Rhodopsin In Native Membranes Research Articles

Chromophore hydrolysis and release from photoactivated rhodopsin in native membranes

For sustained vision, photoactivated rhodopsin (Rho*) must undergo hydrolysis and release of all-trans-retinal, producing substrate for the visual cycle and apo-opsin available for regeneration with 11-cis-retinal. The kinetics of this hydrolysis has yet to be described for rhodopsin in its native membrane environment. We developed a method consisting of simultaneous denaturation and chromophore trapping by isopropanol/borohydride, followed by exhaustive protein digestion, complete extraction, and liquid chromatography-mass spectrometry. Using our method, we tracked Rho* hydrolysis, the subsequent formation of N-retinylidene-phosphatidylethanolamine (N-ret-PE) adducts with the released all-trans-retinal, and the reduction of all-trans-retinal to all-trans-retinol. We found that hydrolysis occurred faster in native membranes than in detergent micelles typically used to study membrane proteins. The activation energy of the hydrolysis in native membranes was determined to be 17.7 ± 2.4 kcal/mol. Our data support the interpretation that metarhodopsin II, the signaling state of rhodopsin, is the primary species undergoing hydrolysis and release of its all-trans-retinal. In the absence of NADPH, free all-trans-retinal reacts with phosphatidylethanolamine (PE), forming a substantial amount of N-ret-PE (∼40% of total all-trans-retinal at physiological pH), at a rate that is an order of magnitude faster than Rho* hydrolysis. However, N-ret-PE formation was highly attenuated by NADPH-dependent reduction of all-trans-retinal to all-trans-retinol. Neither N-ret-PE formation nor all-trans-retinal reduction affected the rate of hydrolysis of Rho*. Our study provides a comprehensive picture of the hydrolysis of Rho* and the release of all-trans-retinal and its reentry into the visual cycle, a process in which alteration can lead to severe retinopathies.

Membrane Lipids and Cellular Water Modulate the G‐Protein–Coupled Receptor Activation

G-protein-coupled receptors (GPCRs) are a pivotal superfamily of seven-helix transmembrane receptors responsible for the transduction of stimuli across cellular membranes. They are the targets of ~30% of the pharmaceuticals currently in the market. Understanding how the soft membrane matter (membrane Lipids and Cellular Water) influences the structural changes during the activation of GPCRs is crucial to discovering and developing novel GPCR-targeted drugs. Their dynamic conformational ensembles encompassing various inactive and active states can be biased by the lipids and surrounding aqueous environments [1]. By using different polyethylene glycol (PEG) solutions, we explored the effect of osmotic pressure and lipid bilayer composition on the metarhodopsin equilibrium of the archetypical GPCR rhodopsin in native membranes and POPC recombinant membranes [2]. Our results show a flood of ~80 water molecules into the rhodopsin interior during photoactivation, forming a solvent-swollen Meta-II active state [3]. Under applied osmotic pressure, the overall equilibrium generally shifted to Meta-I. Dehydrating conditions favor Meta-I through the efflux of water from the protein interior while increasing bilayer thickness and the monolayer spontaneous curvature favor Meta-II. The osmotic effect on the protein is more significant than the effect of the lipid bilayer. However, small osmolytes favored the Meta-II state at lower concentrations because they can penetrate the protein core giving a lower excluded volume, decreasing the osmotic effect on the protein and favoring the Meta-II state. Furthermore, the metarhodopsin equilibrium was shifted towards the Meta-I state in POPC recombinant membranes compared to the native membrane environment. Analysis of transducin C-terminal peptide-binding isotherms revealed that the binding affinity is significantly decreased when the lipid environment is changed from the native lipids to POPC lipids. The POPC lipid membrane has zero-spontaneous curvature that shifts the equilibrium towards the more compact, inactive Meta-I state. By contrast, the native lipid membrane environment has a negative spontaneous curvature that favors the more expanded state of Meta-II. Our results delineate the crucial role of soft matter in regulating the metarhodopsin equilibrium in a membrane environment. [1] M.F. Brown (2017) Annu. Rev. Biophys. 46, 379-410. [2] N. Weerasinghe et al. (2018) Biophys. J. 114, 274a. [3] U. Chawla et al. (2020) Angew. Chem. Int. Ed. doi: https://doi.org/10.1002/ange.202003342.

