Contribution of intra- and intermolecular hydrogen bonds to the conformational stability of human lysozyme

In globular proteins, there are intermolecular hydrogen bonds between prote in and water molecules, and between water molecules, which are bound with t he proteins, in addition to intramolecular hydrogen bonds. To estimate the contribution of these hydrogen bonds to the conformational stability of a p rotein, the thermodynamic parameters for denaturation and the crystal struc tures of five Thr to Val and five Thr to Ala mutant human lysozymes were de termined. The denaturation Gibbs energy (Delta G) 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 co ntribution of hydrogen bonds to the stability (Delta Delta G(HB)) Of the Th r and other mutant human lysozymes previously reported was extracted from t he observed stability changes (Delta Delta G) with correction for changes i n hydrophobicity and side chain conformational entropy between the wild-typ e and mutant structures. The estimation of the Delta Delta G(HB) values of all mutant proteins after removal of hydrogen bonds, including protein-wate r hydrogen bonds, indicates a favorable contribution of the intra- and inte rmolecular hydrogen bonds to the protein stability. The net contribution of an intramolecular hydrogen bond (Delta G(HB[pp])), an intermolecular one b etween protein and ordered water molecules (Delta G(HB[pw])), and an interm olecular one between ordered water molecules (Delta G(HB[ww])) could be est imated to be 8.5, 5.2, and 5.0 kJ/mol, respectively, for a 3 Angstrom long hydrogen bond. This result shows the different contributions to protein sta bility of intra- and intermolecular hydrogen bonds. The entropic cost due t o the introduction of a water molecule (Delta G(H2O)) could be also estimat ed to be about 8 kJ/mol.