Folding and stability of the bacterial membrane protein DsbB

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