title: Room-temperature distance measurements of immobilized spin-labeled protein by DEER/PELDOR.
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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Denver, Denver, Colorado.
authors.forename: Virginia
authors.initials: V
authors.lastname: Meyer
authors.affiliation: Department of Chemistry and Biochemistry, University of Denver, Denver, Colorado.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Swanson
authors.affiliation: Department of Chemistry, University of Nebraska, Lincoln, Nebraska.
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Clouston
authors.affiliation: Department of Chemistry, University of Nebraska, Lincoln, Nebraska.
authors.forename: Przemysław J
authors.initials: PJ
authors.lastname: Boratyński
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Stein
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee.
authors.forename: Hassane S
authors.initials: HS
authors.lastname: Mchaourab
authors.affiliation: Department of Chemistry, University of Nebraska, Lincoln, Nebraska.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Rajca
authors.affiliation: Department of Chemistry and Biochemistry, University of Denver, Denver, Colorado.
authors.forename: Sandra S
authors.initials: SS
authors.lastname: Eaton
authors.affiliation: Department of Chemistry and Biochemistry, University of Denver, Denver, Colorado. Electronic address: geaton@du.edu.
authors.forename: Gareth R
authors.initials: GR
authors.lastname: Eaton
mesh_terms: D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry; D009113:Muramidase / Q000737:chemistry; D013113:Spin Labels; D013696:Temperature; D014764:Viral Proteins / Q000737:chemistry
chemical_list: D004800:Enzymes, Immobilized; D013113:Spin Labels; D014764:Viral Proteins; D009113:Muramidase