[Show abstract][Hide abstract] ABSTRACT: Rhodopsin forms nanoscale domains (i.e., nanodomains) in rod outer segment disc membranes from mammalian species. It is unclear whether rhodopsin arranges in a similar manner in amphibian species, which are often used as a model system to investigate the function of rhodopsin and the structure of photoreceptor cells. Moreover, since samples are routinely prepared at low temperatures, it is unclear whether lipid phase separation effects in the membrane promote the observed nanodomain organization of rhodopsin from mammalian species. Rod outer segment disc membranes prepared from the cold-blooded frog Xenopus laevis were investigated by atomic force microscopy to visualize the organization of rhodopsin in the absence of lipid phase separation effects. Atomic force microscopy revealed that rhodopsin nanodomains form similarly as that observed previously in mammalian membranes. Formation of nanodomains in ROS disc membranes is independent of lipid phase separation and conserved among vertebrates.
[Show abstract][Hide abstract] ABSTRACT: Mutations in rhodopsin can cause misfolding and aggregation of the receptor, which leads to retinitis pigmentosa, a progressive retinal degenerative disease. The structure adopted by misfolded opsin mutants and the associated cell toxicity is poorly understood. Förster resonance energy transfer (FRET) and Fourier transform infrared (FTIR) microspectroscopy were utilized to probe within cells the structures formed by G188R and P23H opsins, which are misfolding mutants that cause autosomal dominant retinitis pigmentosa. Both mutants formed aggregates in the endoplasmic reticulum and exhibited altered secondary structure with elevated β-sheet and reduced α-helical content. The newly formed β -sheet structure may facilitate the aggregation of misfolded opsin mutants. The effects observed for the mutants were unrelated to retention of opsin molecules in the endoplasmic reticulum itself.
[Show abstract][Hide abstract] ABSTRACT: Rhodopsin is the light receptor embedded in rod outer segment (ROS) disc membranes of photoreceptor cells that initiates vision via phototransduction. The relationship between rhodopsin expression and the formation of membrane structures in the ROS is unclear but important to better understand both normal function and pathological conditions. To determine the impact of reduced rhodopsin expression on the structure of ROS discs and the supramolecular organization of rhodopsin, ROS disc membrane samples from heterozygous rhodopsin knockout mice were examined by atomic force microscopy. Similar to rhodopsin in wild-type mice, rhodopsin formed nanodomains in ROS disc membranes of heterozygous knockout mice. The reduced rhodopsin expression in heterozygous knockout mice resulted in ROS disc membranes that were smaller compared to those in wild-type mice at all ages tested. Changes in ROS disc membrane properties were observed between 4 and 6 weeks of age in heterozygous knockout mice that were not present in age-matched wild-type mice. In 4 week-old mice, the number and density of rhodopsin in ROS disc membranes was lower than that in age-matched wild-type mice. In contrast, 6 and 8 week-old mice had more rhodopsin molecules present in disc membranes compared to 4 week-old mice, which resulted in rhodopsin densities similar to those found in age-matched wild-type mice. Thus, mechanisms appear to be present that maintain a constant density of rhodopsin within ROS disc membranes even when reducing the expression of the light receptor by about half. These adaptive mechanisms, however, only occur after 4 weeks of age.
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Feb 2015 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: Biological membranes display distinct domains that organize membrane proteins and signaling molecules to facilitate efficient and reliable signaling. The organization of rhodopsin, a G protein-coupled receptor, in native rod outer segment disc membranes was investigated by atomic force microscopy. Atomic force microscopy revealed that rhodopsin is arranged into domains of variable size, which we refer to herein as nanodomains, in native membranes. Quantitative analysis of 150 disc membranes revealed that the physical properties of nanodomains are conserved in humans and mice and that the properties of individual disc membranes can be variable. Examining the variable properties of disc membranes revealed some of the factors contributing to the size of rod outer segment discs and the formation of nanodomains in the membrane. The diameter of rod outer segment discs was dependent on the number of rhodopsin molecules incorporated into the membrane but independent of the spatial density of rhodopsin. The number of nanodomains present in a single disc was also dependent on the number of rhodopsin molecules incorporated into the membrane. The size of the nanodomains was largely independent of the number or spatial density of rhodopsin in the membrane.
