Entropic Stabilization of a Mutant Human Lysozyme Induced by Calcium Binding

• Published in: Proceedings of the National Academy of Sciences - PNAS 1992-08, Vol.89 (15), p.6803-6807
• Main Authors: Kuroki, Ryota, Kawakita, Shigetsugu, Nakamura, Haruki, Yutani, Katsuhide
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; binding; Binding Sites; Biochemistry; Biological and medical sciences; Calcium; Calcium - metabolism; Calorimeters; Calorimetry, Differential Scanning; differential scanning calorimetry; Enthalpy; Entropy; Enzyme Stability; Enzymes; Fundamental and applied biological sciences. Psychology; Heat measurement; Humans; Kinetics; Ligands; lysozyme; Molecular biophysics; Molecules; Muramidase - chemistry; Muramidase - genetics; Muramidase - metabolism; Mutagenesis, Site-Directed; mutants; Mutation; Physico-chemical properties of biomolecules; Protein Conformation; Protein Denaturation; stabilization; Thermodynamics; Titration
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.89.15.6803

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 (Δ 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⚬C. However, the unfolding Gibbs energy change (Δ G) of the holo mutant was 3.3 kcal/mol greater than the apo type at 85⚬C, indicating a significant decrease of entropy (TΔ 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 Δ G was -8.1 kcal/mol at 85⚬C, which indicates that the binding was caused by a large increase in entropy (TΔ S = 12.6 kcal/mol). From these analyses, the unfolded holo mutant was determined to bind Ca2+ with a binding Δ G of -4.8 kcal/mol (Δ H = -2.6 kcal/mol, TΔ S = 2.2 kcal/mol) at 85⚬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.