LINKAGE OF PROTONATION AND ANION-BINDING TO THE FOLDING OF SAC7D

The temperature, pH, and salt dependence of the folding of recombinant Sac7d from the hyperthermophile Sulfolobus acidocaldarius is mapped u sing multi-dimensional differential scanning calorimetry (DSC) and fol ding progress surfaces followed by circular dichroism. Linkage relatio ns are derived to explain the observed dependencies, and it is shown t hat the data can be explained by the linkage of at least two protonati on reactions and two anion binding sites to a two-state unfolding proc ess. Circular dichroism spectra indicate that a native-like fold is st abilized at acid pH by anion binding. An apparent binding isotherm sur face (folding progress versus pH and salt) is used to obtain intrinsic chloride binding constants as a function of pH for both sites. A sadd le is predicted in the folding progress surface (progress versus tempe rature and pH) at low salt with a minimum near pH 2 and 20 degrees C w ith approximately 25% of the protein folded. The position of the saddl e is sensitive to the intrinsic Delta C-p(o) of unfolding and provides a third measure of Delta C-p(o) independent of that obtained by a Kir chhoff plot of DSC data and chemical denaturation. The observed enthal py of unfolding approaches zero near the saddle making the unfolding l argely invisible to DSC under these conditions. The Linkage analysis d emonstrates that the Delta C-p(o) for unfolding obtained from a Kirchh off plot of DSC data should be distinguished from the intrinsic Delta C-p(o), of unfolding. It is shown that the discrepancy between the fre e energy of unfolding for Sac7d obtained by DSC and that obtained by c hemical denaturation may be explained by the linkage of protonation an d anion binding to protein folding. The linkage analysis demonstrates the limitations of using the Delta H-cal/Delta H-vh ratio as an indica tion of two-state unfolding. (C) 1998 Academic Press Limited.