No preview · Article · Oct 2014 · Biochimica et Biophysica Acta (BBA) - Biomembranes
[Show abstract][Hide abstract] ABSTRACT: Membrane proteins are embedded in lipid bilayers and facilitate the communication between the external environment and the interior of the cell. This communication is often mediated by the binding of ligands to the membrane protein. Understanding the nature of the interaction between a ligand and membrane protein is required to both understand the mechanism of action of these proteins and for the development of novel pharmacological drugs. The highly hydrophobic nature of membrane proteins and the requirement of a lipid bilayer for native function have hampered the structural and molecular characterization of these proteins under physiologically relevant conditions. Atomic force microscopy offers a solution to studying membrane proteins and their interactions with ligands under physiologically relevant conditions and can provide novel insights about the nature of these critical molecular interactions that facilitate cellular communication. In this review, we provide an overview of the atomic force microscopy technique and discuss its application in the study of a variety of questions related to the interaction between a membrane protein and a ligand. This article is part of a Special Issue entitled: Structural and biophysical characterisation of membrane protein-ligand binding.
[Show abstract][Hide abstract] ABSTRACT: Rhodopsin, the photoreceptor pigment of the retina, initiates vision upon photon capture by its covalently linked chromophore 11-cis-retinal. In the absence of light, the chromophore serves as an inverse agonist locking the receptor in the inactive dark state. In the absence of chromophore, the apoprotein opsin shows low-level constitutive activity. Toward revealing insight into receptor properties controlled by the chromophore, we applied dynamic single-molecule force spectroscopy to quantify the kinetic, energetic, and mechanical differences between dark-state rhodopsin and opsin in native membranes from the retina of mice. Both rhodopsin and opsin are stabilized by ten structural segments. Compared to dark-state rhodopsin, the structural segments stabilizing opsin showed higher interaction strengths and mechanical rigidities and lower conformational variabilities, lifetimes, and free energies. These changes outline a common mechanism toward activating G-protein-coupled receptors. Additionally, we detected that opsin was more pliable and frequently stabilized alternate structural intermediates.
[Show abstract][Hide abstract] ABSTRACT: Several point mutations in rhodopsin cause retinal diseases including congenital stationary night blindness and retinitis pigmentosa. The mechanism by which a single amino acid residue substitution leads to dysfunction is poorly understood at the molecular level. A G90D point mutation in rhodopsin causes constitutive activity and leads to congenital stationary night blindness. It is unclear which perturbations the mutation introduces and how they can cause the receptor to be constitutively active. To reveal insight into these mechanisms, we characterized the perturbations introduced into dark state G90D rhodopsin from a transgenic mouse model expressing exclusively the mutant rhodopsin in rod photoreceptor cells. UV-visible absorbance spectroscopy revealed hydroxylamine accessibility to the chromophore-binding pocket of dark state G90D rhodopsin, which is not detected in dark state wild-type rhodopsin but is detected in light-activated wild-type rhodopsin. Single-molecule force spectroscopy suggested that the structural changes introduced by the mutation are small. Dynamic single-molecule force spectroscopy revealed that, compared with dark state wild-type rhodopsin, the G90D mutation decreased energetic stability and increased mechanical rigidity of most structural regions in the dark state mutant receptor. The observed structural, energetic, and mechanical changes in dark state G90D rhodopsin provide insights into the nature of perturbations caused by a pathological point mutation. Moreover, these changed properties observed for dark state G90D rhodopsin are consistent with properties expected for an active state.
Full-text · Article · May 2012 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: G protein-coupled receptors (GPCRs) play critical roles in cellular signal transduction and are important targets for therapeutics. Although these receptors have been intensely studied for quite some time, our understanding about their mechanism of action is still incomplete. GPCR activity has traditionally been viewed within the context of two-state models where the receptor is in equilibrium between a single inactive state and a single active state. This framework is too simple and restrictive to accommodate more recent observations made on these receptors, which instead point to a situation where the receptor can adopt several different active conformational substates with distinct functional effects. Structural and functional evidence for this emerging view is presented in this review. Implications of this emerging view in rationalizing diseased states and in drug discovery are also discussed.
Preview · Article · Feb 2012 · Current Medicinal Chemistry
[Show abstract][Hide abstract] ABSTRACT: Rhodopsin is the light receptor that initiates phototransduction in rod photoreceptor cells. The structure and function of rhodopsin are tightly linked to molecular interactions that stabilize and determine the receptor's functional state. Single-molecule force spectroscopy (SMFS) was used to localize and quantify molecular interactions that structurally stabilize bovine and mouse rhodopsin from native disk membranes of rod photoreceptor cells. The mechanical unfolding of bovine and mouse rhodopsin revealed nine major unfolding intermediates, each intermediate defining a structurally stable segment in the receptor. These stable structural segments had similar localization and occurrence in both bovine and mouse samples. For each structural segment, parameters describing their unfolding energy barrier were determined by dynamic SMFS. No major differences were observed between bovine and mouse rhodopsin, thereby implying that the structures of both rhodopsins are largely stabilized by similar molecular interactions.
