HYPERTHERMOPHILE PROTEIN-FOLDING THERMODYNAMICS - DIFFERENTIAL SCANNING CALORIMETRY AND CHEMICAL DENATURATION OF SAC7D

Recombinant Sac7d protein from the thermoacidophile Sulfolobus acidoca ldarius is shown to be stable towards acid, thermal and chemical denat uration. The protein maintains a compact native fold between pH 0 and 10 in 0.3 M KCl and 25 degrees C as indicated by near and far UV circu lar dichroism spectra. Thermal unfolding followed by differential scan ning calorimetry (DSC) occurs as a reversible, two-state transition fr om pH 0 to 10, with a maximal T-m of 90.7 degrees C between PPI 5 and 9. At pH 0 the protein unfolds with a T-m of 63.3 degrees C. Plots of the enthalpy of unfolding as a function of T-m are linear and yield an anomalously low Delta C-p of 497 (+/- 20) cal deg(-1) mol(-1) using t he Kirchhoff relation. Guanidine hydrochloride and urea-induced chemic al denaturation of Sac7d occur reversibly and can be followed by circu lar dichroism. Global non-linear regression of the chemical denaturati on data constrained by DSC determined values for Delta H-m and T-m yie lds a Delta C-p of unfolding of 858 (+/-21) cal deg(-1) mol(-1). The h igher Delta C-p is in good agreement with that predicted from the buri ed polar and apolar surface areas using the NMR solution structure. It is similar to values reported for mesophile proteins of comparable si ze, indicating that the packing and change in solvent-accessible surfa ce area on unfolding are not unusual. Similarly, guanidine hydrochlori de and urea rn-values are in good agreement with these expected for a protein of 66 residues. Possible explanations for the difference in De lta C-p determined by application of the Kirchhoff relation to DSC dat a and that determined by the global fit are discussed. Protein stabili ty curves defined by either Delta C-p values are similar to those obse rved for smalt mesophile proteins. Although the protein is thermally s table, it is marginally stable thermodynamically with a free energy of unfolding of 1.6 (+/-0.1) kcal mol(-1) at the growth temperature of 8 0 degrees C. The large number of potential ion Fairs on the surface of this hyperthermophile protein do not result in an inordinate increase in stability. Post-translational modification, possibly lysine monome thylation, appears to be the single most important stabilizing factor that distinguishes the native hyperthermophile protein from small meso phile proteins. Additional stabilization in vivo is expected from comp atible osmolytes (polyamines) and DNA-binding. (C) 1996 Academic Press Limited