ION GRADIENTS AND ENERGY COUPLING IN BACTERIA

描述：这项工作解决了三个核心问题 膜转运：(1)什么残基构成转位途径？ (2)这些残基存在于特征结构基序中吗？(3)什么 是这样一种蛋白质疏水核心的整体组织吗？至 回答这些问题，我们将利用诱变技术植入半胱氨酸 在战略位置的残基，否则不含半胱氨酸的版本 UhpT，一种来自大肠杆菌的模型反转载体。vbl.使用 半胱氨酸导向的探针扫描一组这样的变异，我们可以 以单一分辨率生成功能和结构图 残留物。这些目标通过三种类型的实验来实现： 第一个目标是定位UhpT易位上的残基 路径。这将通过探测单半胱氨酸突变体来完成 亲水性和无意义的SH反应剂。途径上的残留物将 被确定为如果被半胱氨酸取代，可以获得 将不必要的SH定向探针添加到膜表面，并且其 底物的存在防止了探针的抑制。这部作品 还将导致关于UhpT拓扑的更准确的信息。 这样的实验将识别出一组围绕着 运输途径。我们的第二个目标是通过以下方式评估当地结构 检测对不同尺寸和形状的亲水性探针(S)的反应性。 探测器的反应性模式将证实或拒绝常见的假设 这种跨膜片段本质上是a螺旋的。这项工作将 也指导关于路径大小、形状和位置的初步决定。 我们的第三个目标是在此信息的基础上建立初步模型 UhpT结构。这些模型将通过植入适当的位置进行测试 成对的半胱氨酸。这些半胱氨酸形成二硫键的能力 将充当分子尺子，显示相邻的片段，给出 为疏水部门制定连贯的计划，并允许我们决定是否 易位途径位于N端和C端结构域之间 分子，正如间接论证所预测的那样。

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.