ION GRADIENTS AND ENERGY COUPLING IN BACTERIA

细菌中的离子梯度和能量耦合

• 批准号: 2444472
• 负责人: PETER C MALONEY
• 金额: $ 24.71万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-08-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2444472
• 关键词: Escherichia coli; antiport; bacterial proteins; bioenergetics; cysteine; gene expression; gene mutation; ion transport; membrane permeability; protein purification; protein sequence; protein structure function; site directed mutagenesis

DESCRIPTION: This work addresses three questions central to the field of membrane transport: (1) What residues constitute a translocation pathway? (2) Are these residues found in characteristic structural motifs? (3) What is the overall organization of the hydrophobic core of such a protein? To answer these questions, we will exploit mutagenesis to implant cysteine residues at strategic locations in an otherwise cysteine-less version of UhpT, a model antiport carrier from Escherichia coli. Using cysteine-directed probes to scan a population of such variants, we can generate functional and structural maps with a resolution of a single residue. These objectives are approached in three kinds of experiments: The first goal is to locate residues that line the UhpT translocation pathway. This will be done by probing single-cysteine mutants with hydrophilic and impermeant SH-reactive agents. Residues on the pathway will be identified as those which, if replaced by cysteine, are accessible to impermeant SH-directed probes added to either membrane surface and whose inhibition by probe is prevented by the presence of substrate. This work will also lead to more precise information on UhpT topology. Such experiments will identify a set of transmembrane segments surrounding the transport pathway. Our second aim is to assess local structure by examining reactivity to hydrophilic probe(s) of varying size and shape. Patterns of probe reactivity will confirm or reject the common assumption that such transmembrane segments are a-helical in nature. This work will also guide preliminary decisions about pathway size, shape and location. Our third objective builds on this information to make preliminary models of UhpT structure. These models will be tested by implanting suitably placed pairs of cysteines. The ability of these cysteines to form disulfide bonds will serve as a molecular ruler, revealing neighboring segments, giving a coherent plan for the hydrophobic sector and allowing us to decide if the translocation pathway lies between N- and C-terminal domains of the molecule, as predicted by indirect arguments.

描述：这项工作解决了三个核心问题， 膜转运：（1）哪些残基构成转运途径？ (2)这些残基是否存在于特征结构基序中？ (3)什么 是这种蛋白质的疏水核心的整体结构？ 到 为了回答这些问题，我们将利用诱变来植入半胱氨酸， 在其他无半胱氨酸版本中的战略位置的残基 UhpT，来自大肠杆菌的模式反向转运载体。 使用 半胱氨酸定向探针扫描这样的变异群体，我们可以 生成具有单个分辨率的功能和结构图 残余物 这些目标通过三种实验来实现： 第一个目标是定位与UhpT易位相关的残基 通路 这将通过用探针探测单半胱氨酸突变体来完成， 亲水性和不渗透性SH反应性试剂。 路径上的残留物将 被鉴定为如果被半胱氨酸替代， 不渗透的SH定向探针添加到任一膜表面，并且其 底物的存在防止了探针的抑制。 这项工作 还将导致关于UhpT拓扑的更精确的信息。 这样的实验将识别一组跨膜片段， 运输途径。 我们的第二个目标是评估当地的结构， 检测对不同大小和形状的亲水性探针的反应性。 探针反应的模式将证实或拒绝共同的假设 这种跨膜片段本质上是α-螺旋。 这项工作将 还可指导关于通道大小、形状和位置的初步决定。 我们的第三个目标建立在这些信息的基础上， UhpT结构。 这些模型将通过植入适当位置进行测试 成对的半胱氨酸。 这些半胱氨酸形成二硫键的能力 将作为一个分子的统治者，揭示相邻的部分，给一个 疏水部门的连贯计划，并允许我们决定 易位途径位于N-和C-末端结构域之间， 分子，正如间接论证所预测的那样。