The scientific focus of the project is to characterize the events at the membrane level that yield the triggering of visual signal transduction by rhodopsin. The significance is that rhodopsin is a prototype for G protein-coupled receptors (GPCRs) that are the targets of 45% of known pharmaceuticals. Mutations of rhodopsin are implicated in macular degeneration and retinitis pigmentosa. In addition polyunsaturated lipids of the retinal disk membranes affect visual diseases. Here we shall test the hypothesis that torsional deformation of the retinal ligand involves specific binding interactions with the opsin apoprotein. The binding cavity for the ligand has three sites, viz. involving the 2-ionone ring, the polyene chain, and the protonated Schiff base. Photolysis triggers activation of the photoreceptor, which is coupled to elastic membrane deformation. The approach will apply a key biophysical technology, deuterium (2H) NMR spectroscopy, to investigate aligned membranes containing rhodopsin with a deuterated retinal chromophore. Solid-state NMR methods will include lineshape simulations for a semi-random distribution of proteins in the membrane. Angular restraints from 2H NMR will illuminate the retinal structure in the dark and photoactivated states of rhodopsin. Further innovation entails ultra-large scale molecular dynamics (MD) simulations and 2H NMR relaxation measurements. These methods will be combined to yield a comprehensive picture of rhodopsin activation at the membrane level.
Our specific aims during the next funding cycle are the following: (1) First, we plan to apply solid-state 2H NMR technology to aligned membrane samples to illuminate the 3D structure and mobility of the retinylidene ligand in the light-activated Meta II state of rhodopsin. The conformation and orientation of retinal in Meta II will be established and compared to the dark-state as obtained in the previous funding cycle. (2) Next, solid-state 2H NMR spectroscopy will investigate conformational distortion of the retinal chromophore in the Bathorhodopsin, Lumi, Meta I, and Meta II states. A new aspect is to investigate progressive relaxation of the distorted retinal ligand in the photolysis pathway. (3) Third, the non-bonded interactions of retinal within the rhodopsin binding cavity that stabilize the activated Meta II state will be investigated with modified retinoids that dramatically shift the Meta I Meta II equilibrium. (4) We will then characterize changes in the retinal molecular dynamics as it undergoes its 11-cis to trans isomerization and progressive relaxation in the bleaching pathway leading to activation of rhodopsin. Non-bonded interactions of the ligand will be investigated through relaxation time measurements and further interpreted by molecular dynamics (MD) simulations. (5) Additional research will illuminate how light activation of rhodopsin is governed by elastic deformation of the membrane lipid bilayer. Here 2H and 31P NMR will investigate the biophysical properties of membrane lipids that influence the photochemical function of rhodopsin. Thus we intend to provide an innovative new view of how rhodopsin together with the bilayer lipids triggers visual perception in the retinal disk membranes as a paradigm for GPCRs and signal transduction in general.
Rhodopsin is a prototype for G protein-coupled receptors (GPCRs) that are implicated in biological signaling and constitute the targets of 45% of all known pharmaceuticals. The project will adopt an innovative approach using solid-state 2H NMR spectroscopy to illuminate visual function at the membrane level. Human hereditary blindness is can be understood through structural studies of rhodopsin, and extension of the approach to other GPCRs can stimulate ligand-based drug discovery. Moreover, investigations of the highly polyunsaturated I-3 lipids of retinal disk membranes are potentially far-reaching in significance. Essential fatty acid (EFA) deficiency affects cognitive skills and learning. Nationwide there is great concern about the loss of intellectual potential due to lack of polyunsaturated fatty acids in the diets of human infants. Understanding the protein and lipid components of visual membranes is vitally significant to the prevention of human diseases.
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|Knepp, Adam M; Sakmar, Thomas P; Huber, Thomas (2013) Homogeneous time-resolved fluorescence assay to probe folded G protein-coupled receptors. Methods Enzymol 522:169-89|
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|Knepp, Adam M; Grunbeck, Amy; Banerjee, Sourabh et al. (2011) Direct measurement of thermal stability of expressed CCR5 and stabilization by small molecule ligands. Biochemistry 50:502-11|
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