CAREER: Measuring Large-Amplitude Motions in Proteins Using Fourier Transform Electron Spin Resonance

产生结构波动的热运动对于调节蛋白质在酶激活等现象中的功能至关重要。在这个项目中，将发展一种傅里叶变换(FT)电子自旋共振(ESR)方法来测量蛋白质中两个位置之间的距离波动的幅度。复杂的FT实验将被用来提取两个电子自旋之间的偶极驰豫，这可能被分析以确定两个自旋之间最小和最大接近的距离。这种方法将扩展目前的光谱方法，这些方法通常只提供区域运动的时间尺度和平均距离。新方法以及目前的ESR方法将应用于一种跨膜蛋白质--甘氨酸受体，这种受体可以瞬间允许氯离子通过膜。结构转变是这种蛋白质离子渗透的显著专一性的基础。将表达几种在不同位置标记了氮氧化物对的突变蛋白。标记位置之间的距离将使用FT-ESR在三个关键构象中进行探测，以获得有关导致离子渗透的结构域运动的身份和幅度的直接信息。更广泛的影响：这项研究将创造新的方法，使蛋白质中许多重要动态事件的量化得到显著改进。该项目的教育方面包括外展、培训和教学活动。外展工作将吸引匹兹堡地区的本科生投身科学职业。将为当地四年制大学的本科生创造机会，在会议上展示他们的研究成果。将与四年制学院的教师建立伙伴关系，使他们的学生能够参与FT-ESR研究。研究生培训将包括系统的专业技能发展计划，以便化学专业的学生能够有效地过渡到跨学科工作环境中的职业。拟议的课程开发将向本科生介绍研究单分子的新技术，而研究生水平的新课程将培训学生使用现代磁共振方法。研究生和本科生将在高度跨学科的研究环境中接受培训。分子和细胞生物科学部的生物分子系统集群和化学部的实验物理化学计划共同资助了这一项目。

Thermal motions that create structural fluctuations are crucial for mediating protein function in phenomena such as enzyme activation. In this project, a Fourier Transform (FT) electron spin resonance (ESR) method will be developed to measure the amplitude of distance fluctuations between two sites in a protein. Sophisticated FT experiments will be used to extract the dipolar relaxation between two electron-spins, which may be analyzed to determine the distances of minimum and maximum approach between the spins. This method will extend current spectroscopic methods that generally provide only the timescales of domain motions and an averaged distance. The new method as well as the current ESR methods will be applied to a membrane-spanning protein, glycine receptor, which transiently permits the permeation of chloride ions across the membrane. Structural transitions underlie the remarkable specificity of ion permeation in this protein. Several mutant proteins with pairs of nitroxides tagged at various sites will be expressed. Distances between tagged sites will be probed in three key conformations using FT-ESR to obtain direct information on the identities and amplitudes of domain movements responsible for ion-permeation. Broader Impacts: The research will create new methods that allow a significant refinement in the quantification of many important dynamic events in proteins. The educational aspects of this project encompass outreach, training, and teaching activities. Outreach efforts will attract undergraduates in the Pittsburgh area to careers in science. Opportunities will be created for undergraduates at local 4-year colleges to present their research at conferences. Partnerships with faculty at 4-year colleges will be developed to enable their students' participation in FT-ESR research. Graduate training will include a systematic professional skills development program, so that Chemistry students can transition effectively into careers in an interdisciplinary job environment. Proposed development in coursework will introduce undergraduates to new techniques for studying single molecules, and new curriculum at the graduate level will train students in modern magnetic resonance methods. Graduate and undergraduate students will be trained in a highly interdisciplinary research environment. The Biomolecular Systems Cluster of the Division of Molecular and Cellular Biosciences and the Experimental Physical Chemistry Program of the Chemistry Division fund this project jointly.