title: Analysis of Nitroxide-Based Distance Measurements in Cell Extracts and in Cells by Pulsed ESR Spectroscopy.
abstract: Measurements of distances in cells by pulsed ESR spectroscopy afford tremendous opportunities to study proteins in native environments that are irreproducible in vitro. However, the in-cell environment is harsh towards the typical nitroxide radicals used in double electron-electron resonance (DEER) experiments. A systematic examination is performed on the loss of the DEER signal, including contributions from nitroxide decay and nitroxide side-chain cleavage. In addition, the possibility of extending the lifetime of the nitroxide radical by use of an oxidizing agent is investigated. Using this oxidizing agent, DEER distance measurements are performed on doubly nitroxide-labeled GB1, the immunoglobulin-binding domain of protein G, at varying incubation times in the cellular environment. It is found that, by comparison of the loss of DEER signal to the loss of the CW spectrum, cleavage of the nitroxide side chain contributes to the loss of DEER signal, which is significantly greater in cells than in cell extracts. Finally, local spin concentrations are monitored at varying incubation times to show the time required for molecular diffusion of a small globular protein within the cellular milieu.
authors.affiliation: Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, PA, 15260, USA.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Lawless
authors.affiliation: Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, PA, 15260, USA.
authors.forename: Amit
authors.initials: A
authors.lastname: Shimshi
authors.affiliation: Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, PA, 15260, USA.|Current address: Department of Chemistry, Hanover College, 484 Ball Dr, Hanover, IN, 47243, USA.
authors.forename: Timothy F
authors.initials: TF
authors.lastname: Cunningham
authors.affiliation: Department of Anesthesiology, University of Pittsburgh School of Medicine, 3501 5th Avenue, Pittsburgh, PA, 15213, USA.|Current address: Division of Basic Sciences, Kansas City University of Medicine and Biosciences, 2901 St. John's Blvd., Joplin, MO, 64804, USA.
authors.forename: Monica N
authors.initials: MN
authors.lastname: Kinde
authors.affiliation: Department of Anesthesiology, University of Pittsburgh School of Medicine, 3501 5th Avenue, Pittsburgh, PA, 15213, USA.
authors.forename: Pei
authors.initials: P
authors.lastname: Tang
authors.affiliation: Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, PA, 15260, USA.
authors.forename: Sunil
authors.identifier: http://orcid.org/0000-0001-9098-6114
authors.initials: S
authors.lastname: Saxena
mesh_terms: D000818:Animals; D002457:Cell Extracts / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D015394:Molecular Structure; D009589:Nitrogen Oxides / Q000032:analysis*; D009865:Oocytes / Q000737:chemistry*; D014982:Xenopus laevis
chemical_list: D002457:Cell Extracts; D009589:Nitrogen Oxides
keywords: ESR spectroscopy; double electron-electron resonance; in-cell measurements; proteins; radicals