Structural and Biochemical Studies of the Metarhodopsin II-Transducin Complex

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DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCRs) regulate a wide range of physiological processes by detecting and transmitting an extracellular signal across a cell membrane to intracellular guanine-nucleotide binding proteins, G proteins. However, our understanding of signal transduction is limited by the absence of high resolution structures of the fully-activated receptor, alone and in complex with G protein. The long-term goal of this study is to resolve these structures using rhodopsin, the dim-light sensing receptor of the retina, as a model GPCR. Determination of these structures will define the conformational triggers that promote G protein activation and G protein specificity. To reach the stated goals I will address the following specific aims: 1. Determine the Crystal Structure of Rhodopsin in the Active State. To form activated receptors I will use a constitutively-active rhodopsin mutant (N2C/E113Q/D282C). Receptors will be expressed in mammalian cells and purified using immunoaffinity chromatography. Purified receptors will be crystallized using vapor-diffusion techniques and diffraction data will be collected at a synchrotron X-ray beamline. 2. Biochemically Characterize the Rhodopsin-G protein Complex. I will isolate the active complex by a modification of the rhodopsin immunoaffinity procedure using purified N2C/E113Q/D282C mutant opsin and G protein from natural sources. I will characterize this complex in regards to stoichiometry, stability, and disassembly. This information will be instrumental in answering questions in the field regarding the rhodopsin-transducin complex. These studies will also guide the crystallization of the complex. 3. Determine the Crystal Structure of the rhodopsin-G protein Complex. Purified N2C/E113Q/D282C mutant-G protein complexes will be crystallized by vapor diffusion and diffraction data will be obtained at a synchrotron X-ray beamline. Structures will be solved using data analysis software. PUBLIC HEALTH RELEVANCE Acquiring the structure of the receptor-G protein complex will impact several aspects of public health by improving our ability to recognize and treat disorders linked to unregulated receptor activity. The spectrum of such diseases includes obesity and stationary night blindness, each caused by mutations in the receptor protein that lead to changes in receptor activity. Treatment for these disorders with small molecules specifically tailored to promote or impede receptor activity will be facilitated by high-resolution structural information.