title: In vivo synthesized proteins with monoexponential fluorescence decay kinetics.
abstract: Tryptophan, when in a protein, typically shows multiexponential fluorescence decay kinetics. Complex kinetics prevents a straightforward interpretation of time-resolved fluorescence protein data, particularly in anisotropy studies or if the effect of a dynamic quencher or a resonance energy transfer (RET) acceptor is investigated. Here, time-resolved fluorescence data are presented of an isosteric tryptophan analogue, 5-fluorotryptophan, which when biosynthetically incorporated in proteins shows monoexponential decay kinetics. Data are presented indicating that the presence of a fluoro atom at the 5-position suppresses the electron transfer rate from the excited indole moiety to the peptide bond. This process has been related to the multiexponential fluorescence decay of tryptophan in proteins. The monoexponential decay of 5-fluorotryptophan makes it possible to measure simultaneously multiple distances between 5-fluorotryptophan and a RET acceptor. We demonstrate that for an oligomeric protein, consisting of two single-tryptophan-containing subunits, the individual distances between 5-fluorotryptophan and the single substrate binding site can be resolved using a substrate harboring a RET acceptor.
authors.affiliation: Department of Biochemistry, Groningen Biomolecular Science and Biotechnology Institute (GBB), and Ultrafast Laser & Spectroscopy Laboratory, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands. j.broos@chem.rug.nl
authors.forename: Jaap
authors.initials: J
authors.lastname: Broos
authors.forename: Francesco
authors.initials: F
authors.lastname: Maddalena
authors.forename: Ben H
authors.initials: BH
authors.lastname: Hesp
mesh_terms: D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D005453:Fluorescence; D007700:Kinetics; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000138:chemical synthesis / Q000737:chemistry*; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry*
chemical_list: D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D011506:Proteins; C026927:5-fluorotryptophan; D014364:Tryptophan; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli