Anion Exchange in Bacteria

该项目涉及OxlT，一种在革兰氏阴性厌氧菌Oxalciumformigenes中发现的不寻常的转运蛋白。 OxlT催化外部二价草酸盐与内部一价甲酸盐的交换。 这种产电交换，沿着细胞内脱羧过程中的质子消耗，构成了一个虚拟的质子泵，产生质子动力，使O。产甲酸菌从草酸盐中提取能量 这种质子动力代谢循环在微生物学中广泛传播，为了在分子水平上理解这些事件，本项目将重点关注OxlT，O.产甲酸菌 针对OxlT N-末端的抗体是可获得的，该基因被克隆，并且在大肠杆菌中异源表达后纯化全功能蛋白。 因此，工具可用于在两个重要领域中研究OxlT的结构-功能关系：（1）主要实验目标将是使用单半胱氨酸和双半胱氨酸变体作为二硫化物捕获和基于半胱氨酸的交联中的试剂来确定螺旋-螺旋接近度。 同时，红外光谱将用于询问是否存在伴随底物结合的螺旋取向的变化。 (2)更雄心勃勃的工作将集中在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的遗传易处理性，以使得能够形成2D晶体用于电子显微镜分析。 初步试验表明，分子尺度的脆弱管的形成，在未来几年的进一步努力应该是有益的。 小分子穿过细胞膜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，考虑红细胞对糖的获取，或微生物抗药性的起源，或植物对高盐度的适应。 所有这些事件都是由主要易化因子超家族内的蛋白质介导的，主要易化因子超家族是进化上相关的转运蛋白的大量集合，包括OxlT作为其更好表征的成员之一。 因此，对OxlT的研究可能有助于描述这一重要基团的基本性质。 这对于计划中的晶体学工作来说尤其如此，因为以前没有任何一个主要易化因子超家族的成员在这个分辨率水平上被可视化。 了解这些转运蛋白在膜内的排列方式将大大加快未来的研究，这些研究可以通过设计这些重要蛋白质的新型抑制剂、激活剂和底物来服务于公众利益。

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.