TRANSPORT ATPASE--ENERGY COUPLING

转运ATP酶--能量耦合

• 批准号: 2734761
• 负责人: PARJIT KAUR
• 金额: $ 10.01万
• 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 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2734761
• 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.

质粒决定了对砷和锑化合物的抗性， 大肠杆菌是由一个阴离子转运ATP酶介导的。 这 ATP酶不属于任何已知种类的溶质- 转移ATP酶它由一个完整的膜蛋白ArsB组成 和外周膜蛋白ArsA， 形成催化亚基。在相互作用时，ArsA和ArsB 蛋白质形成功能泵以将阴离子挤出细胞。 拟议研究的长期目标是了解 ATP水解的能量与阴离子的移位相耦合， Ars ATP酶 催化组分ArsA是阴离子刺激的ATP酶， 在阴离子存在下发生二聚化。ArsA的每个单体具有 两个同源的核苷酸结合结构域，并且似乎 两条多肽链之间反式结构域的相互作用导致 形成一个界面。我们将检验以下假设： ArsA蛋白的同源二聚体中的两个活性位点，并且每个位点 由两条多肽链的残基组成。的组合物 将通过杂交方法检查活性位点，其中 混合两种失活点突变蛋白-每种在一个缺陷 核苷酸结合结构域-预期将导致产生一个 活性部位 野生型蛋白质与 两个结构域都有缺陷的突变体将导致ATP酶活性的丧失 如果活性位点由两个之间的共享残基形成， 多肽链。我们会找出各个小组之间的联系点 并通过体外诱变改变那些区域中的残基， 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作用。 两个ATP结合位点的性质，这将是至关重要的 了解能量转换的机制。 可能的 核苷酸结合结构域的相互作用将与F1 质子转运ATP酶，其中第一位点的催化速率为 通过核苷酸与第二个和第三个核苷酸的结合， 第三个网站。 该泵在结构和功能上与ABC的成员相似 型转运蛋白，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 Ars ATP酶和多种酶的能量耦合的分子机制 其它离子和溶质移位泵。