Molecular Mechanisms of Channels and Transporters

摘要 蛋白质运动的知识对于弥合结构和功能之间的差距和获得洞察力是必要的 蛋白质机器的分子机制。我的实验室研究离子通道和离子偶联的功能 转运蛋白，必须经历结构转换才能调节离子流动的完整的膜蛋白 (通道)或主动泵送底物(转运体)越过生物膜屏障。一组截然不同但 相关项目检查二次主动转运、混杂多药的机制 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 转运体和通道活动，以及小的局部交互如何调节更广泛的动态 膜蛋白。这些研究问题跨越时间和长度尺度，需要一系列 实验方法和一套完善的模型系统，使假设驱动的研究成为可能。 我们的主要工具之一是核磁共振波谱，它可以同时提供结构、热力学 (种群)和具有特定站点分辨率的动力学(转换率)数据。核磁共振化学位移也是 对当地环境的变化高度敏感，提供质子结合的直接读数，这是一个过程 这在其他方面很难通过实验进行监测，但却是剖析质子耦合输运的核心。超过了 在过去的10年里，我的实验室做了必要的艰苦工作，开发出了三个完全独立的 建立传输器和渠道系统模型(EMRE、NAK、Shaker-VSD)并建立实验工具范围 从核磁共振和分子生物物理学到体外和体内功能分析。我们现在已经准备好解决 探索这些特定系统的分子机制的基本研究问题，但也有 对理解蛋白质构象变化如何调控的更广泛的影响，混杂与 底物识别的特异性，质子耦合传输的复杂性，以及 蛋白质功能的变构调节。

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.