Kuroki R, Kawakita S, Nakamura H, Yutani K
Protein Engineering Research Institute, Osaka, Japan.
Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6803-7. 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 (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.
采用量热法研究了具有钙结合位点（D86/92）的突变型人溶菌酶的稳定机制。通过差示扫描量热法发现，在85℃时，全酶-D86/92展开过程中的焓变（ΔH）比野生型和脱辅基-D86/92溶菌酶小6.8 kcal/mol。然而，在85℃时，全酶突变体的展开吉布斯自由能变化（ΔG）比脱辅基型大3.3 kcal/mol，这表明在Ca2+存在下熵显著降低（TΔS = 10.1 kcal/mol）。随后，使用滴定等温量热法分析了突变体折叠状态下的Ca2+结合过程。结合焓变估计为4.5 kcal/mol，在85℃时ΔG为-8.1 kcal/mol，这表明结合是由熵的大幅增加（TΔS = 12.6 kcal/mol）引起的。通过这些分析，确定未折叠的全酶突变体在85℃时结合Ca2+的结合ΔG为-4.8 kcal/mol（ΔH = -2.6 kcal/mol，TΔS = 2.2 kcal/mol）。因此，全酶-D86/92稳定的主要原因是Ca2+与未折叠蛋白结合导致肽链熵的降低。
