ION GRADIENTS AND ENERGY COUPLING IN BACTERIA

这项工作解决了膜运输领域的关键问题：(1)运输蛋白的整体组织是什么？(2)通过转运体的易位途径由哪些残基组成？(3)是什么决定了底物的特异性和选择性？为了回答这些问题，我们将结合生物化学与大肠杆菌模型转运体UhpT的定点定向和选择驱动诱变，我们计划进行四种实验。将通过将半胱氨酸对放置在其他半胱氨酸缺失的UhpT中来测试UhpT的结构模型。暴露于氧化条件下，二硫键的形成将作为识别邻近螺旋的分子标尺，揭示跨膜螺旋阵列的整体组织。同时，我们将开发一个体外系统，用纯化和重组的蛋白质来表征易位途径上的残基。我们将探讨单半胱氨酸变异与亲水性和不意味着硫醇反应剂。途径上的残基，如果被半胱氨酸取代，当探针从蛋白质的细胞外或细胞内表面接近时，就可以接近。探针在大小、形状和电荷上不同的可接近性模式将用于了解底物进入和离开途径的方式。目前的证据表明TM11在UhpT的底物特异性中起关键作用。为了探索这个问题，我们将选择或设计具有新特征的UhpT变体，包括改变的底物特异性和改变的电荷选择性。长期目标将是获得UhpT二维晶体，以便将结构-功能关系置于合理的基础上。因此，我们将建立条件，以产生毫克量的稳定，纯化的UhpT，用于晶体学试验期间的电子衍射分析。

This work addresses key issues in the field of membrane transport: (1) What is the overall organization of a transport protein? (2) What residues comprise the translocation pathway through the transporter? (3) What determines substrate specificity and selectivity? To answer these questions, we will combine biochemistry with site-directed and selection-driven mutagenesis of UhpT, a model transporter in Escherichia coli, We plan four kinds of experiments. A structural model of UhpT will be tested by placing pairs of cysteines in an otherwise cysteine-less UhpT. On exposure to oxidizing conditions, formation of disulfide bonds will serve as a molecular ruler to identify neighboring helices, revealing the overall organization of the transmembrane helical array. Concurrently we will develop an in vitro system, with purified and reconstituted protein to characterize residues that line the translocation pathway. We will probe single-cysteine variants with hydrophilic and impermeant thiol-reactive agents. Residues on the pathway, if replaced by cysteine, are accessible to such probes when they approach from either they extra- or intracellular face of the protein. Patterns of accessibility to probes differing in size, shape and charge will be used to learn about the way(s) in which substrates enter and leave the pathway. Current evidence implicates TM11 as a key player in setting substrate specificity in UhpT. To explore this question, we will select or engineer UhpT variants that have acquired novel features, including an altered substrate specificity and altered charge selectivity. A long term objective will be to obtain UhpT 2-dimensional crystals, so that structure-function relationships can be placed on a rational basis. Accordingly, we will establish conditions that yield milligram quantities of stable, purified UhpT for analysis by electron diffraction during trials of crystallography.