Thermodynamic characterization of the reversible, two-state unfolding of maltose binding protein, a large two-domain protein

The folding and stability of maltose binding protein (MBP) have been investigated as a function of pH and temperature by intrinsic tryptophan fluorescence, far- and near-UV circular dichroism, and high-sensitivity differential scanning calorimetric measurements. MBP is a monomeric, two-domain protein containing 370 amino acids. The protein is stable in the pH range of 4-10.5 at 25 degrees C. The protein exhibits reversible, two-state, thermal and guanidine hydrochloride-mediated denaturation at neutral pH. The thermostability of MBP is maximal at pH 6, with a T-m of 64.9 degrees C and a Delta H-m of 259.7 kcal mol(-1) The linear dependence of Delta H-m on T-m was used to estimate a value of Delta C-p of 7.9 kcal mol(-1) K-1 or 21.3 cal (mol of residue)(-1) K-1. These values are higher than the corresponding Delta C-p's for most globular proteins studied to date. However, the extrapolated values of Delta H and Delta S (per mole of residue) at 110 degrees C are similar to those of other globular proteins. These data have been used to show that the temperature at which a protein undergoes cold denaturation depends primarily on the Delta C-p (per mol of residue) and that this temperature increases with an increase in Delta C-p. The predicted decrease in stability of MBP at low temperatures was experimentally confirmed by carrying out denaturant-mediated unfolding studies at neutral pH at 2 and 28 degrees C.