Molecular Mechanisms of Channels and Transporters

通道和转运蛋白的分子机制

基本信息

批准号：
10608951
负责人：
Katherine Anne Henzler-Wildman
金额：
$ 46.65万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
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

项目摘要

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.

抽象的了解蛋白质运动对于弥合结构和功能之间的差距并获得洞察力是必要的深入研究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，必须经历结构转变以调节离子流的整合膜蛋白（通道）或主动泵送底物（转运蛋白）穿过生物膜屏障。一组不同但相关项目研究了次级主动转运、混杂多种药物的机制识别、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，以及微小的局部相互作用如何调节更广泛的动态膜蛋白。这些研究问题跨越时间和长度尺度，需要一系列的实验方法和一套完善的模型系统可以实现假设驱动的研究。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）和动力学（转变速率）数据，具有特定地点的分辨率。 NMR 化学位移也对当地环境的变化高度敏感，提供质子结合的直接读数，这是一个过程否则很难通过实验监测，但对于剖析质子耦合传输至关重要。超过在过去的10年里，我的实验室做了必要的艰苦工作来开发三个完全独立的模拟转运体和通道系统（EmrE、NaK、Shaker-VSD）并建立实验工具从核磁共振和分子生物物理学到体外和体内功能测定。我们现在准备解决探究这些特定系统的分子机制的基本研究问题，但也有对于理解蛋白质构象变化如何调节、混杂与底物识别的特异性、质子耦合传输的复杂性以及分子基础蛋白质功能的变构调节。