Membranes play a critical role in mediating the protein-protein and protein-lipid interactions of intracellular signaling cascades. Understanding this role in the phototransduction system of vertebrate retinal rods and cones helps us understand the normal molecular mechanisms of vision, and how they are altered in diseases of the eye. It also serves as a model for the most common signal transduction mechanism in our bodies, which is based on G proteins and G protein-coupled receptors. This proposal uses an interdisciplinary approach to uncover these mechanisms at several levels. To understand the molecular structures of membrane associated signal-transducing protein complexes, we will use fluorescence and electron paramagnetic resonance (EPR) spectroscopy, molecular electron cryo-microscopy, and x-ray crystallography. To understand the roles of membranes in signal transduction, we will fractionate the photoreceptor membranes to identify and analyze the lipid and protein components of subdomains such as lipid rafts, and disk rims, as well as total composition of regulatory lipids such as phosphoinositides and sphingolipids. We will also reconstitute the phototransduction components on lipid vesicles containing raft-forming components to assess their effects on signaling efficiency. To understand the dynamics of signaling proteins on disk membranes, we will use transgenic flogs to measure protein diffusion kinetics in intact photoreceptors by fluorescence photobleaching recovery kinetics. Thus we will continue to assemble a comprehensive picture of the disk membrane and its role in G protein signaling and vision: from static structures to millisecond kinetics, and from the roles of individual atoms in protein structures to the proteins' functions and dynamics in intact cells.
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