Anion Exchange in Bacteria

这个项目的重点是一种膜转运蛋白(OxlT)，它可以催化草酸根细菌中二价草酸和一价甲酸的交换。这一反应，与草酸在细胞内的脱羧化一起，产生了质子动力，由福尔马氏菌用来驱动质膜上的离子耦合活动。这种质子动力代谢循环在微生物中广泛传播，这一提议将把最初关于OxlT的工作从早期的关注细胞生理学扩展到了解膜运输的结构相关性。在大肠杆菌中异源表达后，完全功能的OxlT被纯化，使电子结晶学能够提供6.5埃的分辨率结构作为当前工作的指导。结构与功能的关系将从三个方面进行考察。(1)初步研究将通过使用二硫键捕获和基于半胱氨酸的双半胱氨酸变体的交联来确定特定的螺旋-螺旋关系。与单半胱氨酸变异体的平行工作将通过使用不必要的SH活性试剂来探索易位途径的组成。(2)第二条线通过将这些半胱氨酸变体用作定点自旋标记的目标来扩展它们的价值，以通过电子顺磁谱(EPR)来评估动态行为。这将允许评估底物诱导的螺旋-螺旋距离的变化。(3)最后一系列实验建立在较早的结晶学成功的基础上。将做出新的努力来提高2D晶体的质量，并找到适合3D晶体的X射线分析的条件。广泛的影响：导致该项目的过去的工作支持了本科生、研究生和博士后研究员的贡献，他们通过参与这项跨学科的研究丰富了他们的教育经验。在更大的范围内，对膜转运蛋白的了解对于了解所有细胞如何与其直接环境相互作用是至关重要的。因为OxlT代表了最大的膜转运蛋白家族--主要促进器超家族的所有成员，所以从细胞生物学和结构生物学的角度来看，OxlT到目前为止都是一个信息丰富的模型。这种对培训和研究的积极贡献将继续下去。

This project focuses on a membrane transporter (OxlT) that catalyzes the exchange of divalent oxalate and monovalent formate in the bacterium, Oxalobacter formigenes. This reaction, together with the intracellular decarboxylation of oxalate, generates the proton-motive force used by O. formigenes to drive ion-coupled activities at the plasma membrane. Proton-motive metabolic cycles of this sort are widely spread among microbes, and this proposal will extend the original work on OxlT from an early focus on cell physiology to one that deals with understanding the structural correlates of membrane transport. After its heterologous expression in Escherichia coli, fully functional OxlT is purified, enabling electron crystallography that provides a 6.5 angstrom resolution structure as a guide to current work. Structure-function relationships will be examined in three ways. (1) Initial studies will identify specific helix-helix relationships by use of disufide trapping and cysteine-based cross-linking of double-cysteine variants. Parallel work with single-cysteine variants will probe the composition of the translocation pathway by using impermeant SH-active reagents. (2) A second line of work extends the value of these cysteine variants by using them as targets for site-directed spin labeling to assess dynamic behavior by electron paramagnetic spectroscopy (EPR). This will allow an evaluation of substrate-induced changes in helix-helix distance. (3) A final series of experiments builds on the earlier crystallographic success. New efforts will be made to improve the quality of 2D crystals and to find conditions suitable for x-ray analysis of 3D crystals.Broader Impact: The past work leading to this project has supported the contributions of undergraduates, graduate students and postdoctoral fellows, who enriched their educational experience by participation in this interdisciplinary research. On a larger scale, an understanding of membrane transporters is essential to understanding how all cells interact with their immediate environment. Because it is representative of all members in the Major Facilitator Superfamily, the largest collection of membrane transporters, OxlT has so far been an informative model from the perspective of both cell biology and structural biology. Such positive contributions to training and research will continue.