[Show abstract][Hide abstract] ABSTRACT: Vision is initiated in photoreceptor cells of the retina by a set of biochemical events called phototransduction. These events occur via coordinated dynamic processes that include changes in secondary messenger concentrations, conformational changes and post-translational modifications of signaling proteins, and protein-protein interactions between signaling partners. A complete description of the orchestration of these dynamic processes is still unavailable. Described in this work is the first step in the development of tools combining fluorescent protein technology, Förster resonance energy transfer (FRET), and transgenic animals that have the potential to reveal important molecular insights about the dynamic processes occurring in photoreceptor cells. We characterize the fluorescent proteins SCFP3A and SYFP2 for use as a donor-acceptor pair in FRET assays, which will facilitate the visualization of dynamic processes in living cells. We also demonstrate the targeted expression of these fluorescent proteins to the rod photoreceptor cells of Xenopus laevis, and describe a general method for detecting FRET in these cells. The general approaches described here can address numerous types of questions related to phototransduction and photoreceptor biology by providing a platform to visualize dynamic processes in molecular detail within a native context.
Preview · Article · Nov 2010 · Journal of Biomedical Optics
[Show abstract][Hide abstract] ABSTRACT: S-palmitoylation is a conserved feature in many G protein-coupled receptors (GPCRs) involved in a broad array of signaling processes. The prototypical GPCR, rhodopsin, is S-palmitoylated on two adjacent C-terminal Cys residues at its cytoplasmic surface. Surprisingly, absence of palmitoylation has only a modest effect on in vitro or in vivo signaling. Here, we report that palmitoylation-deficient (Palm(-/-)) mice carrying two Cys to Thr and Ser mutations in the opsin gene displayed profound light-induced retinal degeneration that first involved rod and then cone cells. After brief bright light exposure, their retinas exhibited two types of deposits containing nucleic acid and invasive phagocytic macrophages. When Palm(-/-) mice were crossed with Lrat(-/-) mice lacking lecithin:retinol acyl transferase to eliminate retinoid binding to opsin and thereby rendering the eye insensitive to light, rapid retinal degeneration occurred even in 3- to 4-week-old animals. This rapid degeneration suggests that nonpalmitoylated rod opsin is unstable. Treatment of 2-week-old Palm(-/-)Lrat(-/-) mice with an artificial chromophore precursor prevented this retinopathy. In contrast, elimination of signaling to G protein in Palm(-/-)Gnat1(-/-) mice had no effect, indicating that instability of unpalmitoylated opsin lacking chromophore rather than aberrant signal transduction resulted in retinal pathology. Together, these observations provide evidence for a structural role of rhodopsin S-palmitoylation that may apply to other GPCRs as well.
Full-text · Article · May 2010 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Rhodopsin is palmitylated at two cysteine residues in its carboxyl terminal region. We have looked at the effects of palmitylation on the molecular interactions formed by rhodopsin using single-molecule force spectroscopy and the function of rhodopsin using both in vitro and in vivo approaches. A knockin mouse model expressing palmitate-deficient rhodopsin was used for live animal in vivo studies and to obtain native tissue samples for in vitro assays. We specifically looked at the effects of palmitylation on the chromophore-binding pocket, interactions of rhodopsin with transducin, and molecular interactions stabilizing the receptor structure. The structure of rhodopsin is largely unperturbed by the absence of palmitate linkage. The binding pocket for the chromophore 11-cis-retinal is minimally altered as palmitate-deficient rhodopsin exhibited the same absorbance spectrum as wild-type rhodopsin. Similarly, the rate of release of all-trans-retinal after light activation was the same both in the presence and absence of palmitylation. Significant differences were observed in the rate of transducin activation by rhodopsin and in the force required to unfold the last stable structural segment in rhodopsin at its carboxyl terminal end. A 1.3-fold reduction in the rate of transducin activation by rhodopsin was observed in the absence of palmitylation. Single-molecule force spectroscopy revealed a 2.1-fold reduction in the normalized force required to unfold the carboxyl terminal end of rhodopsin. The absence of palmitylation in rhodopsin therefore destabilizes the molecular interactions formed in the carboxyl terminal end of the receptor, which appears to hinder the activation of transducin by light-activated rhodopsin.
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Mar 2008 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Molecular interactions and mechanical properties that contribute to the stability and function of proteins are complex and of fundamental importance. In this study, we used single-molecule dynamic force spectroscopy (DFS) to explore the interactions and the unfolding energy landscape of bovine rhodopsin and bacteriorhodopsin. An analysis of the experimental data enabled the extraction of parameters that provided insights into the kinetic stability and mechanical properties of these membrane proteins. Individual structural segments of rhodopsin and bacteriorhodopsin have different properties. A core of rigid structural segments was observed in rhodopsin but not in bacteriorhodopsin. This core may reflect differences in mechanisms of protein folding between the two membrane proteins. The different structural rigidity of the two proteins may also reflect their adaptation to differing functions.
