Molecular Mechanisms of Channels and Transporters

通道和转运蛋白的分子机制

• 批准号: 10204502
• 负责人: Katherine Anne Henzler-Wildman
• 金额: $ 38.23万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10204502
• 关键词: Active Biological Transport; Address; Allosteric Regulation; Antibiotics; Binding; Biochemical; Biological; Biological Assay; Biological Models; Biological Process; Biophysics; Chemicals; Coupled; Data; Environment; In Vitro; Integral Membrane Protein; Ion Channel; Ions; Kinetics; Knowledge; Length; Membrane; Membrane Proteins; Modeling; Molecular; Molecular Probes; Monitor; Motion; Movement; NMR Spectroscopy; Population; Process; Protein Conformation; Proteins; Protons; Pump; Research; Resolution; Site; Specificity; Structure; System; Temperature Sense; Thermodynamics; Time; Work; in vivo; insight; protein function; small molecule; tool

ABSTRACT Knowledge of protein motion is necessary to bridge the gap between structure and function and gain insight into the molecular mechanism of protein machines. My lab studies the function of ion channels and ion-coupled transporters, integral membrane proteins that must undergo structural transitions to regulate the flow of ions (channels) or actively pump substrates (transporters) across biological membrane barriers. A set of distinct but interrelated projects examines the mechanism of secondary active transport, promiscuous multidrug recognition, ion channel selectivity and gating, molecular basis of temperature sensing, allosteric regulation of transporter and channel activity, and how small localized interactions can regulate broader dynamics in membrane proteins. These research questions span time- and length- scales, requiring an array of experimental approaches and a well-developed set of model systems to enable hypothesis driven research. One of our primary tools is NMR spectroscopy, which can simultaneously provide structural, thermodynamic (populations), and kinetic (rates of transitions) data with site-specific resolution. NMR chemical shifts are also highly sensitive to changes in the local environment, providing a direct readout of proton binding, a process that is otherwise difficult to monitor experimentally but central to dissecting proton-coupled transport. Over the past 10 years, my lab has done the painstaking work necessary to develop three completely independent model transporter and channel systems (EmrE, NaK, Shaker-VSD) and establish experimental tools ranging from NMR and molecular biophysics to in vitro and in vivo functional assays. We are now primed to address essential research questions that probe the molecular mechanism of these specific systems but also have broader implications for understanding how protein conformational change is regulated, promiscuity versus specificity in substrate recognition, the complexity of proton-coupled transport, and molecular basis for allosteric regulation of protein function.

抽象的 蛋白质运动的知识对于弥合结构和功能之间的差距是必要的 进入蛋白质机器的分子机制。我的实验室研究离子通道和离子耦合的功能 转运蛋白，整体膜蛋白，必须进行结构过渡以调节离子的流动 （通道）或主动泵基板（转运蛋白）跨生物膜屏障。一组独特但 相互关联的项目检查了二次主动运输的机制 识别，离子通道选择性和门控，温度传感的分子基础，变构调节 转运蛋白和通道活动，以及小局部相互作用如何调节更广泛的动态 膜蛋白。这些研究问题涵盖了时间和长度，需要一个数组 实验方法和一组开发的模型系统，以实现假设驱动的研究。 我们的主要工具之一是NMR光谱法，它可以同时提供结构性的热力学 （种群）和动力学（过渡速率）数据具有特定地点分辨率。 NMR化学位移也是 对本地环境的变化高度敏感，提供了质子结合的直接读数，一个过程 否则，这很难在实验中监测，但要剖析质子耦合转运的中心。在 过去10年，我的实验室做了必要的艰苦工作，以开发三个完全独立的工作 模型运输商和通道系统（EMRE，NAK，Shaker-VSD）并建立实验工具 从NMR和分子生物物理学到体外和体内功能测定。我们现在已经准备好解决 探测这些特定系统的分子机制但也具有的基本研究问题 了解如何调节蛋白质构象变化，滥交与 底物识别的特异性，质子耦合转运的复杂性以及分子基础的基础 蛋白质功能的变构调节。