Solvation Drives G-Protein-Coupled Receptor Activation

G-protein–coupled receptors (GPCRs) are membrane proteins that transduce information across lipid bilayers. Their dynamic conformational ensembles encompassing various inactive and active states can be biased by the lipids and surrounding aqueous environments. By using different polyethylene glycol (PEG) solutions, we explored the effect of osmotic pressure and lipid bilayer composition on the metarhodopsin equilibrium of the archetypical GPCR rhodopsin in native membranes and POPC recombinant membranes. Our results show a flood of ∼80 water molecules into the rhodopsin interior during photoactivation, forming a solvent-swollen Meta-II active state [1]. Under applied osmotic pressure, the overall equilibrium generally shifted to Meta-I. Dehydrating conditions favor Meta-I through an efflux of water from the protein interior, while increasing bilayer thickness and the monolayer spontaneous curvature favor Meta-II. The osmotic effect on the protein is more significant than the effect of the lipid bilayer. However, small osmolytes favored the Meta-II state at lower concentrations, because they can penetrate the protein core giving a lower excluded volume, decreasing the osmotic effect on the protein and favoring the Meta-II state. Furthermore, the metarhodopsin equilibrium was shifted towards the Meta-I state in POPC recombinant membranes compared to the native membrane environment. Analysis of transducin C-terminal peptide-binding isotherms revealed that the binding affinity is significantly decreased when the lipid environment is changed from the native lipids to POPC lipids. The POPC lipid membrane has zero-spontaneous curvature that shifts the equilibrium towards the more compact, inactive Meta-I state. By contrast, the native lipid membrane environment has a negative spontaneous curvature that favors the more expanded state of Meta-II. Our results delineate the crucial role of soft matter in regulating the metarhodopsin equilibrium in a membrane environment. [1] U. Chawla et al. (2020) Angew. Chem. Int. Ed. DOI: https://doi.org/10.1002/ange.202003342.

Investigating the Nanodomain Organization of Rhodopsin in Native Membranes by Atomic Force Microscopy.

Membrane proteins play an integral role in cellular communication. They are often organized within the crowded cell membrane into nanoscale domains (i.e., nanodomains), which facilitates their function in cell signaling processes. The visualization of membrane proteins and nanodomains within biological membranes under physiological conditions presents a challenge and is not possible using conventional microscopy methods. Atomic force microscopy (AFM) provides an opportunity to study the organization of membrane proteins within biological membranes with sub-nanometer resolution. An example of a membrane protein organized into nanodomains is rhodopsin. Rhodopsin is expressed in photoreceptor cells of the retina and upon photoactivation initiates a series of biochemical reactions called phototransduction, which represents the first steps of vision. AFM has provided an opportunity to directly visualize the packing of rhodopsin in native retinal membranes and the quantitative analysis of AFM images is beginning to reveal insights about the nanodomain organization of rhodopsin in the membrane. In this report, we outline procedures for imaging rhodopsin nanodomains by AFM and the quantitative analysis of those AFM images.

Science Signaling Podcast: 10 March 2015

Structural analysis of rhodopsin in native membranes shows real-time conformational changes induced by light.

Dynamic single-molecule force spectroscopy of rhodopsin in native membranes.

Membrane proteins are an important class of proteins in biology and therapeutics. Understanding the dynamic nature of the molecular interactions that stabilize membrane protein structure is critical to dissect the mechanism of action and dysfunction of these proteins. Single-molecule force spectroscopy (SMFS) and dynamic SMFS (DFS) are emerging nanotechniques that allow the study of membrane proteins under the physiologically relevant conditions of a lipid bilayer and buffer conditions. These techniques directly probe the molecular interactions underlying protein structure and reveal unique insights about their properties. Outlined in this report will be procedures on how to conduct SMFS and DFS on rhodopsin in native retinal membranes. Rhodopsin is a membrane protein belonging to the G protein-coupled receptor family of proteins, one of the largest families of proteins in nature.

