Site-specific cross-linking of in vitro synthesized E. coli preproteins for investigating transmembrane translocation pathways.
A method is described for the preparation and usage of an E. coli cell-free translation system primed to incorporate the commercially available photoreactive analogue of phenyalanine, pBpa, into newly synthesized proteins. Incorporation is achieved by means of an amber suppressor tRNA specifically charged with pBpa. The method is exemplified for the site-specific photocross-linking of the signal sequence of a Tat (twin-arginine translocation) precursor protein to the Tat translocase in the cytoplasmic membrane of E. coli.
Panahandeh
Sascha
S
Institute of Biochemistry and Molecular Biology, ZBMZ, University of Freiburg, Freiburg, Germany.
Müller
Matthias
M
D001345:Autoradiography; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism; D021381:Protein Transport; D015398:Signal Transduction / Q000502:physiology*
D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C408585:twin-arginine translocase complex, E coli