Fluorescence quenching in the DsbA protein from Escherichia coli: Completepicture 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 peripl asm, DsbA contains two tryptophans: W76 and W126, The fluorescence of W76 c hanges upon reduction of the disulfide bridge, as analyzed previously (Henn ecke et al,, Biochemistry 1997;36:6391-6400), The fluorescence of W126 is h ighly quenched. The only two potential side chain quenchers are Q74 and N12 7, 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 co nfirmation of W126 is investigated theoretically by using molecular mechani cs and dynamic simulations and experimentally by using a reaction with N-br omosuccinimide, 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 author s' knowledge, this is the first time that the kinetics of Trp population re shuffling have been measured. Proteins 1999;37:253-263, (C) 1999 Wiley-Liss ,Inc.