Hyperthermophile Protein Folding Thermodynamics: Differential Scanning Calorimetry and Chemical Denaturation of Sac7d

Abstract

Recombinant Sac7d protein from the thermoacidophile Sulfolobus acidocaldariusis shown to be stable towards acid, thermal and chemical denaturation. The protein maintains a compact native fold between pH 0 and 10 in 0.3 M KCl and 25 °C as indicated by near and far UV circular dichroism spectra. Thermal unfolding followed by differential scanning calorimetry (DSC) occurs as a reversible, two-state transition from pH 0 to 10, with a maximal T mof 90.7 °C between pH 5 and 9. At pH 0 the protein unfolds with a T mof 63.3 °C. Plots of the enthalpy of unfolding as a function of T mare linear and yield an anomalously low Δ C pof 497 (±20) cal deg −1mol −1using the Kirchhoff relation. Guanidine hydrochloride and urea-induced chemical denaturation of Sac7d occur reversibly and can be followed by circular dichroism. Global non-linear regression of the chemical denaturation data constrained by DSC determined values for Δ H mand T myields a Δ C pof unfolding of 858 (±21) cal deg −1mol −1. The higher Δ C pis in good agreement with that predicted from the buried polar and apolar surface areas using the NMR solution structure. It is similar to values reported for mesophile proteins of comparable size, indicating that the packing and change in solvent-accessible surface area on unfolding are not unusual. Similarly, guanidine hydrochloride and urea m-values are in good agreement with those expected for a protein of 66 residues. Possible explanations for the difference in Δ C pdetermined by application of the Kirchhoff relation to DSC data and that determined by the global fit are discussed. Protein stability curves defined by either Δ C pvalues are similar to those observed for small mesophile proteins. Although the protein is thermally stable, it is marginally stable thermodynamically with a free energy of unfolding of 1.6 (±0.1) kcal mol −1at the growth temperature of 80 °C. The large number of potential ion pairs on the surface of this hyperthermophile protein do not result in an inordinate increase in stability. Post-translational modification, possibly lysine monomethyla tion, appears to be the single most important stabilizing factor that distinguishes the native hyperthermophile protein from small mesophile proteins. Additional stabilization in vivois expected from compatible osmolytes (polyamines) and DNA-binding.