TY  - JOUR
A1  - Ketter, Sophie
A1  - Gopinath, Aathira
A1  - Rogozhnikova, Olga
A1  - Trukhin, Dmitrii
A1  - Tormyshev, Victor M.
A1  - Bagryanskaya, Elena
A1  - Joseph, Benesh
T1  - In situ labeling and distance measurements of membrane proteins in E. coli using Finland and OX063 trityl labels
T2  - Chemistry
N2  - 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.
KW  - DEER or PELDOR
KW  - FTAM and OX063
KW  - membrane protein
KW  - spin labeling
KW  - β-barrel assembly machinery (BAM) complex
Y1  - 2020
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/63864
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-638645
SN  - 1521-3765
N1  - 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.
VL  - 27
IS  - 7
SP  - 2299
EP  - 2304
PB  - Wiley-VCH
CY  - Weinheim
ER  -