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是主要易化因子超家族（MFS）的成员。MFS的其他成员在人体生理学中起着重要作用，使大多数细胞能够吸收糖和氨基酸，并使中枢神经系统中的神经元能够循环神经递质。这些人的例子中没有一个可以用OxlT详细研究，因此使用该模型的工作对于（a）转运蛋白在底物结合和转运期间采用的各种构象和（B）这些转运蛋白如何建立其底物特异性应该是有指导意义的。这些信息应使疾病诊断和治疗干预措施的设计更加容易。规划了三条总的研究路线。(1)初步工作已经建立了产生稳定、单分散和低脂质含量的OxlT的高水平生产的条件。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射线晶体学研究，从手动和基于机器人的屏幕开始，以已经产生小晶体的条件为中心。(2)OxlT的结构分析将通过生物化学工作进行，该生物化学工作集中于（a）确定可溶性和膜包埋形式的OxlT寡聚状态;（B）对渗透途径中的残基进行定点诱变，以便了解设定底物特异性/选择性的因素;和（c）鉴定显示与它们在渗透途径的打开和关闭中的作用一致的底物触发的邻近变化的残基。(3)我们的第三个计划涉及合作工作，将荧光探针对放置在OxlT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采用的OxlT构象的目录，并将连接结构和动力学模型。公共卫生相关性：这项工作旨在揭示转运蛋白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非常类似于在肠道和肾脏中移动糖，在神经系统中循环神经递质并赋予病原菌耐药性的系统。

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.