Bimolecular quenching of tryptophan fluorescence in a membrane protein: Evolution of local solvation and environment during folding into a bilayer

[Display omitted] •Desolvation kinetics of the folding of a membrane protein, OmpA, were probed with Stern-Volmer experiments.•Time-resolved Stern-Volmer analysis utilized sphere-of-action quenching model.•Single tryptophan OmpA mutants provided site-specific changes in the kinetics of desolvation.•Evolution of tryptophan environment and desolvation occur on different timescales. Fluorescence spectroscopy, including Stern-Volmer quenching, is a valuable tool for the study of protein dynamics. Changes in protein solvation during the folding reaction of a membrane protein, Outer membrane protein A (OmpA), into lipid bilayers was probed with bimolecular fluorescence quenching with acrylamide quencher. Six single-tryptophan OmpA mutants (W7, W15, W57, W102, W129, and W143) allowed for site-specific investigations at varying locations within the transmembrane β-barrel domain. A sphere-of-action quenching model that combines both static and dynamic components gave rise to Stern-Volmer quenching constants, KD, for OmpA denatured in 8.0 M urea, aggregated in 0.5 M urea, adsorbed onto small unilamellar vesicles (SUVs), and folded in SUVs (t = 6 hrs). The average KD values were KDdenatured(6.4M-1)>KDaggregated5.9M-1>KDadsorbed(1.9M-1)>KDfolded(0.6M-1). With knowledge of the fluorescence lifetimes in the absence of quencher, the bimolecular quenching constants, kq, were derived; the evolution of kq (and therefore KD)during the folding reaction into SUVs (t = 0 hr to t = 6 hrs) revealed desolvation timescales, τdesolv of 41–46 min (W7, W15, W57, W102), 27 min (W129), and 15 min (W143). The evolution of λmax during folding revealed fast and slow components, τenvironmentfast and τenvironmentslow of 7–13 min and 25–84 min, respectively, for all mutants. For the five lipid- facing mutants (W7, W15, W57, W129, and W143), the general trend was τenvironmentfast7-13min