Room-temperature distance measurements of immobilized Spin-labeled Protein by DEER/PELDOR

Virginia Meyer, Michael A. Swanson, Laura J. Clouston, Przemysław J. Boratyński, Richard A. Stein, Hassane S. McHaourab, Andrzej Rajca, Sandra S. Eaton, Gareth R. Eaton

Research output: Contribution to journalArticle

60 Scopus citations

Abstract

Nitroxide spin labels are used for double electron-electron resonance (DEER) measurements of distances between sites in biomolecules. Rotation of gem-dimethyls in commonly used nitroxides causes spin echo dephasing times (Tm) to be too short to perform DEER measurements at temperatures between ∼80 and 295 K, even in immobilized samples. A spirocyclohexyl spin label has been prepared that has longer Tm between 80 and 295 K in immobilized samples than conventional labels. Two of the spirocyclohexyl labels were attached to sites on T4 lysozyme introduced by site-directed spin labeling. Interspin distances up to ∼4 nm were measured by DEER at temperatures up to 160 K in water/glycerol glasses. In a glassy trehalose matrix the Tm for the doubly labeled T4 lysozyme was long enough to measure an interspin distance of 3.2 nm at 295 K, which could not be measured for the same protein labeled with the conventional 1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-(methyl)methanethio-sulfonate label.

Original languageEnglish (US)
Pages (from-to)1213-1219
Number of pages7
JournalBiophysical journal
Volume108
Issue number5
DOIs
StatePublished - Mar 10 2015

ASJC Scopus subject areas

  • Biophysics

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    Meyer, V., Swanson, M. A., Clouston, L. J., Boratyński, P. J., Stein, R. A., McHaourab, H. S., Rajca, A., Eaton, S. S., & Eaton, G. R. (2015). Room-temperature distance measurements of immobilized Spin-labeled Protein by DEER/PELDOR. Biophysical journal, 108(5), 1213-1219. https://doi.org/10.1016/j.bpj.2015.01.015