Cells from more than 30% of human cancers have been shown to contain point mutants of ras proteins. Residues 12, 13, 59, 61, 63, 116, 117, 119 and 146 of the proteins belong to all of the known mutation sties identified either in mammalian tumor or in transformed mammalian cells. The overall objective is to correlate biochemical and biological consequences of the oncogenic mutations with the structural changes associated with the mutations. We propose to construct, to determine the crystal structures of, and to characterize the mutants that are either the same as those found in various mammalian tumors or important for illuminating the structural basis for understanding biochemical and biological properties of the mutants. Experimental methods used will involve (a) x-ray crystallographic methods to determine the crystal structures of the normal and the mutants complexed with GDP, non-hydrolyzable GTP analogs, or a transition state analog; (b) in vitro biochemical characterization of GTP hydrolysis and GDP/GTP exchange rates using radiolabelled guanine nucleotides; and (c) microinjection of purified proteins into frog oocytes for in vivo assay of the transforming activity of the various mutant ras proteins.
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