No abstract

A series of 24 mutants was made in the buried core of chicken lysozyme at positions 40, 5 5 , and 91. The midpoint temperature of thermal denaturation transition (T,) values of these core constructs range from 60.9 to 77.3 "C, extending an earlier, more limited investigation on thermostability. The T,,, values of variants containing conservative replacements for the wild type (WT) (Thr 40-Ile 55-Ser 91) triplet are linearly correlated with hydrophobicity (r = 0.81) and, to a lesser degree, with combined side-chain volume (r = 0.75). The X-ray structures of the S91A (1.9A) and 155L/S91T/DlOlS (1.7 A) mutants are presented. The former amino acid change is found in duck and mammalian lysozymes, and the latter contains the most thermostable core triplet. A network of four conserved, buried water molecules is associated with the core. It is postulated that these water molecules significantly influence the mutational tolerance at the individual triplet positions. The pH dependence of T, for the S91D mutant was compared with that of WT enzyme. The pK, of S91D is 1.2 units higher in the native than in the denatured state, corresponding to AAG298 = 1.7 kcal/mol. This is a low value for charge burial and likely reflects the moderating influence of the buried water molecules or a conformational change. Thermal and chemical denaturation and far UV CD spectroscopy were used to characterize the in vitro properties of I55T. This variant, which buries a hydroxyl group, has similar properties to those of the human amyloidogenic variant I56T.

A hyperstable (hs) variant of chicken egg-white lysozyme with enhanced thermal (AT, = + 10.5 "C) and chemical (AC,n for guanidine hydrochloride denaturation = + 1.3 M) stabilities relative to wild-type (WT) was constructed by combining several individual stabilizing substitutions. The free energy difference between the native and denatured states of the hs variant is 3.1 (GdnHCl, 25 "C) to 4.0 (differential scanning calorimetry, 74 "C) kcal mol" greater than that of WT. The specific activity of the hs variant is 2.5-fold greater than that of WT. The choice of mutations came from diverse sources: (1) The 155L/S91T core construct with AT, = 3.3 "C from WT was available from the accompanying study (Shih P, Holland DR, Kirsch JF, 1995, Protein Sci 4:2050-2062. (2) The A31V mutation was suggested by the better atomic packing in the human lysozyme structure where the Ala 3 1 equivalent is Leu. (3) The H15L and R114H substitutions were selected on the basis of sequence comparisons with pheasant lysozymes that are more stable than the chicken enzyme. (4) The DlOlS variant was identified from a screen of mutants previously prepared in this laboratory. The effects of the individual mutations on stability are cumulative and nearly additive.Keywords: a-helix propensity; avian lysozyme sequences; chicken lysozyme; human lysozyme; interior packing; protein design; structures; thermodynamic stability A challenging goal of protein engineering is the design of proteins with significantly enhanced thermo-and chemical stabilities. A variety of successful routes to this objective has been undertaken. These include: (1) introducing disulfide bridges (Matsumura et al., 1989b); (2) increasing internal hydrophobic packing (Malcolm et al., 1990); (3) reducing solvent-exposed nonpolar surface area (Pakula & Sauer, 1990); (4) incorporating a metal association site (Kuroki et al., 1989); and (5) adding charged side chains to interact with the a-helix dipoles (Nicholson et al., 1988). However, the above strategies are not always successful and their pursuit has often yielded proteins that are less stable than the WT progenitors for reasons that are not fully understood (e.g., Karpusas et al., 1989;Matsumura et al., 1989a;Eijsink et al., 1992;Leontiev et al., 1993 Abbreviations: hs, a hyperstable variant of chicken lysozyme; GdnHCI, guanidine hydrochloride; T,, the midpoint temperature of the thermal denaturation transition; C,, the midpoint concentration of the GdnHCl denaturation profile; DSC, differential scanning calorimetry; AH,,,, calorimetrically determined enthalpy of denaturation; AC,, the difference between the heat capacities of the native and the denatured states; WT or wt, wild type.