ION GRADIENTS AND ENERGY COUPLING IN BACTERIA

这项工作解决了膜转运领域的关键问题：（1）转运蛋白的整体结构是什么？ (2)哪些残基构成通过转运蛋白的转运途径？ (3)什么决定底物的特异性和选择性？ 为了回答这些问题，我们将联合收割机与生物化学相结合，对大肠杆菌转运蛋白UhpT进行定点突变和选择驱动突变。将通过将成对的半胱氨酸放置在另外无半胱氨酸的UhpT中来测试UhpT的结构模型。 在暴露于氧化条件下时，二硫键的形成将作为识别相邻螺旋的分子标尺，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，纯化和重建的蛋白质，以表征残留物，线的易位途径。 我们将用亲水性和不渗透性的巯基反应剂探测单半胱氨酸变体。 如果被半胱氨酸取代，则该途径上的残基在它们从蛋白质的细胞外或细胞内表面接近时可被这样的探针接近。 不同大小、形状和电荷的探针的可及性模式将用于了解底物进入和离开通路的方式。目前的证据表明TM 11是UhpT中设置底物特异性的关键参与者。 为了探索这个问题，我们将选择或工程化UhpT变体，这些变体具有新的特征，包括改变的底物特异性和改变的电荷选择性。一个长期的目标将是获得UhpT二维晶体，使结构-功能关系可以放置在一个合理的基础上。 因此，我们将建立条件，产生毫克量的稳定的，纯化的UhpT的分析，在晶体学试验期间的电子衍射。

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.