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.
Kuroki
R
R
Protein Engineering Research Institute, Osaka, Japan.
Kawakita
S
S
Nakamura
H
H
Yutani
K
K
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D004795:Enzyme Stability; D006801:Humans; D007700:Kinetics; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation
D009113:Muramidase; D002118:Calcium