Folding of DsbB in mixed micelles: a kinetic analysis of the stability of a bacterial membrane protein.
Measuring the stability of integrated membrane proteins under equilibrium conditions is hampered by the nature of the proteins' amphiphilic environment. While intrinsic fluorescence is a useful probe for structural changes in water-soluble proteins, the fluorescence of membrane proteins is sensitive to changes in lipid and detergent composition. As an attempt to overcome this problem, I present a kinetic analysis of the folding of a membrane protein, disulfide bond reducing protein B (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 on the second to millisecond timescale and involve only one relaxation phase, when monitored by conventional stopped-flow. The kinetic data indicate that denaturation occurs around 0.3 mole fraction of SDS, in agreement with CD analysis and acrylamide quenching data. The rate constants have been fit to a three-state folding scheme involving the SDS-denatured state, the native state and an unfolding intermediate that accumulates only under unfolding conditions at high mole fractions of SDS. 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 concentrations of SDS and DM, as well as a framework for the future characterization of the DsbB folding mechanism.
Otzen
Daniel E
DE
Department of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark. daniel.otzen@bio.auc.dk
D001426:Bacterial Proteins / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry*; D002942:Circular Dichroism; D004220:Disulfides; D004926:Escherichia coli / Q000378:metabolism; D005960:Glucosides / Q000494:pharmacology; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry*; D008823:Micelles; D008856:Microscopy, Fluorescence; D017510:Protein Folding; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology
D001426:Bacterial Proteins; D004220:Disulfides; C079503:DsbB protein, Bacteria; D005960:Glucosides; D008565:Membrane Proteins; D008823:Micelles; D012967:Sodium Dodecyl Sulfate; C040358:dodecyl maltoside