In Situ Labeling and Distance Measurements of Membrane Proteins in E. coli Using Finland and OX063 Trityl Labels
Observing membrane proteins in the cellular environment is a difficult task and PELDOR/DEER spectroscopy is a versatile tool for this purpose. The nitroxide spin labels suffer from poor stability and sensitivity in cellular environments. Trityls are emerging as promising tags for in situ PELDOR/DEER. Here we show that FTAM and OX063 labels provide superior sensitivity and selectivity for distance measurements of membrane proteins in E. coli.
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.
Wiley: Chemistry – A European Journal: Table of Contents
Authors: Sophie Ketter, Aathira Gopinath, Olga Rogozhnikova, Dmitrii Trukhin, Victor M. Tormyshev, Elena G. Bagryanskaya, Benesh Joseph