Ion Gradients and Energy Coupling in Bacteria

描述（由申请人提供）：这项工作的长期目标是在基础水平上了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，是负责的摄取和运输己糖磷酸的土方chia大肠杆菌，也代表其亲属的主要促进剂超家族，膜运输系统的最大的集合之一。这些相关系统（i）促进糖穿过所有哺乳动物细胞膜的运动，包括参与对胰岛素的反应的那些细胞膜;（ii）被募集以组织中枢神经系统中神经递质的分泌;以及（iii）在病原菌的耐药性中充当中心角色。由于这些原因，理解我们模型（UhpT）中的运输机制也将有助于我们理解大量与人类健康抗病相关的系统。四行实验研究计划，以了解我们的模型蛋白质，UhpT的结构/功能关系。(1)通过使用特别构建的双半胱氨酸变体，螺旋-螺旋接近的一般特征将通过二硫键捕获和其他交联方案来探测。(2)在一项合作努力中，半胱氨酸取代诱变将用于植入支持电子顺磁光谱（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)继续的研究将利用定点和选择性诱变来揭示易位途径中残基的性质。这将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2D和3D晶体学的研究应该允许直接和具体的证据支持通过更间接的方法得出的结论。总之，这种遗传，生物化学和生物物理方法的组合应该揭示膜运输现象背后的结构相关性。

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.