Ion Gradients and Energy Coupling in Bacteria

这项工作的长期目标是了解膜结构之间的关系 转运蛋白及其功能的机制特征。我们正在研究的模型蛋白质OxIT携带 草酸与甲酸盐的外交换，作为主要促进剂超家族的成员，起着 许多其他运输系统的模型，例如(I)那些促进糖在 哺乳动物细胞膜，包括胰岛素反应转运体；(Ii)循环的转运体 中枢神经系统中的神经递质；以及(Iii)介导致病菌耐药性的系统。 细菌。因此，对OxIT的研究对于了解与人类健康相关的转运蛋白和 疾病。 计划了四条学习路线。(1)以电子和X射线结构模型为指导 结晶学方面，我们将使用定点突变来探测渗透途径，从而操纵 底物特异性/选择性，并揭示了可能的构象灵活性。(2)在协同工作中，我们将 在OxIT上的关键位置放置成对的荧光探针，并使用单对荧光共振 能量转移(SpFRET)用于测量分离距离在单分子水平上的变化。这将是 给出在底物结合和运输过程中采用的OxIT构象的目录，以及连接结构和 动力学模型。(3)前期工作为x射线结晶学研究奠定了良好的条件。 以稳定、单分散和低脂含量的形式高水平生产OxIT。我们将继续追查 在合适合作者的建议和帮助下，从基于机器人的开始，对OxIT的X射线结晶学进行研究 初始条件的筛选。(4)最后，我们将开发一个遗传系统，以应用基于选择的 突变以更充分地筛选具有信息特性的OxIT突变体。 我们的工作探索了一个模型膜转运蛋白OxIT的结构和机制。作为该协会的成员 主要的促进者超级家族，OxIT的近亲在人类生理学中发挥着至关重要的作用，使 大多数细胞对糖和氨基酸的吸收和神经元对神经递质的循环 中枢神经系统。这些人类的例子都不能用OxIT进行详细的研究，所以 使用该模型系统对于(A)这种转运体如何建立它们的底物应该是有指导意义的 特异性，以及(B)转运体在底物结合和运输过程中采用的各种构象。 这将使诊断疾病和设计用于治疗的干预药物变得更容易。

The long-term objective of this work is to understand the relationship between the structure of a membrane transport protein and mechanistic features of its function. The model protein we are studying, OxIT, carries out exchange of oxalate with formate, and as a member of the Major Facilitator Superfamily, serves as a model for many other transporter systems, such as (i) those that facilitate sugar movements across mammalian cell membranes, including the insulin-responsive transporters; (ii) the transporters that cycle neurotransmitters in the central nervous system; and (iii) systems that mediate drug resistance in pathogenic bacteria. Thus, study of OxIT is important to understanding transporters relevant to human health and disease. Four lines of study are planned. (1) Guided by structural models derived from electron and x-ray crystallography, we will use site-directed mutagenesis to probe the permeation pathway, so as to manipulate substrate specificity/selectivity and reveal possible conforrmational flexibility. (2) In collaborative work, we will place pairs of fluorescent probes at strategic positions on OxIT and use single-pair fluorescence resonance energy transfer (spFRET) to measure separation distances changes at the single-molecuule level. This will give a catalog of OxIT conformations adopted during substrate binding and transport and link structural and kinetic models. (3) Early work has established favorable conditions for x-ray crystallography by providing high level production of OxIT in a form that is stable, monodisperse and of low lipid content. We will pursue x-ray crystallography of OxIT with the advice and help of suitable collaborators, beginning with robotic-based screens of initial conditions. (4) Finally, we will develop a genetic system so as to apply selection-based mutagenesis to more adequately screen for OxIT mutants with informative properties. Our work probes the structure and mechanism of a model membrane transporter, OxIT. As a member of the Major Facilitator Superfamily, close relatives of OxIT 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 OxIT, so work with this model system should be instructive as to (a) how such transporters establish their substrate specificity, and (b) the various conformations adopted by transporters during substrate binding and transport. This should make it easier to both diagnose disease and design interventins for treatment.