In Situ Labeling and Distance Measurements of Membrane Proteins in E. coli Using Finland and OX063 Trityl Labels.

Chemistry : a European Journal
Sophie KetterBenesh Joseph

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 β-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 (TM ) of ≈5 μs. All the trityls enabled distance measurements between BtuB and an orthogonally labeled substrate with high selectivity and sensitivity down to a few μm concentration. Our data corroborate the BtuB and BamA conformations in the cellular environment of E. coli.

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Citations

Jan 7, 2021·Chemistry : a European Journal·Nico FleckOlav Schiemann
Apr 4, 2021·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Ken-Ichiro MatsumotoMurali C Krishna
Apr 9, 2021·The Journal of Physical Chemistry Letters·Svetlana KucherEnrica Bordignon
May 14, 2021·The Journal of Physical Chemistry. B·Zikri HasanbasriSunil Saxena
Nov 19, 2021·Chemical Communications : Chem Comm·Anandi KugeleMalte Drescher

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Methods Mentioned

BETA
ESR
NMR

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