Contribution of amino acid substitutions at two different interior positions to the conformational stability of human lysozyme

To elucidate correlative relationships between structural changeand thermodynamic stability in proteins, a series of mutanthuman lysozymes modified at two buried positions (Ile56 andIle59) were examined. Their thermodynamic parameters of denaturationand crystal structures were studied by calorimetry and X-raycrystallography. The mutants at positions 56 and 59 exhibiteddifferent responses to a series of amino acid substitutions.The changes in stability due to substitutions showed a linearcorrelation with changes in hydrophobicity of substituted residues,having different slopes at each mutation site. However, thestability of each mutant was found to be represented by a uniqueequation involving physical properties calculated from mutantstructures. By fitting present and previous stability data formutant human lysozymes substituted at various positions to theequation, the magnitudes of the hydrophobicity of a carbon atomand the hydrophobicity of nitrogen and neutral oxygen atomswere found to be 0.178 and –0.013 kJ/mol.Ų, respectively.It was also found that the contribution of a hydrogen bond witha length of 3.0 Å to protein stability was 5.1 kJ/moland the entropy loss of newly introduction of a water moleculeswas 7.8 kJ/mol.