Anion Exchange in Bacteria

这个项目涉及OxlT，一种在革兰氏阴性厌氧菌，草酸杆菌formigenes中发现的不寻常的运输蛋白。OxlT催化外部二价草酸交换为内部一价甲酸。这种生电交换，以及细胞内脱羧过程中的质子消耗，构成了一个虚拟质子泵，产生质子动力，允许O. formigenes从草酸盐中提取能量。这种质子驱动的代谢循环在微生物学中广泛存在，为了在分子水平上理解这些事件，本项目将重点研究O. formigenes中处于这一过程中心的反port蛋白OxlT。获得了OxlT n端抗体，克隆了该基因，在大肠杆菌中外源表达后纯化出功能齐全的蛋白。因此，研究OxlT结构-功能关系的工具在两个重要领域是可用的：(1)一个主要的实验目标将是确定螺旋-螺旋接近度，使用单半胱氨酸和双半胱氨酸变体作为试剂在二氟化捕获和半胱氨酸交联中。同时，红外光谱将用于询问是否有螺旋方向的变化伴随底物的结合。(2)更有雄心的工作将集中在OxlT的二维晶体学上。在这种情况下，目标是利用OxlT-底物复合物的显著稳定性，以及OxlT的遗传可追溯性，以形成二维晶体进行电子显微镜分析。初步的试验显示，形成了分子尺度的易碎管，未来几年的进一步努力应该会提供信息。小分子在细胞膜上移动的方式是生物学许多方面的核心，包括生物技术和植物生物学。例如，红细胞对糖的获取，微生物耐药性的起源，植物对高盐度的适应。所有这些事件都是由主要促进者超家族（Major Facilitator Superfamily）中的蛋白质介导的，这是一个巨大的进化相关转运蛋白集合，其中包括OxlT作为其较好表征的成员之一。因此，对OxlT的研究可能有助于表征这一重要基团的基本性质。这对于投影晶体学工作来说尤其正确，因为在此之前，没有任何主要促进者超家族的成员在这个分辨率水平上被可视化。了解这些转运蛋白是如何在膜内排列的，将极大地加速未来的研究，通过设计这些重要蛋白质的新型抑制剂、激活剂和底物，为公众利益服务。

This project concerns OxlT, an unusual transport protein found in the Gram-negative anaerobe, Oxalobacter formigenes. OxlT catalyzes the exchange of external divalent oxalate for internal monovalent formate. This electrogenic exchange, along with proton consumption during intracellular decarboxylation, constitutes a virtual proton pump that generates a proton-motive force allowing O. formigenes to extract energy from oxalate. Such proton-motive metabolic cycles are widely spread in microbiology, and to understand these events at a molecular level this project will focus on OxlT, the antiport protein at the center of this process in O. formigenes. Antibody against the OxlT N-terminus is available, the gene is cloned, and fully functional protein is purified after heterologous expression in Escherichia coli. Therefore, tools are available to study structure-function relationships for OxlT in two important areas: (1) A major experimental goal will be the determination of helix-helix proximity, using single-cysteine and double-cysteine variants as reagents in disufide trapping and cysteine-based cross-linking. In parallel, infrared spectroscopy will be used to ask whether there are changes in helix orientation accompanying the binding of substrate. (2) The more ambitious work will focus on 2-dimensional crystallography of OxlT. In this case, the goal is to capitalize on the remarkable stability of the OxlT-substrate complex, as well as the genetic tractability of OxlT, to enable formation of 2D crystals for analysis by electron microscopy. Preliminary trials show formation of fragile tubes of molecular dimension, and further effort over the next few years should be informative. The way in which small molecules move across cell membranes is central to many aspects of biology, including biotechnology and plant biology. As examples, consider the acquisition of sugar by the red blood cell, or the origin of drug resistance in microorganisms, or the adaptation to high salinity by plants. All these events are mediated by proteins within the Major Facilitator Superfamily, a vast collection of evolutionarily related transporters that includes OxlT as one of its better-characterized members. Study of OxlT may therefore help characterize the fundamental properties of this important group. This is especially true of the projected crystallographic work, since no member of the Major Facilitator Superfamily has been visualized at this level of resolution before. Understanding how such transporters are arranged within the membrane would greatly accelerate future studies that can serve the public interest by the design of novel inhibitors, activators, and substrates of these important proteins.