title: A triarylmethyl spin label for long-range distance measurement at physiological temperatures using T1 relaxation enhancement.
abstract: Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy has become an important tool for measuring distances in proteins on the order of a few nm. For this purpose pairs of spin labels, most commonly nitroxides, are site-selectively introduced into the protein. Recent efforts to develop new spin labels are focused on tailoring the intrinsic properties of the label to either extend the upper limit of measurable distances at physiological temperature, or to provide a unique spectral lineshape so that selective pairwise distances can be measured in a protein or complex containing multiple spin label species. Triarylmethyl (TAM) radicals are the foundation for a new class of spin labels that promise to provide both capabilities. Here we report a new methanethiosulfonate derivative of a TAM radical that reacts rapidly and selectively with an engineered cysteine residue to generate a TAM containing side chain (TAM1) in high yield. With a TAM1 residue and Cu(2+) bound to an engineered Cu(2+) binding site, enhanced T1 relaxation of TAM should enable measurement of interspin distances up to 50Å at physiological temperature. To achieve favorable TAM1-labeled protein concentrations without aggregation, proteins are tethered to a solid support either site-selectively using an unnatural amino acid or via native lysine residues. The methodology is general and readily extendable to complex systems, including membrane proteins.
authors.affiliation: Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Zhongyu
authors.initials: Z
authors.lastname: Yang
authors.affiliation: Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Bridges
authors.affiliation: Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Carlos J
authors.initials: CJ
authors.lastname: López
authors.affiliation: N.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Novosibirsk 630090, Russia; Novosibirsk State University, Novosibirsk 630090, Russia.
authors.forename: Olga Yu
authors.initials: OY
authors.lastname: Rogozhnikova
authors.affiliation: N.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Novosibirsk 630090, Russia; Novosibirsk State University, Novosibirsk 630090, Russia.
authors.forename: Dmitry V
authors.initials: DV
authors.lastname: Trukhin
authors.affiliation: Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Evan K
authors.initials: EK
authors.lastname: Brooks
authors.affiliation: N.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Novosibirsk 630090, Russia; Novosibirsk State University, Novosibirsk 630090, Russia. Electronic address: torm@nioch.nsc.ru.
authors.forename: Victor
authors.initials: V
authors.lastname: Tormyshev
authors.affiliation: The Center for EPR Imaging in vivo Physiology, Department of Radiation and Cellular Oncology, University of Chicago, Chicago, IL 60637, USA. Electronic address: h-halpern@uchicago.edu.
authors.forename: Howard J
authors.initials: HJ
authors.lastname: Halpern
authors.affiliation: Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address: hubbellw@jsei.ucla.edu.
authors.forename: Wayne L
authors.initials: WL
authors.lastname: Hubbell
mesh_terms: D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D011506:Proteins / Q000737:chemistry*; D013113:Spin Labels; D013696:Temperature
chemical_list: D011506:Proteins; D013113:Spin Labels
keywords: Distance measurement at physiological temperature; Relaxation enhancement; Saturation recovery; Spin labeling; Triarylmethyl radical