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

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 beta-barrel domain. A sphere-of-action quenching model that combines both static and dynamic components gave rise to Stern-Volmer quenching constants, K-D, 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 K-D values were K-D(denatured) (6.4 M-1) > K-D(aggregated) (5.9 M-1) > K-D(adsorbed) (1.9 M-1) > K-D(folded) (0.6 M-1). With knowledge of the fluorescence lifetimes in the absence of quencher, the bimolecular quenching constants, kq, were derived; the evolution of k(q) (and therefore K-D)during the folding reaction into SUVs (t = 0 hr to t = 6 hrs) revealed desolvation timescales, tau(desolv) of 41-46 min (W7, W15, W57, W102), 27 min (W129), and 15 min (W143). The evolution of lambda(max) during folding revealed fast and slow components, tau(fast)(environment) and tau(slow)(environment) 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 sfast environmento7 13 minTHORN < tau(desolv) (15 - 46 min <= tau(slow)(environment) (25 - 84 min). These results suggest that there is an initial fast step in which there is a large change in polarity to a hydrophobic environment, followed by a slower desolvation process during evolution within the hydrophobic environment. These results complement previous mechanisms of concerted folding and provide insights into site-specific changes in solvation during formation of native b-barrel structure. (C) 2021 Elsevier B.V. All rights reserved.