Anion Exchange in Bacteria

细菌中的阴离子交换

• 批准号: 0235305
• 负责人: Peter Maloney
• 金额: $ 62.43万
• 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: 2003
• 资助国家: 美国
• 起止时间: 2003-05-15 至 2006-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0235305&HistoricalAwards=false
• 关键词: Anion; Exchange; Bacteria

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.

这个项目的重点是膜转运蛋白（OxlT）催化二价草酸盐和一价甲酸盐在细菌中的交换，草酸草杆菌formigenes。这个反应，连同细胞内草酸的脱羧作用，产生了O. formigenes用来驱动质膜上离子偶联活性的质子动力。这种质子驱动的代谢循环在微生物中广泛存在，这一提议将把OxlT的原始工作从早期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，纯化了功能齐全的OxlT，使提供6.5埃分辨率结构的电子晶体学可以指导当前的工作。结构-功能关系将以三种方式进行检验。(1)初步研究将通过双半胱氨酸变异的二聚体捕获和基于半胱氨酸的交联来确定特定的螺旋-螺旋关系。与单半胱氨酸变异的平行工作将通过使用非必需sh活性试剂来探测易位途径的组成。(2)第二项工作扩展了这些半胱氨酸变异的价值，利用它们作为定位自旋标记的靶标，通过电子顺磁谱（EPR）评估其动态行为。这将允许评估底物引起的螺旋-螺旋距离的变化。最后的一系列实验建立在早先晶体学成功的基础上。将作出新的努力来提高二维晶体的质量，并找到适合三维晶体的x射线分析的条件。更广泛的影响：本项目的前期工作支持了本科生、研究生和博士后的贡献，他们通过参与这一跨学科的研究，丰富了他们的教育经验。在更大的范围内，了解膜转运蛋白对于理解所有细胞如何与其直接环境相互作用至关重要。因为它代表了主要促进者超家族（最大的膜转运体集合）的所有成员，OxlT迄今为止从细胞生物学和结构生物学的角度来看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。