CAREER: The Evolution of Phosphoryl Transfer and Protein Sequence Analysis

9984919 sanders在这项提案中，PI希望通过研究一个古老而多样的磷酸转移酶家族的进化来更深入地了解蛋白质初级结构的信息含量。他将集中于嗜热甲烷菌醋酸激酶结构的x射线晶体学测定。探讨金属离子与核苷酸结合的化学细节、催化机制以及各保守氨基酸残基在蛋白质结构和功能中的作用。通过这些分析，他希望深入了解糖激酶/肌动蛋白/Hsc70转运酶超家族分化过程中底物结合和催化模式的变化。二级结构元素的重要性，已添加到核心蛋白折叠在不同的家庭成员将被调查。通过结构、生化和诱变方法的结合，我们还将研究丁酸激酶和外多磷酸酶对磷酸化转移的催化作用。一个检查是否热toga海事丁酸激酶催化磷酸化转移通过一个或多个共价磷酸酶中间体将进行。我们将进一步发展一门为高年级本科生介绍蛋白质序列分析的课程。它将包括如何解释一个蛋白质序列取决于两个不同序列的比较和氨基酸残基在蛋白质的上下文依赖的化学理解的检查。学生将有机会探索计算机生成的蛋白质结构表示，并执行和探索蛋白质序列分析的原理。将编制和传播可能对其他研究人员和学生有益的课程材料。

9984919SandersIn 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.