MECHANISM OF DOMAIN MOVEMENT IN PHOSPHOGLYCERATE KINASE

拟议研究的目标是检查结构基础 关于底物诱导磷酸甘油酸酯中结构域移动的机制 蛋白激酶(PGK)。提出了一种实验方法，它涉及一种 基因工程和物理化学方法相结合。PGK 由两个由铰链连接的球状域组成。角色 二级结构元素和单个氨基酸位于 PGK的铰链区和表面环的铰链区域将使用 寡核苷酸定向诱变。氨基酸取代和/或 将介绍基于酵母的计算机图形分析的缺失 PGK.构象变化的离域程度将是 通过在距铰链不同距离处引入突变进行评估。这个 底物结合事件和相关结构域之间的耦合 运动将通过研究这些突变对 构象、柔性、催化活性和配体结合性质 突变的酶。引入的突变对基因的影响 稳定性和折叠性能也将进行评估。结构和 突变的磷酸甘油酸激酶的功能将用 物理化学方法(镉、荧光、紫外光谱、化学 修改和动力学)。

The objective of the proposed research is to examine the structural basis for the mechanism of substrate-induced domain movement in phosphoglycerate kinase (PGK). An experimental approach is proposed which involves a combination of genetic engineering and physico-chemical methods. PGK consists of two globular domains which are connected by a hinge. The role of the secondary structure elements and individual amino acids situated in the hinge region of PGK, and of the surface loops, will be studied using oligonucleotide-directed mutagenesis. Amino acid substitutions and/or deletions will be introduced, based on computer graphics analysis of yeast PGK. The extent of delocalization of the conformational changes will be assessed by introducing mutations at various distances from the hinge. The coupling between the substrate binding event and the relative domain movement will be examined by studying the effect of these mutations on conformation, flexibility, catalytic activity and ligand binding properties of the mutant enzymes. The effects of the introduced mutations on the stability and folding properties will also be evaluated. Structure and function of the mutant phosphoglycerate kinases will be studied using physico-chemical methods (CD, fluorescence, UV spectroscopy, chemical modifications and kinetics).