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

University Of Pittsburgh, Pittsburgh PA

Investigators

Abstract

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.