In Situ Labeling and Distance Measurements of Membrane Proteins in E. coli Using Finland and OX063 Trityl Labels.
Chemistry
; 27(7): 2299-2304, 2021 Feb 01.
Article
en En
| MEDLINE
| ID: mdl-33197077
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 ß-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 ≈5â
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.
Palabras clave
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Compuestos de Sulfhidrilo
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Proteínas de Escherichia coli
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Escherichia coli
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Proteínas de la Membrana
País/Región como asunto:
Europa
Idioma:
En
Revista:
Chemistry
Asunto de la revista:
QUIMICA
Año:
2021
Tipo del documento:
Article
País de afiliación:
Alemania