Folding of DsbB in Mixed Micelles: A Kinetic Analysis of the Stability of a Bacterial Membrane Protein

摘要: Measuring the stability of integrated membrane proteins under equilibrium conditions is hampered by nature proteins' amphiphilic environment. While intrinsic fluorescence a useful probe for structural changes in water-soluble proteins, sensitive to lipid and detergent composition. As an attempt overcome this problem, I present kinetic analysis folding protein, disulfide bond reducing protein B (DsbB), mixed micelle system consisting varying molar ratios sodium dodecyl sulfate (SDS) maltoside (DM). This incorporates both unfolding rates, making it possible determine native state process which folds. Refolding occur on second millisecond timescale involve only one relaxation phase, when monitored conventional stopped-flow. The data indicate that denaturation occurs around 0.3 mole fraction SDS, agreement with CD acrylamide quenching data. rate constants have been fit three-state scheme involving SDS-denatured state, intermediate accumulates at high fractions SDS. DsbB 4.4 kcal/mol DM, halved upon reduction two periplasmic bonds, mutagenesis. With caveat are always open alternative interpretations, time-resolved studies micelles provide approach measure over wide range concentrations SDS as well framework future characterization mechanism.