THERMAL-STABILITY DETERMINANTS OF CHICKEN EGG-WHITE LYSOZYME CORE MUTANTS - HYDROPHOBICITY, PACKING VOLUME, AND CONSERVED BURIED WATER-MOLECULES

A series of 24 mutants was made in the buried core of chicken lysozyme at positions 40, 55, and 91. The midpoint temperature of thermal dena turation transition (T-m) values of these core constructs range from 6 0.9 to 77.3 degrees C, extending an earlier, more limited investigatio n on thermostability. The T-m values of variants containing conservati ve replacements for the wild type (WT) (Thr 40-Ile 55-Ser 91) triplet are linearly correlated with hydrophobicity (r = 0.81) and, to a lesse r degree, with combined side-chain volume (r = 0.75). The X-ray struct ures of the S91A (1.9 Angstrom) and I55L/S91T/D101S (1.7 Angstrom) mut ants are presented. The former amino acid change is found in duck and mammalian lysozymes, and the latter contains the most thermostable cor e triplet. A network of four conserved, buried water molecules is asso ciated with the core. It is postulated that these water molecules sign ificantly influence the mutational tolerance at the individual triplet positions. The pH dependence of T-m for the S91D mutant was compared with that of WT enzyme. The pK(a) of S91D is 1.2 units higher in the n ative than in the denatured state, corresponding to Delta Delta G(298) = 1.7 kcal/mol. This is a low value for charge burial and likely refl ects the moderating influence of the buried water molecules or a confo rmational change. Thermal and chemical denaturation and far UV CD spec troscopy were used to characterize the in vitro properties of I55T. Th is variant, which buries a hydroxyl group, has similar properties to t hose of the human amyloidogenic variant I56T.