CONTRIBUTION OF HYDROPHOBIC RESIDUES TO THE STABILITY OF HUMAN LYSOZYME - CALORIMETRIC STUDIES AND X-RAY STRUCTURAL-ANALYSIS OF THE 5-ISOLEUCINE TO VALINE MUTANTS

In order to understand the contribution of hydrophobic residues to the conformational stability of human lysozyme, five Ile mutants (Ile --> Val) in the interior of the protein were constructed. The thermodynam ic parameters characterizing the denaturation of these mutant proteins were determined by scanning calorimetry, and the three-dimensional st ructure of each mutant protein was solved at high resolution by X-ray crystallography. The thermodynamic analyses at 64.9 degrees C and at p H 2.7 revealed the following. (1) The stabilities of all the mutant pr oteins were decreased as compared with that of the wild-type protein. (2) The changes in the calorimetric enthalpies were larger than those in the Gibbs energies, and were compensated by entropy changes. (3) Th e destabilization mechanism of the mutant proteins differs, depending on the location of the mutation sites. X-ray analyses showed that the overall structures of all the mutant human lysozymes examined were ide ntical to that of the wild-type protein, and only small structural rea rrangements were observed locally around some of the mutation sites. T he most striking change among the mutant proteins was found in the mut ant protein, 159V, which contains a new water molecule in the cavity c reated by the mutation. The thermodynamic stabilities of the mutant pr oteins are discussed in light of the high-resolution X-ray structures of the wild-type and five mutant human lysozymes examined. (C) 1995 Ac ademic Press Limited