Ion Gradients and Energy Coupling in Bacteria

描述（由申请人提供）：我们的工作探讨了细菌膜转运蛋白OxlT的结构和机制，OxlT是主要促进者超家族（MFS）的成员。MFS的其他成员在人体生理学中发挥着重要作用，使大多数细胞吸收糖和氨基酸，并使中枢神经系统神经元的神经递质循环。这些人类的例子都不能用OxlT进行详细的研究，所以使用这个模型应该对(a)转运体在底物结合和运输过程中采用的各种构象以及(b)这些转运体如何建立它们的底物特异性具有指导意义。这些信息将使诊断疾病和设计治疗干预措施变得更加容易。计划了三条一般的研究路线。(1)初步工作已经建立了稳定、单分散、低脂含量的高水平生产OxlT的条件。我们现在将在感兴趣的合作者的建议和帮助下进行OxlT的x射线晶体学，从人工和机器人屏幕开始，以已经产生小晶体的条件为中心。(2) OxlT的结构分析将与生化工作并行，重点是(a)测定可溶性和膜包埋形式的OxlT寡聚物状态；(b)对渗透通路上的残基进行定点诱变，以了解决定底物特异性/选择性的因素；(c)鉴定显示底物触发的接近性变化与其在渗透途径的打开和关闭中的作用一致的残基。(3)我们的第三个倡议涉及合作工作，将荧光探针对放置在OxlT上的战略位置，并使用单对荧光共振能量转移（spFRET）来测量单分子水平上的分离距离变化。这将给出在底物结合和运输过程中所采用的OxlT构象的目录，并将结构和动力学模型联系起来。公共卫生相关性：这项工作旨在揭示转运体OxlT促进底物跨膜运动的机制。这项工作与人类健康有关，因为OxlT与肠道和肾脏中的糖移动系统、神经系统中的神经递质循环系统以及赋予致病菌耐药性的系统非常相似。

DESCRIPTION (provided by applicant): Our work probes the structure and mechanism of a bacterial membrane transporter, OxlT, a member of the Major Facilitator Superfamily (MFS). Other members of the MFS play essential roles in human physiology, enabling the absorption of sugars and amino acids by most cells and the cycling of neurotransmitters by neurons in the central nervous system. None of these human examples can be studied in the detail possible with OxlT, so work with this model should be instructive as to (a) the various conformations adopted by transporters during substrate binding and transport and (b) how such transporters establish their substrate specificity. Such information should make it easier to both diagnose disease and design interventions for treatment. Three general lines of study are planned. (1) Preliminary work has established conditions yielding high level production of OxlT that is stable, monodisperse and of low lipid content. We will now pursue x-ray crystallography of OxlT with the advice and help of interested collaborators, beginning with manual and robotic-based screens centered on conditions that have already produced small crystals. (2) The structural analysis of OxlT will be paralleled by biochemical work focused on (a) determination of the OxlT oligomeric state in both soluble and membrane-embedded forms; (b) site-directed mutagenesis of residues lining the permeation pathway, so as to understand factors that set substrate specificity/selectivity; and (c) identification of residues that show substrate-triggered changes in proximity consistent with their role(s) in the opening and closing of the permeation pathway. (3) Our third initiative involves collaborative work to place pairs of fluorescent probes at strategic positions on OxlT and use single-pair fluorescence resonance energy transfer (spFRET) to measure separation distances changes at the single-molecule level. This will give a catalog of OxlT conformations adopted during substrate binding and transport and will link structural and kinetic models. PUBLIC HEALTH RELEVANCE: This work seeks to reveal the mechanism by which a transporter, OxlT, facilitates substrate movement across membranes. The work relates to human health, since OxlT closely resembles systems that move sugars in the gut and kidney, cycle neurotransmitters in the nervous system and confer drug resistance to pathogenic bacteria.