Ion Gradients and Energy Coupling in Bacteria

细菌中的离子梯度和能量耦合

• 批准号: 7087705
• 负责人: PETER C MALONEY
• 金额: $ 37.63万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7087705
• 关键词: Escherichia coli; X ray crystallography; antiport; bacterial proteins; bioenergetics; cysteine; disulfide bond; electron spin resonance spectroscopy; gene mutation; ion transport; membrane channels; membrane permeability; membrane transport proteins; protein structure function; protein transport; site directed mutagenesis; sugar phosphates

DESCRIPTION (provided by applicant): The long-term objective of this work is to understand, at a fundamental level, the relationship between the structure of a membrane transport protein and the mechanistic features of its function. The model protein we are studying, known as UhpT, is responsible for the uptake and transport of hexose phosphates by Escherchia coli and serves also to represent its relatives in the Major Facilitator Superfamily, one of the largest collections of membrane transport systems. Such related systems (i) facilitate movement of sugar across all mammalian cell membranes, including those involved in the response to insulin; (ii) are recruited to organize the secretion of neurotransmitters in the central nervous system; and (iii) act as central players in the drug resistance of pathogenic bacteria. For these reasons, understanding the mechanism of transport in our model (UhpT) will also help us understand a large number of systems relevant to human health antidisease. Four lines of experimental study are planned to understand structure/function relationships in our model protein, UhpT. (1) By using specially constructed double-cysteine variants, the general features of helix-helix proximity will be probed by disulfide trapping and other cross-linking protocols. (2) In a collaborative effort, cysteine-substitution mutagenesis will be used to implant probes supporting electron paramagnetic spectroscopy (EPR) to precisely judge changes in helix-helix distance during substrate binding. (3) Continuing studies will exploit both site- and selection-directed mutagenesis to reveal the properties of residues lining the translocation pathway. This should illuminate principles that underlie substrate specificity and selectivity. (4) Finally, in an ongoing collaboration with colleagues at the National Institutes of Health, studies aimed at both 2D and 3D crystallography of two UhpT-related proteins (GIpT and UhpC) should allow direct and concrete evidence supporting conclusions drawn by the more indirect approaches. Together, this combination of genetic, biochemical and biophysical approaches should reveal the structural correlates underlying the phenomenon of membrane transport.

描述（由申请人提供）：这项工作的长期目标是在基础水平上理解膜转运蛋白的结构与其功能的机制特征之间的关系。我们正在研究的模型蛋白被称为UhpT，它负责大肠杆菌对磷酸己糖的摄取和运输，也代表了它在主要促进物超家族中的亲戚，这是最大的膜运输系统集合之一。这些相关系统(1)促进糖在所有哺乳动物细胞膜上的运动，包括那些参与胰岛素反应的细胞膜；（ii）被招募来组织中枢神经系统的神经递质的分泌；（三）在致病菌的耐药性中发挥核心作用。由于这些原因，了解我们模型中的转运机制（UhpT）也将有助于我们了解与人类健康抗疾病相关的大量系统。我们计划进行四项实验研究，以了解我们的模型蛋白UhpT的结构/功能关系。(1)利用特殊构建的双半胱氨酸变体，通过二硫捕获和其他交联协议来探测螺旋-螺旋接近的一般特征。(2)在合作的努力下，半胱氨酸替代诱变将用于植入支持电子顺磁谱（EPR）的探针，以精确判断底物结合过程中螺旋-螺旋距离的变化。(3)后续的研究将利用位点导向和选择导向诱变来揭示易位通路内残基的特性。这应该阐明底物特异性和选择性的基本原理。(4)最后，在与美国国立卫生研究院的同事正在进行的合作中，针对两种uhpt相关蛋白（GIpT和UhpC）的二维和三维晶体学研究应该允许直接和具体的证据支持更间接方法得出的结论。总之，遗传、生化和生物物理方法的结合应该揭示膜运输现象背后的结构关联。