In situ labeling and distance measurements of membrane proteins in E. coli using Finland and OX063 trityl labels

  • 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.
Author:Sophie Ketter, Aathira Gopinath, Olga Rogozhnikova, Dmitrii Trukhin, Victor M. Tormyshev, Elena Bagryanskaya, Benesh Joseph
Parent Title (English):Chemistry
Place of publication:Weinheim
Document Type:Article
Date of Publication (online):2020/11/16
Date of first Publication:2020/11/16
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2022/03/15
Tag:DEER or PELDOR; FTAM and OX063; membrane protein; spin labeling; β-barrel assembly machinery (BAM) complex
Page Number:6
First Page:2299
Last Page:2304
This work was financially supported through the Emmy Noether program (JO 1428/1-1, B.J.), the Science Funding from Johanna Quandt Young Academy at Goethe (B.J.), and SFB807 (B.J.) from the Deutsche Forschungsgemeinschaft. E.B., V.T., O.R., and D.T., thank the financial support by the Ministry of Science and High Education of the Russian Federation (state contract No.14.W03.31.0034). Open access funding enabled and organized by Projekt DEAL.
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 53 Physik / 530 Physik
Licence (German):License LogoCreative Commons - Namensnennung 4.0