title: A Reactive, Rigid GdIIILabeling Tag for In-Cell EPR Distance Measurements in Proteins.
abstract: The cellular environment of proteins differs considerably from in vitro conditions under which most studies of protein structures are carried out. Therefore, there is a growing interest in determining dynamics and structures of proteins in the cell. A key factor for in-cell distance measurements by the double electron-electron resonance (DEER) method in proteins is the nature of the used spin label. Here we present a newly designed GdIIIspin label, a thiol-specific DOTA-derivative (DO3MA-3BrPy), which features chemical stability and kinetic inertness, high efficiency in protein labelling, a short rigid tether, as well as favorable spectroscopic properties, all are particularly suitable for in-cell distance measurements by the DEER method carried out at W-band frequencies. The high performance of DO3MA-3BrPy-GdIIIis demonstrated on doubly labelled ubiquitin D39C/E64C, both in vitro and in HeLa cells. High-quality DEER data could be obtained in HeLa cells up to 12 h after protein delivery at in-cell protein concentrations as low as 5-10 μm.
authors.affiliation: Department of Chemical Physics, Weizmann Institute of Science, Rehovot, 76100, Israel.
authors.forename: Yin
authors.initials: Y
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Elemento-organic Chemistry, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin, 300071, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Elemento-organic Chemistry, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin, 300071, China.
authors.forename: Yan-Jun
authors.initials: YJ
authors.lastname: Gong
authors.affiliation: State Key Laboratory of Elemento-organic Chemistry, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin, 300071, China.
authors.forename: Jia-Liang
authors.initials: JL
authors.lastname: Chen
authors.affiliation: Department of Chemical Physics, Weizmann Institute of Science, Rehovot, 76100, Israel.
authors.forename: Daniella
authors.initials: D
authors.lastname: Goldfarb
authors.affiliation: State Key Laboratory of Elemento-organic Chemistry, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin, 300071, China.
authors.forename: Xun-Cheng
authors.initials: XC
authors.lastname: Su
mesh_terms: D056831:Coordination Complexes / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D005682:Gadolinium / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D015394:Molecular Structure; D025801:Ubiquitin / Q000032:analysis*
chemical_list: D056831:Coordination Complexes; D025801:Ubiquitin; D005682:Gadolinium
keywords: EPR spectroscopy; protein dynamics; protein structures; spin labels; structure elucidation