title: Orthogonal spin labeling using click chemistry for in vitro and in vivo applications.
abstract: Site-directed spin labeling for EPR- and NMR spectroscopy has mainly been achieved exploiting the specific reactivity of cysteines. For proteins with native cysteines or for in vivo applications, an alternative coupling strategy is required. In these cases click chemistry offers major benefits by providing a fast and highly selective, biocompatible reaction between azide and alkyne groups. Here, we establish click chemistry as a tool to target unnatural amino acids in vitro and in vivo using azide- and alkyne-functionalized spin labels. The approach is compatible with a variety of labels including reduction-sensitive nitroxides. Comparing spin labeling efficiencies from the copper-free with the strongly reducing copper(I)-catalyzed azide-alkyne click reaction, we find that the faster kinetics for the catalyzed reaction outrun reduction of the labile nitroxide spin labels and allow quantitative labeling yields within short reaction times. Inter-spin distance measurements demonstrate that the novel side chain is suitable for paramagnetic NMR- or EPR-based conformational studies of macromolecular complexes.
authors.affiliation: Department of Physics, University of Osnabrück, Barbarastr. 7, 49076 Osnabrück, Germany.
authors.forename: Svetlana
authors.initials: S
authors.lastname: Kucher
authors.affiliation: Department of Biology & Chemistry, Barbarastr. 11, 49076 Osnabrück, Germany.
authors.forename: Sergei
authors.initials: S
authors.lastname: Korneev
authors.affiliation: Structural and Computational Biology Unit, Cell Biology and Biophysics Unit, EMBL, Meyerhofstr. 1, 69117 Heidelberg, Germany.
authors.forename: Swati
authors.initials: S
authors.lastname: Tyagi
authors.affiliation: Physical and Theoretical Chemistry, Technical University of Braunschweig, Hans-Sommer-Str. 10, 38106 Braunschweig, Germany.
authors.forename: Ronja
authors.initials: R
authors.lastname: Apfelbaum
authors.affiliation: Physical and Theoretical Chemistry, Technical University of Braunschweig, Hans-Sommer-Str. 10, 38106 Braunschweig, Germany.
authors.forename: Dina
authors.initials: D
authors.lastname: Grohmann
authors.affiliation: Structural and Computational Biology Unit, Cell Biology and Biophysics Unit, EMBL, Meyerhofstr. 1, 69117 Heidelberg, Germany.
authors.forename: Edward A
authors.initials: EA
authors.lastname: Lemke
authors.affiliation: Department of Physics, University of Osnabrück, Barbarastr. 7, 49076 Osnabrück, Germany.
authors.forename: Johann P
authors.initials: JP
authors.lastname: Klare
authors.affiliation: Department of Physics, University of Osnabrück, Barbarastr. 7, 49076 Osnabrück, Germany. Electronic address: hsteinho@uos.de.
authors.forename: Heinz-Jürgen
authors.initials: HJ
authors.lastname: Steinhoff
authors.affiliation: Department of Physics, University of Osnabrück, Barbarastr. 7, 49076 Osnabrück, Germany. Electronic address: daniel.klose@phys.chem.ethz.ch.
authors.forename: Daniel
authors.initials: D
authors.lastname: Klose
mesh_terms: D000480:Alkynes / Q000737:chemistry; D001386:Azides / Q000737:chemistry; D002384:Catalysis; D057930:Click Chemistry / Q000379:methods*; D003300:Copper / Q000737:chemistry; D061565:Cycloaddition Reaction; D003545:Cysteine / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008968:Molecular Conformation; D009589:Nitrogen Oxides / Q000737:chemistry; D013113:Spin Labels / Q000138:chemical synthesis*
chemical_list: D000480:Alkynes; D001386:Azides; D009589:Nitrogen Oxides; D013113:Spin Labels; D003300:Copper; D003545:Cysteine
keywords: Click chemistry; DEER; EPR spectroscopy; In vivo EPR; Orthogonal labeling; Site-directed spin labeling