ION GRADIENTS AND ENERGY COUPLING IN BACTERIA

这项工作解决了膜转运蛋白领域的关键问题：(1)转运蛋白的整体组织是什么？(2)通过转运蛋白的转位途径包括什么残基？(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，我们将把生物化学与UhpT的定点和选择驱动突变相结合，UhpT是大肠杆菌中的模型转运蛋白，我们计划进行四种实验。UhpT的结构模型将通过将成对的半胱氨酸放置在原本不含半胱氨酸的UhpT中进行测试。在氧化条件下，二硫键的形成将作为分子标尺来识别相邻的螺旋，揭示跨膜螺旋阵列的整体结构。同时，我们将开发一个体外系统，用纯化和重组的蛋白来表征沿着易位途径的残基。我们将探索具有亲水性和非亲水性的硫醇反应剂的单半胱氨酸变体。如果途径上的残基被半胱氨酸取代，当它们从蛋白质的胞外或胞内表面接近时，这些探针就可以接触到它们。不同大小、形状和电荷的探头的可及性模式将被用来了解底物进入和离开通路的方式(S)。目前的证据表明，TM11在UhpT的底物特异性设定中起关键作用。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，包括改变的底物专一性和改变的电荷选择性。一个长期的目标将是获得UhpT的二维晶体，以便结构-功能关系可以在合理的基础上放置。因此，我们将建立生产毫克量稳定的、纯化的UhpT的条件，以便在结晶学试验期间通过电子衍射进行分析。

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.