors through the same signaling cascade to reduce the generation and utilization of 11-cis-retinal.     

Molecular dynamics simulations of retinal in rhodopsin: from the dark-adapted state towards lumirhodopsin

The formation of photointermediates and conformational changes observed in the retinal chromophore of bilayer-embedded rhodopsin during the early steps of the protein activation have been studied by molecular dynamics (MD) simulation. In particular, the lysine-bound retinal has been examined, focusing on its conformation in the dark-adapted state (10 ns) and on the early steps after the isomerization of the 11-cis bond to trans (up to 10 ns). The parametrization for the chromophore is based on a recent quantum study [Sugihara, M., Buss, V., Entel, P., Elstner, M., and Frauenheim, T. (2002) Biochemistry 41, 15259-15266] and shows good conformational agreement with recent experimental results. The isomerization, induced by switching the function governing the dihedral angle for the C11=C12 bond, was repeated with several different starting conformations. From the repeated simulations, it is shown that the retinal model exhibits a conserved activation pattern. The conformational changes are sequential and propagate outward from the C11=C12 bond, starting with isomerization of the C11=C12 bond, then a rotation of methyl group C20, and followed by increased fluctuations at the beta-ionone ring. The dynamics of these changes suggest that they are linked with photointermediates observed by spectroscopy. The exact moment when these events occur after the isomerization is modulated by the starting conformation, suggesting that retinal isomerizes through multiple pathways that are slightly different. The amplitudes of the structural fluctuations observed for the protein in the dark-adapted state and after isomerization of the retinal are similar, suggesting a subtle mechanism for the transmission of information from the chromophore to the protein.     

Oxygen diffusion-concentration product in rhodopsin as observed by a pulse ESR spin labeling method.

Permeation of molecular oxygen in rhodopsin, an integral membrane protein, has been investigated by monitoring the bimolecular collision rate between molecular oxygen and the nitroxide spin label using a pulse electron spin resonance (ESR) T1 method. Rhodopsin was labeled by regeneration with the spin-labeled 9-cis retinal analogue in which the beta-ionone ring of retinal is replaced by the nitroxide tetramethyl-oxypyrrolidine ring. The bimolecular collision rate was evaluated in terms of an experimental parameter W(x), defined as T1(-1)(air,x)--T1(-1)(N2,x) where T1's are the spin-lattice relaxation times of the nitroxide in samples equilibrated with atmospheric air and nitrogen respectively, which is proportional to the product of local oxygen concentration and local diffusion coefficient (transport). W-values at the beta-ionone binding site in spin-labeled rhodopsin are in the range of 0.02-0.13 microseconds-1, which are 10-60 times smaller than W's in water and 1.1-20 times smaller than in model membranes in the gel phase, indicating that membrane proteins create significant permeation resistance to transport of molecular oxygen inside and across the membrane. W(thereby the oxygen diffusion-concentration product) is larger in the meta II-enriched sample than in rhodopsin, indicating light-induced conformational changes of opsin around the beta-ionone binding site. W decreases with increase of temperature for both rhodopsin and meta II-enriched samples, suggesting that temperature-induced conformational changes take place in both samples. These changes were not observable using conventional ESR spectroscopy. It is concluded that W is a sensitive monitor of conformational changes of proteins.