THE UNFOLDING THERMODYNAMICS OF C-TYPE LYSOZYMES - A CALORIMETRIC STUDY OF THE HEAT DENATURATION OF EQUINE LYSOZYME

The energetics of the temperature-induced unfolding of equine lysozyme was studied calorimetrically and compared with that of two structurally homologous proteins: hen egg white lysozyme and alpha-lactalbumin. The structure of each of these proteins is characterized by the presence of a deep cleft that divides the molecule into two regions called the alpha and beta domains. In equine lysozyme and alpha-lactalbumin the latter domain specifically binds Ca2+. It is shown that, in contrast to hen egg white lysozyme in which the alpha and beta domains unfold as a single cooperative unit, in equine lysozyme the two domains unfold in two separate cooperative stages even in the presence of excess Ca2+. The calcium binding beta-domain unfolds at a lower temperature and with more extensive heat absorption than the alpha-domain. Binding of Ca2+ increases the stability of the beta-domain, but even in the hole form it is less stable than the alpha-domain. The thermodynamic characteristics of Ca2+ binding have been determined, and indicate that it is an entropically driven process. The unfolding of equine lysozyme largely resembles the unfolding of alpha-lactalbumin, which also unfolds in two stages, but in the latter case the second stage is much less cooperative and proceeds with a smaller and diffuse heat absorption. As a result, the total enthalpy of unfolding of equine lysozyme is significantly larger than that of alpha-lactalbumin, being almost of the same magnitude as the enthalpy of egg white lysozyme unfolding, which proceeds as a single two-stare transition. Analyses of the unfolding enthalpy function of various lysozymes, which bind or do not bind Ca2+, and unfold in one or two stages, have led us to the conclusion that the main reason for the loss of interdomain cooperativity in equine lysozyme is not the cluster of negative charges forming the calcium binding site, but the difference in atomic packing in the interior and at the interface between the alpha and beta domains. (C) 1995 Academic Press Limited