Thermodynamic Characterization of the Reversible, Two-State Unfolding of Maltose Binding Protein, a Large Two-Domain Protein

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Abstract
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 °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 Tm of 64.9 °C and a ΔHm of 259.7 kcal mol-1. The linear dependence of ΔHm on Tm was used to estimate a value of ΔCp 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 ΔCp's for most globular proteins studied to date. However, the extrapolated values of ΔH and ΔS (per mole of residue) at 110 °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 ΔCp (per mol of residue) and that this temperature increases with an increase in ΔCp. 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 °C.