THIS IS A SHANNON AWARD PROVIDING PARTIAL SUPPORT FOR THE RESEARCH PROJECTS THAT FALL SHORT OF THE ASSIGNED INSTITUTE'S FUNDING RANGE BUT ARE IN THE MARGIN OF EXCELLENCE. THE SHANNON AWARD IS INTENDED TO PROVIDE SUPPORT TO TEST THE FEASIBILITY OF THE APPROACH; DEVELOP FURTHER TESTS AND REFINE RESEARCH TECHNIQUES; PERFORM SECONDARY ANALYSIS OF AVAILABLE DATA SETS; OR CONDUCT DISCRETE PROJECTS THAT CAN DEMONSTRATE THE PI'S RESEARCH CAPABILITIES OR LEAD ADDITIONAL WEIGHT TO AN ALREADY MERITORIOUS APPLICATION. THE APPLICATION BELOW IS TAKEN FROM THE ORIGINAL DOCUMENT SUBMITTED BY THE PRINCIPAL INVESTIGATOR. The primary goal of the research proposed here is to further develop and apply a new approach for the construction of reliable three dimensional models of the delta, mu, and kappa opioid receptors as typical examples of the rhodopsin-like family of G-protein coupled receptors (GPCR). The approach assumes spatial proximity for residues with similar conservation patterns and posits that all polar sidechains within the transmembrane core will participate in intramolecular hydrogen bonding. In the first stage of the modeling, the sequential assignment of 7 GPCR transmembrane alpha-helices to the peaks of the 9 Angstroms resolution electron microscopy projection map of bovine rhodopsin can be deduced from mutagenesis and cross-linking data and from analysis of helix inner arcs occupied by evolutionary conserved and hydrophilic residues in 5 sequence alignments of 427 GPCRs. Then, the """"""""maximum hydrogen bonding participation"""""""" criterion and the inferred spatial proximity of residues conserved in a correlated fashion in sequence alignments of GPCR are used to find rotational orientations of the alpha-helices, to align the helices in the direction perpendicular to the membrane plane, and to derive constraints for refinement of the obtained receptor structure with distance geometry calculations. Although only opioid receptors are initially targeted, the approach is generally applicable for all members of the large (>480 unique sequences) rhodopsin-like subset of the GPCR superfamily. In preliminary studies we have constructed an approximate model of the transmembrane core of the delta opioid receptor and calculated a preliminary set of its conformations (with coordinate r.m.s.d. of Calpha atoms about 1.5 Angstroms) using distance constraints.derived from analysis of the sequence alignment of 105 peptide
