CAREER: Elucidating conformational landscapes in proteins using high-sensitivity pulse EPR spectroscopy

在这个由化学部化学测量和成像项目资助的项目中，华盛顿大学的Stefan Stoll教授正在开发用于测定柔性蛋白质纳米级结构的高灵敏度仪器和方法。蛋白质的柔韧性是其活性的先决条件，但在实验上很难量化。该项目开发的方法提供了在分子水平上精确探测生命基础化学过程的工具。更广泛的影响包括对人类健康的潜在益处，因为通过该项目开发的方法获得的分子水平知识进一步提高了我们合理设计新药和疗法的能力。本项目主要研究生物结构应用中高灵敏度脉冲电子顺磁共振（EPR）光谱的仪器开发，特别是双电子-电子共振（DEER）光谱。DEER是一种脉冲EPR技术，通过测量附着在蛋白质上的自旋标签之间的纳米尺度距离，可以定量地确定蛋白质的构象分布和灵活性。研究方法包括三个重点：(1)开发具有更高灵敏度的脉冲EPR光谱仪和相关方法，即使在非常低的浓度下也能准确测量蛋白质和复合物的构象景观。(2)量化一个小的可溶性配体结合蛋白的蛋白质构象变化，以确定方法的基准，并深入了解配体结合的构象动力学和平衡。(3)阐明心脏离子通道片段的构象景观，以便在更复杂的膜蛋白系统上测试和验证该方法。

In this project funded by the Chemical Measurement and Imaging program of the Chemistry Division, Professor Stefan Stoll of the University of Washington is developing high-sensitivity instrumentation and methodology for the determination of the nanoscale structure of flexible proteins. The flexibility of proteins is a prerequisite for their activity, but is difficult to quantify experimentally. The methods developed in this project provide tools to accurately probe fundamental chemical processes underlying life at the molecular level. The broader impacts include potential benefits to human health, as the molecular-level knowledge attainable through the methods developed in this project furthers our ability to rationally design new drugs and therapies.This project focuses on instrumentation development of high-sensitivity pulse electron paramagnetic resonance (EPR) spectroscopies for biostructural applications, specifically double electron-electron resonance (DEER) spectroscopy. DEER is a pulse EPR technique that can quantitatively determine protein conformational distributions and flexibility by measuring nanometer-scale distances between spin labels attached to proteins. The research approach consists of three thrusts: (1) Develop a pulse EPR spectrometer with improved sensitivity for DEER and related methods to be able to accurately measure conformational landscapes in proteins and complexes even at very low concentrations. (2) Quantify protein conformational change in a small soluble ligand-binding protein to benchmark the methodology and to gain insight into conformational dynamics and equilibria involved in ligand binding. (3) Elucidate the conformational landscape in fragments of a cardiac ion channel, in order to test and validate the methodology on a more complex membrane protein system.