Long Distance Measurements up to 160 angstrom in the GroEL Tetradecamer Using Q-Band DEER EPR Spectroscopy

摘要

Current distance measurements between spin-labels on multimeric protonated proteins using double electron-electron resonance (DEER) EPR spectroscopy are generally limited to the 15-60 angstrom range. Here we show how DEER experiments can be extended to dipolar evolution times of ca. 80s, permitting distances up to 170 angstrom to be accessed in multimeric proteins. The method relies on sparse spin-labeling, supplemented by deuteration of protein and solvent, to minimize the deleterious impact of multispin effects and substantially increase the apparent spin-label phase memory relaxation time, complemented by high sensitivity afforded by measurements at Q-band. We demonstrate the approach using the tetradecameric molecular machine GroEL as an example. Two engineered surface-exposed mutants, R268C and E315C, are used to measure pairwise distance distributions with mean values ranging from 20 to 100 angstrom and from 30 to 160 angstrom, respectively, both within and between the two heptameric rings of GroEL. The measured distance distributions are consistent with the known crystal structure of apo GroEL. The methodology presented here should significantly expand the use of DEER for the structural characterization of conformational changes in higher order oligomers.