ENTROPIC STABILIZATION OF A MUTANT HUMAN LYSOZYME INDUCED BY CALCIUM-BINDING

The stabilization mechanism of the mutant human lysozyme with a calcium binding site (D86/92) was investigated by using calorimetric approaches. By differential scanning calorimetry, the enthalpy change (DELTA-H) in the unfolding of holo-D86/92 was found to be 6.8 kcal/mol smaller than that of the wild-type and apo-D86/92 lysozymes at 85-degrees-C. However, the unfolding Gibbs energy change (DELTA-G) of the holo mutant was 3.3 kcal/mol greater than the apo type at 85-degrees-C, indicating a significant decrease of entropy (T-DELTA-S = 10.1 kcal/mol) in the presence of Ca2+. Subsequently, the Ca2+ binding process in the folded state of the mutant was analyzed by using titration isothermal calorimetry. The binding enthalpy change was estimated to be 4.5 kcal/mol, and DELTA-G was -8.1 kcal/mol at 85-degrees-C, which indicates that the binding was caused by a large increase in entropy (T-DELTA-S = 12.6 kcal/mol). From these analyses, the unfolded holo mutant was determined to bind Ca2+ with a binding DELTA-G of -4.8 kcal/mol (DELTA-H = -2.6 kcal/mol, T-DELTA-S = 2.2 kcal/mol) at 85-degrees-C. Therefore, the major cause of stabilization of holo-D86/92 is the decrease in entropy of the peptide chain due to Ca2+ binding to the unfolded protein.