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Analysis of Nitroxide‐Based Distance Measurements in Cell Extracts and in Cells by Pulsed ESR Spectroscopy
Author(s) -
Lawless Matthew J.,
Shimshi Amit,
Cunningham Timothy F.,
Kinde Monica N.,
Tang Pei,
Saxena Sunil
Publication year - 2017
Publication title -
chemphyschem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.016
H-Index - 140
eISSN - 1439-7641
pISSN - 1439-4235
DOI - 10.1002/cphc.201700115
Subject(s) - nitroxide mediated radical polymerization , spectroscopy , analytical chemistry (journal) , materials science , nuclear magnetic resonance , chemistry , photochemistry , chromatography , physics , polymer , copolymer , radical polymerization , quantum mechanics
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.