title: Membrane protein-ligand interactions in Escherichia coli vesicles and living cells monitored via a biosynthetically incorporated tryptophan analogue.
abstract: This paper presents a deceptively straightforward experimental approach to monitoring membrane protein-ligand interactions in vesicles and in living Escherichia coli cells. This is achieved via the biosynthetic incorporation of 7-azatryptophan, a tryptophan analogue with a red-shifted absorption spectrum, allowing collection of the emission signal of the target protein in a high tryptophan background via red-edge excitation. The approach is demonstrated for the mannitol permease of E. coli (EII(mtl)), an integral membrane protein of 637 amino acids, including four tryptophans, and single-tryptophan mutants of EII(mtl). By using a tryptophan auxotroph, a high level of 7-azatryptophan incorporation in EII(mtl) was achieved. The change in emission signal of the purified enzyme upon mannitol binding (-28%) was 4-fold larger than with EII(mtl) containing tryptophan, demonstrating the known higher sensitivity of this analogue for changes in the microenvironment [Schlesinger, R. (1968) J. Biol. Chem. 243, 3877-3883]. Changes in emission signal could also be monitored (-5%) when the enzyme was situated in vesicles, although it constituted only 10-15% of the total cytoplasmic membrane fraction. Of the five single-tryptophan mutants, the emission signal of the mutant with 7-azatryptophan at position 198 was the most sensitive for mannitol binding. Changes in emission signal not only were observed in vesicles (-18%) but also could be monitored in viable cells (-5%). The fact that only modest expression levels and no protein purification are needed makes this a useful approach for the characterization of numerous protein systems under in vitro and in vivo conditions.
authors.affiliation: Department of Biochemistry, Groningen Biomolecular Science and Biotechnology Institute, University of Groningen, The Netherlands. J.Broos@chem.rug.nl
authors.forename: J
authors.initials: J
authors.lastname: Broos
authors.forename: F
authors.initials: F
authors.lastname: ter Veld
authors.forename: G T
authors.initials: GT
authors.lastname: Robillard
mesh_terms: D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D008024:Ligands; D008353:Mannitol / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D019897:Periplasm / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
chemical_list: D008024:Ligands; D008565:Membrane Proteins; C006657:7-azatryptophan; D008353:Mannitol; D014364:Tryptophan