Efficient Coupling of Transducin to Monomeric Rhodopsin in a Phospholipid Bilayer

G protein-coupled receptors (GPCRs) are seven transmembrane domain proteins that transduce extracellular signals across the plasma membrane and couple to the heterotrimeric family of G proteins. Like most intrinsic membrane proteins, GPCRs are capable of oligomerization, the function of which has only been established for a few different receptor systems. One challenge in understanding the function of oligomers relates to the inability to separate monomeric and oligomeric receptor complexes in membrane environments. Here we report the reconstitution of bovine rhodopsin, a GPCR expressed in the retina, into an apolipoprotein A-I phospholipid particle, derived from high density lipoprotein (HDL). We demonstrate that rhodopsin, when incorporated into these 10 nm reconstituted HDL (rHDL) particles, is monomeric and functional. Rhodopsin.rHDL maintains the appropriate spectral properties with respect to photoactivation and formation of the active form, metarhodopsin II. Additionally, the kinetics of metarhodopsin II decay is similar between rhodopsin in native membranes and rhodopsin in rHDL particles. Photoactivation of monomeric rhodopsin.rHDL also results in the rapid activation of transducin, at a rate that is comparable with that found in native rod outer segments and 20-fold faster than rhodopsin in detergent micelles. These data suggest that monomeric rhodopsin is the minimal functional unit in G protein activation and that oligomerization is not absolutely required for this process.

Constraints on the conformation of the cytoplasmic face of dark-adapted and light-excited rhodopsin inferred from antirhodopsin antibody imprints.

Rhodopsin is the best-understood member of the large G protein-coupled receptor (GPCR) superfamily. The G-protein amplification cascade is triggered by poorly understood light-induced conformational changes in rhodopsin that are homologous to changes caused by agonists in other GPCRs. We have applied the "antibody imprint" method to light-activated rhodopsin in native membranes by using nine monoclonal antibodies (mAbs) against aqueous faces of rhodopsin. Epitopes recognized by these mAbs were found by selection from random peptide libraries displayed on phage. A new computer algorithm, FINDMAP, was used to map the epitopes to discontinuous segments of rhodopsin that are distant in the primary sequence but are in close spatial proximity in the structure. The proximity of a segment of the N-terminal and the loop between helices VI and VIII found by FINDMAP is consistent with the X-ray structure of the dark-adapted rhodopsin. Epitopes to the cytoplasmic face segregated into two classes with different predicted spatial proximities of protein segments that correlate with different preferences of the antibodies for stabilizing the metarhodopsin I or metarhodopsin II conformations of light-excited rhodopsin. Epitopes of antibodies that stabilize metarhodopsin II indicate conformational changes from dark-adapted rhodopsin, including rearrangements of the C-terminal tail and altered exposure of the cytoplasmic end of helix VI, a portion of the C-3 loop, and helix VIII. As additional antibodies are subjected to antibody imprinting, this approach should provide increasingly detailed information on the conformation of light-excited rhodopsin and be applicable to structural studies of other challenging protein targets.

Organization of the G Protein-coupled Receptors Rhodopsin and Opsin in Native Membranes

G protein-coupled receptors (GPCRs), which constitute the largest and structurally best conserved family of signaling molecules, are involved in virtually all physiological processes. Crystal structures are available only for the detergent-solubilized light receptor rhodopsin. In addition, this receptor is the only GPCR for which the presumed higher order oligomeric state in native membranes has been demonstrated (Fotiadis, D., Liang, Y., Filipek, S., Saperstein, D. A., Engel, A., and Palczewski, K. (2003) Nature 421, 127-128). Here, we have determined by atomic force microscopy the organization of rhodopsin in native membranes obtained from wild-type mouse photoreceptors and opsin isolated from photoreceptors of Rpe65-/- mutant mice, which do not produce the chromophore 11-cis-retinal. The higher order organization of rhodopsin was present irrespective of the support on which the membranes were adsorbed for imaging. Rhodopsin and opsin form structural dimers that are organized in paracrystalline arrays. The intradimeric contact is likely to involve helices IV and V, whereas contacts mainly between helices I and II and the cytoplasmic loop connecting helices V and VI facilitate the formation of rhodopsin dimer rows. Contacts between rows are on the extracellular side and involve helix I. This is the first semi-empirical model of a higher order structure of a GPCR in native membranes, and it has profound implications for the understanding of how this receptor interacts with partner proteins.

Relative orientation between the beta-ionone ring and the polyene chain for the chromophore of rhodopsin in native membranes.

