Takano K, Yamagata Y, Funahashi J, Hioki Y, Kuramitsu S, Yutani K
Institute for Protein Research, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan, Graduate School of Pharmaceutical Sciences, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan.
Biochemistry. 1999 Sep 28;38(39):12698-708. doi: 10.1021/bi9910169.
In globular proteins, there are intermolecular hydrogen bonds between protein and water molecules, and between water molecules, which are bound with the proteins, in addition to intramolecular hydrogen bonds. To estimate the contribution of these hydrogen bonds to the conformational stability of a protein, the thermodynamic parameters for denaturation and the crystal structures of five Thr to Val and five Thr to Ala mutant human lysozymes were determined. The denaturation Gibbs energy (DeltaG) of Thr to Val and Thr to Ala mutant proteins was changed from 4.0 to -5.6 kJ/mol and from 1.6 to -6.3 kJ/mol, respectively, compared with that of the wild-type protein. The contribution of hydrogen bonds to the stability (DeltaDeltaG(HB)) of the Thr and other mutant human lysozymes previously reported was extracted from the observed stability changes (DeltaDeltaG) with correction for changes in hydrophobicity and side chain conformational entropy between the wild-type and mutant structures. The estimation of the DeltaDeltaG(HB) values of all mutant proteins after removal of hydrogen bonds, including protein-water hydrogen bonds, indicates a favorable contribution of the intra- and intermolecular hydrogen bonds to the protein stability. The net contribution of an intramolecular hydrogen bond (DeltaG(HB[pp])), an intermolecular one between protein and ordered water molecules (DeltaG(HB[pw])), and an intermolecular one between ordered water molecules (DeltaG(HB[ww])) could be estimated to be 8. 5, 5.2, and 5.0 kJ/mol, respectively, for a 3 A long hydrogen bond. This result shows the different contributions to protein stability of intra- and intermolecular hydrogen bonds. The entropic cost due to the introduction of a water molecule (DeltaG(H)()2(O)) could be also estimated to be about 8 kJ/mol.
在球状蛋白质中，除了分子内氢键外，蛋白质与水分子之间以及与蛋白质结合的水分子之间还存在分子间氢键。为了评估这些氢键对蛋白质构象稳定性的贡献，测定了5个苏氨酸突变为缬氨酸和5个苏氨酸突变为丙氨酸的人溶菌酶突变体的变性热力学参数和晶体结构。与野生型蛋白质相比，苏氨酸突变为缬氨酸和苏氨酸突变为丙氨酸的突变体蛋白质的变性吉布斯自由能（ΔG）分别从4.0 kJ/mol变为 -5.6 kJ/mol和从1.6 kJ/mol变为 -6.3 kJ/mol。从观察到的稳定性变化（ΔΔG）中提取氢键对苏氨酸及先前报道的其他人溶菌酶突变体稳定性（ΔΔG(HB)）的贡献，并校正野生型和突变体结构之间疏水性和侧链构象熵的变化。去除包括蛋白质 - 水氢键在内的所有氢键后，对所有突变体蛋白质的ΔΔG(HB)值进行估算，结果表明分子内和分子间氢键对蛋白质稳定性有有利贡献。对于一个3 Å长的氢键，分子内氢键（ΔG(HB[pp])）、蛋白质与有序水分子之间的分子间氢键（ΔG(HB[pw])）以及有序水分子之间的分子间氢键（ΔG(HB[ww])）的净贡献分别估计为8.5、5.2和5.0 kJ/mol。该结果显示了分子内和分子间氢键对蛋白质稳定性的不同贡献。引入一个水分子所导致的熵成本（ΔG(H₂O)）也可估计约为8 kJ/mol。
