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 ß-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<τdesolv15-46min≤τenvironmentslow(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 ß-barrel structure.

Effects of Single Dose Rifampin on the Pharmacokinetics of Fluvastatin in Healthy Volunteers.

The objective of this study was to determine the effects of the OATP inhibitor rifampin on pharmacokinetic of Biopharmaceutics Drug Disposition Classification System Class 1 compound fluvastatin. A crossover study was carried out in 10 healthy subjects who were randomized to 2 phases to receive fluvastatin 20 mg orally alone and following a 30-minute 600 mg i.v. infusion of rifampin. The results demonstrated that i.v. rifampin increased the mean area under the plasma fluvastatin concentration-time curve (AUC0-∞ ) by 255%, mean peak plasma concentration (Cmax ) by 254%, decreased oral volume of distribution by 71%, whereas the mean elimination terminal half-life (T1/2 ), mean absorption time (MAT), and time to peak concentration (Tpeak ) of fluvastatin did not significantly change. The study demonstrated that rifampin exhibited a significant drug interaction with fluvastatin. The mechanism of the increased plasma concentrations is likely due to inhibition of OATP transporters in hepatocytes.