Thermodynamics of unfolding of an integral membrane protein in mixed micelles

Abstract

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 maltoside 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 degrees and 45 degrees 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.

Figure 1.

(A) Kinetic plots of the refolding and unfolding rate constants of DsbB in mixed SDS/DM micelles measured between 15° and 45°C in steps of 5°C. Data are fitted to Equation 2. (B) Temperature dependence of the refolding rate of DsbB k_f extrapolated to 0 mole fraction SDS. Data are fitted to Equation 3 (summarized in Table 1). (C) Temperature dependence of the unfolding rate of DsbB k_u extrapolated to 0 mole fraction SDS (•) and at 0.4 mole fraction SDS (○). Data are fitted to Equation 3 (summarized in Table 1). (D) Temperature dependence of the equilibrium constant K_I extrapolated to 0 mole fraction SDS (•) and at 0.4 mole fraction SDS (○). Data at 0 mole fraction are fitted to Equation 3 without the term ln(3356T), while data at 0.4 mole fraction are fitted to Equation 4.

Figure 2.

(A) Temperature dependence of unfolding of S6 in 0 molar GdmCl (•) (data from Otzen and Oliveberg 2004) and in 20 mM SDS (○) and 100 mM SDS (×). Data fitted to Equation 3 and summarized in Table 1. (Inset) k_u as a function of SDS concentration in 20 mM Tris (pH 8) (○) and 100 mM NaCl (data from Otzen 2002) and in PN buffer in the presence of 5 mM DM (•). Data are fitted to a second order polynomial. (B) Temperature dependence of refolding of S6 from SDS into DM (○) and at 0 molar GdmCl (•) (data from Otzen and Oliveberg 2004). Data fitted to Equation 3 and summarized in Table 1. (C) Variation of heat capacity of unfolding of S6 in SDS with SDS concentration in the presence of 5 mM DM. For each SDS concentration and temperature, the unfolding rate constant was interpolated from second order polynomials (see inset in A). (D) Time profile of unfolding of S6 into 20 mM SDS and 5 mM DM at 45°C. The inset highlights the 0.2-sec lag phase. The line represents the best fit of the data after 0.2 sec to a single exponential decay with drift.

Figure 3.

(A) Far UV CD scans of the thermal denaturation of DsbB between 20° and 100°C. (Inset) Change in ellipticity at 222 nm as a function of temperature. Data fitted to Equation 1. (B) ΔH_D−N of DsbB as a function of T_m. Data obtained by fitting scans from A to Equation 1 at different mole fractions of SDS and DM (•) or SDS and UM (○). (C) ΔH_D−N of S6 as a function of T_m in 20 mM NaOAc (pH 4) at different mole fractions of SDS in SDS-DM mixed micelles. (D) T_m as a function of SDS mole fraction for DsbB (○) and S6 (•). Data points are joined for clarity. (E) Kinetic m-values for DsbB as a function of temperature. (•) m_f, (○) m_u, (×) m_I.