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Protein Science (2006), 15:890-899. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 The Protein Society
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Thermodynamics of unfolding of an integral membrane protein in mixed micelles

Pankaj Sehgal and Daniel E. Otzen

Department of Life Sciences, Aalborg University, DK-9000 Aalborg, Denmark

(RECEIVED December 12, 2005; FINAL REVISION December 12, 2005; ACCEPTED January 10, 2006)

Quantitative studies of membrane protein folding and unfolding can be difficult because of difficulties with efficient refolding as well as a pronounced propensity to aggregate. However, mixed micelles, consisting of the anionic detergent sodium dodecyl sulfate and the nonionic detergent dodecyl malto-side facilitate reversible and quantitative unfolding and refolding. The 4-transmembrane helix protein DsbB from the inner membrane of Escherichia coli unfolds in mixed micelles according to a three-state mechanism involving an unfolding intermediate I. The temperature dependence of the kinetics of this reaction between 15° and 45°C supports that unfolding from I to the denatured state D is accompanied by a significant decrease in heat capacity. For water-soluble proteins, the heat capacity increases upon unfolding, and this is generally interpreted as the increased binding of water to the protein as it unfolds, exposing more surface area. The decrease in DsbB's heat capacity upon unfolding is confirmed by independent thermal scans. The decrease in heat capacity is not an artifact of the use of mixed micelles, since the water soluble protein S6 shows conventional heat-capacity changes in detergent. We speculate that it reflects the binding of SDS to parts of DsbB that are solvent-exposed in the native DM-bound state. This implies that the periplasmic loops of DsbB are relatively unstructured. This anomalous thermodynamic behavior has not been observed for beta-barrel membrane proteins, probably because they do not bind SDS so extensively. Thus the thermodynamic behavior of membrane proteins appears to be intimately connected to their detergent-binding properties.

Keywords: membrane protein; folding kinetics; detergents; temperature; heat capacity

Abbreviations: D, denatured stateDM, dodecyl maltosideDsbB, disulfide bond forming protein BI, Intermediatekf, refolding rate constantku, unfolding rate constantKI, equilibrium constant for formation of I from NN, native stateSDS, sodium dodecyl maltosideTm, melting temperatureUM, undecyl maltoside


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M. M. Nielsen, K. K. Andersen, P. Westh, and D. E. Otzen
Unfolding of {beta}-Sheet Proteins in SDS
Biophys. J., May 15, 2007; 92(10): 3674 - 3685.
[Abstract] [Full Text] [PDF]


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