Rotational resonance solid state nuclear magnetic resonance has been used to determine the relative orientation of the beta-ionone ring and the polyene chain of the chromophore 11-Z-retinylidene of rhodopsin in rod outer segment membranes from bovine retina. The bleached protein was regenerated with either 11-Z-[8,18-(13)C(2)]retinal or 11-Z-[8,16/17(13)C(2)]retinal, the latter having only one (13)C label at either of the chemically equivalent positions 16 and 17. Observation of (13)C selectively enriched in the ring methyl groups, C16/17, revealed alternative conformational states for the ring. Minor spectral components comprised around 26% of the chromophore. The major conformation (approximately 74%) has the chemical shift resolution required for measuring internuclear distances to (13)C in the retinal chain (C8) separately from each of these methyl groups. The resulting distance constraints, C8 to C16 and C17 (4.05 +/- 0.25 A) and from C8 to C18 (2.95 +/- 0.15 A), show that the major portion of retinylidene in rhodopsin has a twisted 6-s-cis conformation. The more precise distance measurement made here between C8 and C18 (2.95 A) predicts that the chain is twisted out-of-plane with respect to the ring by a modest amount (C5-C6-C7-C8 torsion angle = -28 +/- 7 degrees ).

Light-induced conformational changes of rhodopsin probed by fluorescent alexa594 immobilized on the cytoplasmic surface.

A novel fluorescence method has been developed for detecting the light-induced conformational changes of rhodopsin and for monitoring the interaction between photolyzed rhodopsin and G-protein or arrestin. Rhodopsin in native membranes was selectively modified with fluorescent Alexa594-maleimide at the Cys(316) position, with a large excess of the reagent Cys(140) that was also derivatized. Modification with Alexa594 allowed the monitoring of fluorescence changes at a red excitation light wavelength of 605 nm, thus avoiding significant rhodopsin bleaching. Upon absorption of a photon by rhodopsin, the fluorescence intensity increased as much as 20% at acidic pH with an apparent pK(a) of approximately 6.8 at 4 degrees C, and was sensitive to the presence of hydroxylamine. These findings indicated that the increase in fluorescence is specific for metarhodopsin II. In the presence of transducin, a significant increase in fluorescence was observed. This increase of fluorescence emission intensity was reduced by addition of GTP, in agreement with the fact that transducin enhances the formation of metarhodopsin II. Under conditions that favored the formation of a metarhodopsin II-Alexa594 complex, transducin slightly decreased the fluorescence. In the presence of arrestin, under conditions that favored the formation of metarhodopsin I or II, a phosphorylated, photolyzed rhodopsin-Alexa594 complex only slightly decreased the fluorescence intensity, suggesting that the cytoplasmic surface structure of metarhodopsin II is different in the complex with arrestin and transducin. These results demonstrate the application of Alexa594-modified rhodopsin (Alexa594-rhodopsin) to continuously monitor the conformational changes in rhodopsin during light-induced transformations and its interactions with other proteins.

Influence of pH on the MI-MII equilibrium of rhodopsin in recombinant membranes

Rhodopsin in native rod membranes and incorporated into egg phosphatidylcholine (egg PC) vesicles was studied at pH 5 and 7 at 28°C. Rhodopsin function, as monitored by the formation of metarhodopsin II (MII) from metarhodopsin I (MI) following an actinic flash, was found to be largely blocked in egg PC vesicles at pH 7. When the pH was lowered to 5, however, rhodopsin showed essentially equal activity in both native and egg PC membranes. This activity exceeded that found for rhodopsin in native membranes at pH 7. Phospholipid composition is thus shown to directly affect the MI ⇌ MII equilibrium, which in turn is linked to visual function.

Organization of rhodopsin in photoreceptor membranes. 1. Proteolysis of bovine rhodopsin in native membranes and the distribution of sulfhydryl groups in the fragments.

Papain and thermolysin are shown to cleave bovine rhodopsin in native membranes in two temporally distinct steps at room temperature. The final product of the proteolysis consists of two membrane-bound fragments of molecular weights 27 000 (Rh27) and 12 500 (Rh12). The molecular weights are not changed by reduction with dithiothreitol. The two fragments remain closely associated in both the membrane and nondenaturing detergents before and after bleaching and can be selectively cross-linked with carbodiimides. The sulfhydryl chemistry of the cleaved protein in nearly indistinguishable from native rhodopsin, and of the total of six sulfhydryl groups, two are located on Rh12 and four on Rh27. In the membrane-bound protein, two sulfhydryl groups are accessible for modification, one on Rh12 and the other on Rh27. The sulfhydryl on Rh12 is particularly reactive and may be selectively labeled with maleimides. Continuous irradiation with white light induces additional sulfhydryl reactivity on Rh27.