THERMODYNAMIC CHARACTERIZATION OF THE REVERSIBLE, 2-STATE UNFOLDING OF MALTOSE-BINDING PROTEIN, A LARGE 2-DOMAIN PROTEIN

The folding and stability of maltose binding protein (MBP) have been i nvestigated as a function of pH and temperature by intrinsic tryptopha n fluorescence, far- and near-UV circular dichroism, and high-sensitiv ity differential scanning calorimetric measurements. MBP is a monomeri c, two-domain protein containing 370 amino acids. The protein is stabl e in the pH range of 4-10.5 at 25 degrees C. The protein exhibits reve rsible, two-state, thermal and guanidine hydrochloride-mediated denatu ration at neutral pH. The thermostability of MBP is maximal at pH 6, w ith 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 simila r to those of other globular proteins. These data have been used to sh ow 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 de crease in stability of MBP at low temperatures was experimentally conf irmed by carrying out denaturant-mediated unfolding studies at neutral pH at 2 and 28 degrees C.