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

Abstract

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, 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(6.4M^-1)>K_D^aggregated5.9M^-1>K_D^adsorbed(1.9M^-1)>K_D^folded(0.6M^-1). With knowledge of the fluorescence lifetimes in the absence of quencher, the bimolecular quenching constants, k_q, 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, τ_desolv of 41-46 min (W7, W15, W57, W102), 27 min (W129), and 15 min (W143). The evolution of λ_max during folding revealed fast and slow components, τ_environment^fast and τ_environment^slow 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 τ_environment^fast7-13min<τ_desolv15-46min≤τ_environment^slow(25-84min). 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 β-barrel structure.

Keywords: Desolvation; Kinetics; Membrane protein folding; Stern-Volmer; Vesicles.