Paramagetic metal ion based ESR distance methods for measuring ion-channel conformations in cells and in vitro

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Proteins are large biopolymers made up of amino acids and participate in most processes within cells. Their structure and the dynamics or flexibility of that structure is essential to protein function. This project will develop Electron Spin Resonance (ESR) methodology to measure precise distance constraints that are reporters of protein backbone structure and flexibility. In this project, a Copper ion based spin probe will be developed for the ESR measurements. The copper spin probe can be assembled without modification of the protein, and is much smaller that conventional probes and thus will enable more precise distance measurements. This project will measure changes in protein structure directly in cells under conditions where functional measurements can be performed. While training a number of graduate and undergraduate students, this project will expand access to pulsed ESR instrumentation to undergraduates and graduate students at regional colleges in Pennsylvania including those serving predominantly African American populations. This project will also provide hands on science experience, career awareness and academic advising to assist pre-college historically under-represented students in STEM fields.This project will develop strategies to exploit native protein residues to site-specifically and selectively attach Copper(II) for pulsed ESR distance measurements both in-vitro and in cells; and probe initial steps in the gating mechanism of ELIC, a prokaryotic, pentameric, ligand-gated ion channel from Erwinia chrysanthemi in lipid vesicles and in cells. A Copper(II)-based spin probe that can be assembled in situ from natural amino acid residues and a metal salt, without the need for post-expression synthetic modification, will be developed and used. This probe will be used to determine distance constraints that are more narrowly defined and overcome the inherent limitation of the current technology which relies on a spin label with a highly flexible side-chain. Conformational changes in the extracellular domain of ELIC that lead to the opening of the pore upon ligand binding will be determined and such changes will then be measured directly in cells under functional conditions. This project is supported by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Directorate for Biological Sciences.