CRYSTAL STRUCTURE AND THERMODYNAMICS STABILITY OF TYR TO PHE MUTANTS OF HUMAN LYSOZYME. ¹S. Fujii, ¹Y. Yamagata, ¹Y. Sumikawa, ²K. Takano, ²M. Kubota, ²K. Yutani, ¹Faculty of Pharmaceutical Sciences, Osaka University, ²Institute for Protein Research, Osaka University

In order to clarify the contribution of hydroxy group of tyrosine residues to the conformational stability of human lysozyme, six Tyr mutants (TYR->PHE; Y20F, Y38F, Y45F, Y54F, Y63F and Y124F) were constructed. The thermodynamic parameters for the denaturation were investigated using DSC and the crystal structures were solved at high resolution (resolution=1.8Å; R-factor=0.159-0.178). Small structural arrangements were observed locally around the mutation sites. The most striking change of hydration structure was found in the Y38F protein, which presents a decreased value in [[Delta]]G for the denaturation. The OH group of Y38 of wild protein doesn't adopt the hydrogen bonding with any side chain. Two heavily hold water molecules are lost by the removal of OH group of Y38. In the case of Y54F protein which presents a most large decreasing of the denaturation [[Delta]]G, the removal of OH group causes the breakdown of the hydrogen bonds to OD2 of D67 and the bridged water as expected. The hydration structure around the mutation site is maintained, but the thermal parameters, B-factors of water molecules and aromatic moiety of F54 are larger than that of the wild protein. Then we expected that the removal of tyrosyl hydroxy group in Y54F mutant doesn't cause the drastic change of water structure but cause the subtle destabilities of structural motion, large B-factor' s, of the water molecules and aromatic ring moiety. We estimate the structural characteristics such as ASA value and cavity volume, and analyze the correlations to the thermodynamic properties.