Long Distance Measurements up to 160 Å in the GroEL Tetradecamer Using Q-Band DEER EPR Spectroscopy.
AUTHORS
- PMID: 27860003 [PubMed].
- PMCID: PMC5166617.
- NIHMSID: NIHMS833495
ABSTRACT
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 Å range. Here we show how DEER experiments can be extended to dipolar evolution times of ca. 80 μs, permitting distances up to 170 Å 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 Å and from 30 to 160 Å, 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.
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 Å range. Here we show how DEER experiments can be extended to dipolar evolution times of ca. 80 μs, permitting distances up to 170 Å 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 Å and from 30 to 160 Å, 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.
Tags: 2016