CAREER: The Evolution of Phosphoryl Transfer and Protein Sequence Analysis

Purdue Research Foundation, West Lafayette IN

Investigators

Abstract

9984919 Sanders In this proposal, the PI wishes to achieve a deeper understanding of the information content of the primary structure of a protein by studies of the evolution of an ancient and diverse family of phosphotransferases. He will concentrate on the X-ray crystallographic determination of the structure of acetate kinase from Methanosarcina thermophila. The chemical details of metal-ion and nucleotide binding, the mechanism of catalysis and the roles of each conserved amino-acid residue in protein structure and function will be explored. From these analyses, he wishes to gain insight into the changes in the modes of substrate binding and catalysis that occurred during the divergence of the sugar kinase/actin/Hsc70 super-family of phosphotransferases. The significance of the elements of secondary structure that have been added to the core protein fold in various members of the family will be investigated. Catalysis of phosphoryl transfer by butyrate kinases and an exopolyphosphatase will also be examined through a combination of structural, biochemical, and mutagenesis approaches. An examination of whether the Thermotoga maritima butyrate kinase catalyzes phosphoryl transfer through one or more covalent phosphoenzyme intermediates will be undertaken. A course designed for the introduction of upper level undergraduates to protein sequence analysis will be developed further. It will encompass an examination of how the interpretation of a protein sequence depends upon both comparison of different sequences and an understanding of the context-dependent chemistry of the amino-acid residues in proteins. The students will have the opportunity to explore computer-generated representations of protein structures and to perform and probe the principles of protein-sequence analyses. Course materials that may be beneficial to other researchers and students will be constructed and disseminated.