[Show abstract][Hide abstract] ABSTRACT: Transformation of G protein-coupled receptors (GPCRs) from a quiescent to an active state initiates signal transduction. All GPCRs share a common architecture comprising seven transmembrane-spanning alpha-helices, which accommodates signal propagation from a diverse repertoire of external stimuli across biological membranes to a heterotrimeric G protein. Signal propagation through the transmembrane helices likely involves mechanistic features common to all GPCRs. The structure of the light receptor rhodopsin may serve as a prototype for the transmembrane architecture of GPCRs. Early biochemical, biophysical, and pharmacological studies led to the conceptualization of receptor activation based on the context of two-state equilibrium models and conformational changes in protein structure. More recent studies indicate a need to move beyond these classical paradigms and to consider additional aspects of the molecular character of GPCRs, such as the oligomerization and dynamics of the receptor.
Preview · Article · Feb 2008 · Annual Review of Pharmacology
[Show abstract][Hide abstract] ABSTRACT: M2 muscarinic receptor extracted from Sf9 cells in cholate-NaCl differs from that extracted from porcine sarcolemma. The latter has been shown to exhibit an anomalous pattern in which the capacity for N-[3H]methylscopolamine (NMS) is only 50% of that for [3H]quinuclidinylbenzilate (QNB), yet unlabeled NMS exhibits high affinity for all of the sites labeled by [3H]QNB. The effects can be explained in terms of cooperativity within a receptor that is at least tetravalent [Park PS, Sum CS, Pawagi AB, Wells JW. Cooperativity and oligomeric status of cardiac muscarinic cholinergic receptors. Biochemistry 2002;41:5588-604]. In contrast, M2 receptor extracted from Sf9 membranes exhibited no shortfall in the capacity for [3H]NMS at either 30 or 4 degrees C, although there was a time-dependent inactivation during incubation with [3H]NMS at 30 degrees C; also, any discrepancies in the affinity of NMS were comparatively small. The level of cholesterol in Sf9 membranes was only 4% of that in sarcolemmal membranes, and it was increased to about 100% by means of cholesterol-methyl-beta-cyclodextrin. M2 receptors extracted from treated Sf9 membranes were stable at 30 and 4 degrees C and resembled those from heart. Cholesterol induced a marked heterogeneity detected in the binding of both radioligands, including a shortfall in the apparent capacity for [3H]NMS, and there were significant discrepancies in the apparent affinity of NMS as estimated directly and via the inhibition of [3H]QNB. The data can be described quantitatively in terms of cooperative effects among six or more interacting sites. Cholesterol therefore appears to promote cooperativity in the binding of antagonists to the M2 muscarinic receptor.
No preview · Article · Aug 2007 · Biochemical Pharmacology
[Show abstract][Hide abstract] ABSTRACT: The rod outer segment (ROS) of photoreceptor cells houses all components necessary for phototransduction, a set of biochemical reactions that amplify and propagate a light signal. Theoretical approaches to quantify this process require precise information about the physical boundaries of the ROS. Dimensions of internal structures within the ROS of mammalian species have yet to be determined with the precision required for quantitative considerations. Cryoelectron tomography was utilized to obtain reliable three-dimensional morphological information about this important structure from murine retina. Vitrification of samples permitted imaging of the ROS in a minimally perturbed manner and the preservation of substructures. Tomograms revealed the characteristic highly organized arrangement of disc membranes stacked on top of one another with a surrounding plasma membrane. Distances among the various membrane components of the ROS were measured to define the space available for phototransduction to occur. Reconstruction of segments of the ROS from single-axis tilt series images provided a glimpse into the three-dimensional architecture of this highly differentiated neuron. The reconstructions revealed spacers that likely maintain the proper distance between adjacent discs and between discs and the plasma membrane. Spacers were found distributed throughout the discs, including regions that are distant from the rim region of discs.
Preview · Article · Jul 2007 · The Journal of Cell Biology
[Show abstract][Hide abstract] ABSTRACT: Single-molecule force spectroscopy (SMFS) is a powerful tool to dissect molecular interactions that govern the stability and function of proteins. We applied SMFS to understand the effect of Zn2+ on the molecular interactions underlying the structure of rhodopsin. Force-distance curves obtained from SMFS assays revealed the strength and location of molecular interactions that stabilize structural segments within this receptor. The inclusion of ZnCl2 in SMFS assay buffer increased the stability of most structural segments. This effect was not mimicked by CaCl2, CdCl2, or CoCl2. Thus, Zn2+ stabilizes the structure of rhodopsin in a specific manner.
Full-text · Article · May 2007 · Journal of Biological Chemistry