title: Probing protein conformation in cells by EPR distance measurements using Gd3+ spin labeling.
abstract: Protein structure investigations are usually carried out in vitro under conditions far from their native environment in the cell. Differences between in-cell and in vitro structures of proteins can be generated by crowding effects, local pH changes, specific and nonspecific protein and ligand binding events, and chemical modifications. Double electron-electron resonance (DEER), in conjunction with site-directed spin-labeling, has emerged in the past decade as a powerful technique for exploring protein conformations in frozen solutions. The major challenges facing the application of this methodology to in-cell measurements are the instabilities of the standard nitroxide spin labels in the cell environment and the limited sensitivity at conventional X-band frequencies. We present a new approach for in-cell DEER distance measurement in human cells, based on the use of: (i) reduction resistant Gd(3+) chelates as spin labels, (ii) high frequency (94.9 GHz) for sensitivity enhancement, and (iii) hypo-osmotic shock for efficient delivery of the labeled protein into the cell. The proof of concept is demonstrated on doubly labeled ubiquitin in HeLa cells.
authors.affiliation: Department of Chemical Physics and ‡Department of Materials and Interfaces, Weizmann Institute of Science , Rehovot, Israel 7610001.
authors.forename: Andrea
authors.initials: A
authors.lastname: Martorana
authors.forename: Giuliano
authors.initials: G
authors.lastname: Bellapadrona
authors.forename: Akiva
authors.initials: A
authors.lastname: Feintuch
authors.forename: Enza
authors.initials: E
authors.lastname: Di Gregorio
authors.forename: Silvio
authors.initials: S
authors.lastname: Aime
authors.forename: Daniella
authors.initials: D
authors.lastname: Goldfarb
mesh_terms: D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D005682:Gadolinium / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D011487:Protein Conformation; D013113:Spin Labels; D025801:Ubiquitin / Q000737:chemistry*
chemical_list: D013113:Spin Labels; D025801:Ubiquitin; D005682:Gadolinium