Abstract: In situ investigation of membrane proteins is a challenging task. Previously we demonstrated that nitroxide labels combined with pulsed ESR spectroscopy is a promising tool for this purpose. However, the nitroxide labels suffer from poor stability, high background labeling, and low sensitivity. Here we show that Finland (FTAM) and OX063 based labels enable labeling of the cobalamin transporter BtuB and BamA, the central component of the beta-barrel assembly machinery (BAM) complex, in E coli. Compared to the methanethiosulfonate spin label (MTSL), trityl labels eliminated the background signals and enabled specific in situ labeling of the proteins with high efficiency. The OX063 labels show a long phase memory time (T-M) of approximate to 5 mu s. All the trityls enabled distance measurements between BtuB and an orthogonally labeled substrate with high selectivity and sensitivity down to a few mu m concentration. Our data corroborate the BtuB and BamA conformations in the cellular environment of E. coli.

Cite: Ketter S. , Gopinath A. , Rogozhnikova O. , Trukhin D. , Tormyshev V.M. , Bagryanskaya E.G. , Joseph B.
In Situ Labeling and Distance Measurements of Membrane Proteins in E. coli Using Finland and OX063 Trityl Labels
Chemistry - A European Journal. 2021. V.27. N7. P.2299-2304. DOI: 10.1002/chem.202004606 WOS Scopus РИНЦ

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Dates:

Published online:	Jan 14, 2021
Published print:	Feb 1, 2021

Identifiers:

Web of science	WOS:000607578700001
Scopus	2-s2.0-85099378059
Elibrary	45006810
OpenAlex	W3101656168

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