Room-Temperature Distance Measurements of Immobilized Spin-Labeled Protein by DEER/PELDOR

Authors

Virginia Meyer, University of Denver
Michael A. Swanson, University of Denver
Laura J. Clouston, University of Nebraska-Lincoln
Przemysław J. Boratyński, University of Nebraska-Lincoln
Richard A. Stein, Vanderbilt UniversityFollow
Hassane S. Mchaourab, Vanderbilt University
Andrzej Rajca, University of Nebraska - LincolnFollow
Sandra S. Eaton, University of Denver
Gareth R. Eaton, University of Denver

Date of this Version

2015

Citation

Biophys J. 108(5): 1213-1219

Comments

© 2015 by the Biophysical Society.

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 (T_m) 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 T_m 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 T_m 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.