Anion Exchange in Bacteria

细菌中的阴离子交换

• 批准号: 9986617
• 负责人: Peter Maloney
• 金额: $ 39万
• 依托单位: Johns Hopkins University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9986617&HistoricalAwards=false
• 关键词: Anion; Exchange; Bacteria

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.

这个项目涉及OxlT，一种在革兰氏阴性厌氧菌中发现的不同寻常的运输蛋白，即形成草酸杆菌。OxlT催化外部二价草酸盐与内部单价甲酸盐的交换。这种生电交换，以及细胞内脱羧基过程中的质子消耗，构成了一个虚拟的质子泵，该泵产生质子动力，允许产甲氧菌从草酸中提取能量。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，该项目将专注于OxlT，它是形成乳杆菌这一过程的中心反向转运蛋白。获得了针对OxlT N-末端的抗体，克隆了该基因，并在大肠杆菌中进行了异源表达，纯化了全功能蛋白。因此，可以在两个重要领域研究OxlT的结构-功能关系：(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二硫键捕获和基于半胱氨酸的交联剂。同时，红外光谱将被用来询问在底物结合的过程中是否有螺旋取向的变化。(2)更加雄心勃勃的工作将集中在OxlT的二维结晶学上。在这种情况下，目标是利用OxlT-底物复合体的非凡稳定性以及OxlT的遗传可操作性，以形成供电子显微镜分析的2D晶体。初步试验表明，分子尺寸的脆弱管的形成，未来几年的进一步努力应该是有意义的。小分子穿过细胞膜的方式是生物学的许多方面的核心，包括生物技术和植物生物学。举个例子，比如红血球获取糖分，微生物产生抗药性，或者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中的蛋白质介导的，该超家族是一个进化上相关的转运蛋白的巨大集合，其中包括OxlT作为其特征更好的成员之一。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。这对于被预测的结晶学工作来说尤其如此，因为以前还没有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，将极大地促进未来的研究，这些研究可以通过设计新的抑制剂、激活剂和这些重要蛋白质的底物来服务于公众利益。