The proposal addresses critical questions in understanding biological regulation at two principal levels: 1) Neurochemical signaling by, and mechanisms of ligand and voltage gated ion conducting channels, and 2) The mechanisms of selectivity in protein phosphorylation, and the mechanisms of phosphorylation in one of the most studied paradigms of small molecule phosphorylation. 1.1. New approaches to structure determination of eukaryotic membrane proteins are to be applied to crystallize the Acetylcholine receptor, a fundamental element of neuronal and neuromuular signaling, and of voltage gated K+ channels. 1.2. The crystal structures of bacterial channels Colicin la, a voltage gated channel with similar conductance as the acetylcholine receptor channel is to be determined to 2.5 Angstrom resolution to elucidate principles of transmembrane conductance, transmembrane translocation, insertion into membranes, and mechanisms of host immunity. Mutagenesis will be used to complement the structure analysis. Five heavy metal derivatives have been prepared, some using mutagenesis, and solved using synchrotron radiation data recorded at room temperature. A 3.4 Angstrom resolution density map into which 70% of the backbone structure has been built in this extensively alpha-helical, tripartite molecule provides a powerful basis for completion of the analysis, and extension in resolution. Crystallographic data has recently been recorded to 2.5 Angstroms from frozen crystals using synchrotron X-ray sources. 1.3. The structure of a unique 28 kDa. bacillus thuringiensis israeliensis porin-like insect toxin that forms transmembrane pores is to be completed at 2.3 Angstrom resolution from several derivatives coupled with mutagenesis. 2.1. With the goal of understanding how specificity is determined in the now over 200 kinases that regulate cellular activities, the structure of the first complex between a protein kinase and its target protein is to be determined from crystals that diffract to 2.7 Angstrom resolution. The target protein, isocitrate dehydrogenase is regulated by phosphorylation at the active site, but in a cleft that is inaccessible to even ATP. Issues addressed are how protein kinases can achieve their exquisite specificity within cells, and to resolve whether the mechanisms involve structure specific unfolding of the target protein, or cooperation of the target as part of the catalytic site. 2.2. To define mechanisms of phosphorylation in one of the most studied paradigms of phosphorylation, the structure of creatinine kinase is to be determined from crystals that contain a transition state analog, and diffract to 2.3 Angstroms resolution when preserved in a cold liquid nitrogen temperature stream.
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