Ion Gradients and Energy Coupling in Bacteria

细菌中的离子梯度和能量耦合

• 批准号: 7464847
• 负责人: PETER C MALONEY
• 金额: $ 51.55万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7464847
• 关键词: Address; Adopted; Amino Acids; Bacteria; Binding; Binding Sites; Biochemical; Biological Models; Cataloging; Catalogs; Cell Cycle; Cell Survival; Cell membrane; Condition; Coupling; Crystal Formation; Crystallization; Crystallography; Culicidae; Cysteine; Decarboxylation; Devices; Diagnosis; Disease; Drug resistance; Electrons; Energy Transfer; Escherichia coli; Event; Fluorescence Resonance Energy Transfer; Fluorescent Probes; Formates; Frequencies; Future; Genetic; Goals; Health; Homology Modeling; Human; Insulin; Ions; Kinetics; Label; Lead; Libraries; Ligand Binding; Link; Lipids; Maleimides; Mammalian Cell; Measures; Mediating; Membrane; Membrane Transport Proteins; Modeling; Modification; Molecular; Molecular Conformation; Movement; Mutagenesis; Neuraxis; Neurons; Neurotransmitters; Outcome; Oxalates; Pathway interactions; Pattern; Physiology; Play; Pliability; Positioning Attribute; Production; Property; Proteins; Range; Recruitment Activity; Relative (related person); Resolution; Robot; Robotics; Role; Screening procedure; Site-Directed Mutagenesis; Sorting - Cell Movement; Specificity; Spectrum Analysis; Structural Models; Structure; Substrate Specificity; Surface; Surveys; System; Target Populations; Techniques; Time; Transmembrane Transport; Work; absorption; base; design; experience; falls; fluorophore; interest; member; mutant; nanofluidic; nanolitre; pathogenic bacteria; periplasm; response; single molecule; sugar; thymidine 5' -triphosphate; tool

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.

这项工作的长期目标是了解膜的结构之间的关系， 转运蛋白及其功能的机制特征。我们正在研究的模型蛋白OxIT携带 草酸与甲酸的交换，作为主要促进剂超家族的成员， 模型的许多其他转运系统，如（i）那些促进糖运动， 哺乳动物细胞膜，包括胰岛素应答转运蛋白;（ii）循环转运蛋白 中枢神经系统中的神经递质;和（iii）介导病原体中的药物抗性的系统。 细菌因此，OxIT的研究对于了解与人类健康相关的转运蛋白以及 疾病 计划进行四项研究。(1)由电子和X射线导出的结构模型指导 晶体学，我们将使用定点突变来探测渗透途径，以便操纵 底物特异性/选择性，并揭示可能的构象灵活性。(2)在合作中，我们将 将荧光探针对置于OxIT上的关键位置，并使用单对荧光共振 能量转移（spFRET），以测量在单分子水平的分离距离的变化。这将 给出了在底物结合和运输过程中采用的OxIT构象的目录， 动力学模型(3)早期的工作为X射线晶体学建立了有利的条件， 以稳定、单分散和低脂质含量形式高水平生产OxIT。我们将奉行 OxIT的X射线晶体学，在合适的合作者的建议和帮助下，从基于机器人的 屏幕初始条件(4)最后，我们将开发一个遗传系统，以便应用基于选择的 诱变以更充分地筛选具有信息性质的OxIT突变体。 我们的工作探讨了模型膜转运蛋白OxIT的结构和机制。成员的 主要促进剂超家族，OxIT的近亲在人类生理学中起着重要作用， 大多数细胞对糖和氨基酸的吸收以及脑内神经元对神经递质的循环 中枢神经系统这些人类的例子中没有一个可以用OxIT进行详细的研究， 使用该模型系统的工作应该对以下方面具有指导意义：（a）这些转运蛋白如何建立它们的底物 特异性，和（B）在底物结合和转运过程中转运蛋白所采用的各种构象。 这将使诊断疾病和设计治疗干预措施更容易。