This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Our current research is focused on functional descriptions of a membrane-embedded, well characterized G-protein coupled neuroreceptor (GPCR) for which no high resolution structure is available. This receptor, the neurotensin receptor type 11 (NTS1;MW ~ 40kDa) is implicated in Parkinson's, dietary control and is a marker in colon cancer ?GPCRs are a major focus for all major pharma in view of their importance in all sensory and neurological control but lack of structures and functional descriptions, preclude rational design of new therapies, despite their potential for contributing to human well-being and wealth creation. In part of this wider program of research, the Watts group is using ESR spin label methods to: i) determine structural constraints and secondary structure elements for NTS1;ii) characterize the conformational changes in NTS1 on agonist binding;iii) determine any specific lipid protein interactions required for ligand binding;iv) collate the structural and dynamic data accumulated into structural and functional models for GPCR signaling;and, v) understand how lipid specificity for NTS1 may be coupled to G-protein activation. NTS1 is expressed by us in E. coli, and we can manipulate cys residues and at the same time retain ligand binding capacity and G-protein activation ?the wild type protein has 9 cys residues, four of which are buried in the receptor and not accessible to modification. The ESR methods the Watts group are using rely on DEER and spin-exchange for longer and shorter range distance methods respectively, with nitroxides at well-defined positions (from computer-generated models) at the end of trans-membrane helices of the receptor into which we have engineered reporter cys residues. Our collaboration with ACERT could help is in (i) providing much higher than X-band sensitivity for this work in which receptor in reconstituted, function-supporting membranes, is limiting (nM max);(ii) enable double quantum filtering (DQF) as an approach for resolving distances in conformationally sensitive NTS1 upon ligand binding (iii) input for proper analysis of data using various analytical methods appropriate to the data;(iv) translate the ESR data into structural information using CNS, developed at ACERT.
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