BIOCHEMICAL STUDIES OF ATP-DRIVEN BACTERIAL TRANSPORT

这个项目的长期目标是描述 K-转运蛋白KDP的结构、功能、组装与调控 大肠埃希菌ATPase。这一时期的目标是： A.提高KDP的纯度。在…中获得高表达 所有KDP结构基因都以很高的速度被翻译 将准备和测试结构基因的其他结构 表达载体。将进一步探索提纯方法 获得高纯度、高质量活性酶的方案 投降。 B.KDP的生化研究。的基本结构特征 KDP复合体(确定B亚基的N-末端，C-末端 所有3个亚基，酰基磷酸化位置，存在前导 复合体中的多肽)将被确定，详细的 ATPase的动力学性质。突变复合体的分析和 将使用交联性研究来确定KDP的大小 很复杂。 C.KDP的拓扑学研究。我们将从检查开始 复合体的不同区域对蛋白水解酶和 使用洗涤剂纯化的酶标记试剂，然后 使用来自细胞的材料进行类似的原位研究 过度表达KDP。通过检查Kaback和 从内到外的囊泡，并将它们与 可溶性络合物KDP的结构的部分图像可以 浮出水面。 D.功能改变突变体的分析。DNA序列和 突变体运移动力学分析会降低钾的亲和力 将会被追查。我们将把这一分析扩展到复杂的 也降低了我们没有的最大速率的突变体 到目前为止已经检查了很多。这项工作将帮助我们告诉我们在哪里以及如何 KDP结合K进行运输，并可能泄露有关亚基的信息 互动。 E.对KDP的监管。在这个标题下，将有四个方面 目的：1)完成kdpD和kdpE基因的DNA序列。 2)分析kdpABC启动子区域以确定 并寻找与启动子结合的蛋白质。3) KdpD蛋白的细胞定位及结合试验 KdpE到KdpD。4)确定疏水先导的功能 调节中的多肽。

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.