Analysis of Nitroxide-Based Distance Measurements in Cell Extracts and in Cells by Pulsed ESR Spectroscopy.

Autor: Lawless MJ; Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, PA, 15260, USA., Shimshi A; Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, PA, 15260, USA., Cunningham TF; 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., Kinde MN; 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., Tang P; Department of Anesthesiology, University of Pittsburgh School of Medicine, 3501 5th Avenue, Pittsburgh, PA, 15213, USA., Saxena S; Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, PA, 15260, USA.
Jazyk: angličtina
Zdroj: Chemphyschem : a European journal of chemical physics and physical chemistry [Chemphyschem] 2017 Jun 20; Vol. 18 (12), pp. 1653-1660. Date of Electronic Publication: 2017 May 02.
DOI: 10.1002/cphc.201700115
Abstrakt: 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.
(© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)
Databáze: MEDLINE
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