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Chemphyschem. 2016 Oct 5;17(19):2987-2991. doi: 10.1002/cphc.201600726. Epub 2016 Aug 2.

Quantitative Resolution of Monomer-Dimer Populations by Inversion Modulated DEER EPR Spectroscopy.

Author information

1
Laboratory of Chemical Physics, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892-0520, USA.
2
Laboratory of Chemical Physics, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892-0520, USA. mariusc@mail.nih.gov.

Abstract

A simple method, based on inversion modulated double electron-electron resonance electron paramagnetic resonance (DEER EPR) spectroscopy, is presented for determining populations of monomer and dimer in proteins (as well as any other biological macromolecules). The method is based on analysis of modulation depth versus electron double resonance (ELDOR) pulse flip angle. High accuracy is achieved by complete deuteration, extensive sampling of a large number of ELDOR pulse flip angle values, and combined analysis of differently labeled spin samples. We demonstrate the method using two different proteins: an obligate monomer exemplified by the small immunoglobulin binding B domain of protein A, and the p66 subunit of HIV-1 reverse transcriptase which exists as an equilibrium mixture of monomer and dimer species whose relative populations are affected by glycerol content. This information is crucial for quantitative analysis of distance distributions involving proteins that may exist as mixtures of monomer, dimer and high order multimers under the conditions of the DEER EPR experiment.

KEYWORDS:

HIV-1 reverse transcriptase; double electron-electron resonance (deer); electron paramagnetic resonance (epr) spectroscopy; population distributions; proteins

PMID:
27442455
PMCID:
PMC5590656
DOI:
10.1002/cphc.201600726
[Indexed for MEDLINE]
Free PMC Article

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