TRANSPORT ATPASE--ENERGY COUPLING

转运ATP酶--能量耦合

• 批准号: 2444855
• 负责人: PARJIT KAUR
• 金额: $ 9.58万
• 依托单位: GEORGIA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2444855
• 关键词: Escherichia coli; active sites; adenosine triphosphate; adenosinetriphosphatase; antimony; arsenic; drug resistance; hydrolysis; intermolecular interaction; point mutation; protein structure function; site directed mutagenesis; suppressor mutations

The plasmid determined resistance to arsenic and antimony compounds in Escherichia coli is mediated by an anion-translocating ATPase. This ATPase does not fall into any of the known classes of the solute- translocating ATPases. It consists of an integral membrane protein ArsB that functions as a carrier, and a peripheral membrane protein ArsA that forms the catalytic subunit. Upon interaction, the ArsA and the ArsB proteins form a functional pump to extrude the anions out of the cell. The long term objective of the proposed research is to understand how the energy of ATP hydrolysis is coupled to the translocation of anions by the Ars ATPase. The catalytic component ArsA is an anion-stimulated ATPase and it undergoes dimerization in presence of the anion. Each monomer of ArsA has two homologous nucleotide binding domains, and it appears that an interaction of domains in trans between two polypeptide chains results in the formation of an interface. We will test the hypothesis that there are two active sites in the homodimer of the ArsA protein, and that each site is composed of residues from two polypeptide chains. The composition of the active sites will be examined by a hybridization approach, where mixing of two inactive point mutant proteins- each defective in one nucleotide binding domain- is expected to result in generation of one active site. Negative complementation between the wild type protein and a mutant defective in both domains will result in loss of ATPase activity if the active site is formed of shared residues between the two polypeptide chains. We will identify sites of contact between the subunits and alter residues in those regions by in vitro mutagenesis to determine the role of these interactions in the overall functioning of the pump. Analogs of ATP will be used to study the environment and the catalytic nature of the two ATP binding sites, which will be critical for understanding the mechanism of energy transduction. The possible interaction of nucleotide binding domains will have relevance to the F1 proton translocating ATPase, where rate of catalysis at the first site is increased several fold by binding of nucleotide to the second and the third site. This pump bears structural and functional similarity to members of the ABC type transporters such as the P-glycoprotein and the CFTR protein in the mammalian cells. Hence, these studies will give an insight into the molecular mechanism of energy coupling by Ars ATPase and by a variety of other ion and solute translocating pumps.

质粒确定对砷和锑化合物的抗性 大肠杆菌是由阴离子转换ATPase介导的。 这 ATPase不会属于溶质的任何已知类别 易位ATPases。它由整体膜蛋白ARSB组成 该功能充当载体和外围膜蛋白ARSA 形成催化亚基。互动后，ARSA和ARSB 蛋白质形成功能泵，将阴离子从细胞中挤出。 拟议研究的长期目标是了解 ATP水解的能量与阴离子的易位 ARS ATPase。 催化成分ARSA是一种阴离子刺激的ATPase，IT 在阴离子的存在下进行二聚化。每个ARSA的单体都有 两个同源核苷酸结合结构域，看来一个 两个多肽链之间反式的域的相互作用导致 界面的形成。我们将检验有这样的假设 ARSA蛋白同二聚体中的两个活性位点，每个位点 由两个多肽链中的残基组成。的组成 主动地点将通过杂交方法进行检查，其中 混合两个非活性点突变蛋白 - 一个在一个中有缺陷 核苷酸结合结构域有望产生一个 活性站点。 野生型蛋白质和 两个域中的突变体有缺陷将导致ATPase活性的损失 如果有效位点是由两者之间的共享残留物形成的 多肽链。我们将确定亚基之间的接触部位 并通过体外诱变来改变这些区域的残留物以确定 这些相互作用在泵的整体功能中的作用。 ATP的类似物将用于研究环境和催化 两个ATP绑定位点的性质，这对 了解能量转导的机理。 可能 核苷酸结合结构域的相互作用将与F1相关 质子转移ATPase，第一个位置的催化速率为 通过结合核苷酸与第二个，增加了几倍 第三站点。 该泵与ABC成员具有结构和功能相似性 类型转运蛋白，例如P-糖蛋白和CFTR蛋白 哺乳动物细胞。因此，这些研究将深入了解 通过ARS ATPase和多种 其他离子和溶质易位泵。