Fluorescence quenching in the DsbA protein from Escherichia coli: complete picture of the excited-state energy pathway and evidence for the reshuffling dynamics of the microstates of tryptophan.
The disulfide oxidoreductase DsbA is a strong oxidant of protein thiols and is required for efficient disulfide bond formation in the bacterial periplasm. DsbA contains two tryptophans: W76 and W126. The fluorescence of W76 changes upon reduction of the disulfide bridge, as analyzed previously (Hennecke et al., Biochemistry 1997;36:6391-6400). The fluorescence of W126 is highly quenched. The only two potential side chain quenchers are Q74 and N127, and these were replaced by alanine, resulting in a threefold increase in fluorescence intensity. The fluorescence intensity increase is not due to the removal of dynamic quenchers but to an increase in the population with the longest lifetime. In this report, the possibility of a change in the conformation of W126 is investigated theoretically by using molecular mechanics and dynamic simulations and experimentally by using a reaction with N-bromosuccinimide. This reacts preferably with the most exposed microstate of tryptophan, which is responsible for the longest lifetime. The simulations and the experimental results reveal that the amino acid replacements allow W126 to increase the population of its antiperpendicular conformation. The selectivity of the N-bromosuccinimide reaction allows the visualization of the reshuffling kinetics at exhausting reagent concentration. To the authors' knowledge, this is the first time that the kinetics of Trp population reshuffling have been measured.
Sillen
A
A
Laboratory of Biomolecular Dynamics, University of Leuven, Belgium.
Hennecke
J
J
Roethlisberger
D
D
Glockshuber
R
R
Engelborghs
Y
Y
D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases / Q000737:chemistry* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*
D014364:Tryptophan; D019704:Protein Disulfide-Isomerases