BIOCHEMICAL STUDIES OF ATP-DRIVEN BACTERIAL TRANSPORT

该项目的长期目标是表征 钾转运蛋白Kdp结构、功能、组装和调控 大肠杆菌的ATP酶。 这一期间的目标是： A. 改进KDP的纯化。 为了获得高表达， 所有的kdp结构基因都以高速率翻译， 将准备和测试其他结构基因的构建， 表达载体。 将进一步探索纯化方法 得到了一种获得高纯度、高纯度活性酶的方案， 产率 B。 KDP的生化研究 基本结构特点 Kdp复合物（确定B亚基的N-末端， 所有3个亚基，酰基磷酸化位点，前导序列的存在 复合物中的肽）将被确定，如将详细描述的那样。 ATP酶的动力学性质。 突变体复合物的分析和 交联研究将用于确定Kdp的大小 复杂. C. KDP的拓扑研究 我们将开始检查 复合物的不同区域对蛋白酶的可及性， 使用去污剂纯化的酶标记试剂，然后 使用细胞材料进行类似的原位研究， 过量表达Kdp。 通过检查卡巴克和 由内而外的囊泡，并将其与 可溶性复合物Kdp结构的部分图像可以 出现。 D. 功能改变突变体的分析。 DNA序列和 突变体的运输动力学分析将降低对K 将被追究。 我们将把这种分析扩展到复杂的 突变体，也降低了最大速率， 到目前为止，检查了很多。 这项工作将帮助我们了解 Kdp结合K转运，并可能揭示亚基的信息， 交互. E. KDP的监管。 在这一标题下， 1）完成kdpD和kdpE基因的DNA序列测定。 2)分析kdpABC启动子区域以确定 启动子并寻找与启动子结合的蛋白质。第三章 KdpD蛋白的细胞定位和结合试验 KdpE到KdpD。 4)确定疏水前导序列的功能 肽的调节作用。

The long term goal of this project is the characterization of the structure, function, assembly and regulation of Kdp, a K-transport ATPase of Escherichia coli. Aims for this period are: A. IMPROVE PURIFICATION OF KDP. To obtain high expression in which all kdp structural genes are translated at a high rate we will prepare and test other constructs of the structural genes in expression vectors. Purification methods will be explored further to obtain a scheme to obtain active enzyme in high purity and good yield. B. BIOCHEMICAL STUDIES OF KDP. Basic structural features of the Kdp complex (determine N-terminus of the B subunit, C-termini of all 3 subunits, site of acylphosphorylation, presence of leader peptide in complex) will be determined, as will the detailed kinetic properties of the ATPase. Analysis of mutant complexes and crosslinking studies will be used to establish size of the Kdp complex. C. TOPOLOGIC STUDIES OF KDP. We will begin by examining accessibility of different regions of the complex to proteases and to labeling reagents using the detergent-purified enzyme, and then proceed to similar studies in situ using material froms cells that overexpress Kdp. By examining accessibility in Kaback and in inside-out vesicles and comparing these with accessibility in the soluble complex a partial picture of the structure of Kdp may emerge. D. ANALYSIS OF ALTERED FUNCTION MUTANTS. DNA sequence and transport kinetic analysis of mutants will reduced affinity for K will be pursued. We will extend this analysis to the complicated mutants that also reduce the maximum rate which we have not examined much to date. This work will help tell us where and how Kdp binds K for transport, and may reveal information about subunit interactions. E. REGULATION OF KDP. Under this heading four aspects will be pursued: 1) Complete the DNA sequence of the kdpD and kdpE genes. 2) Analysis of the kdpABC promoter region to determine extent of the promoter and look for protein binding to the promoter. 3) Cellular localization of the KdpD protein and tests for binding of KdpE to KdpD. 4) Determine the function of the hydrophobic leader peptide in regulation.