The Unfolding Thermodynamics of c-Type Lysozymes: A Calorimetric Study of the Heat Denaturation of Equine Lysozyme

Abstract

The energetics of the temperature-induced unfolding of equine lysozyme was studied calorimetricaly and compared with that of two structurally homologous proteins: hen egg white lysozyme and α-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 α and β domains. In equine lysozyme and α-lactalbumin the latter domain specifically binds Ca 2+. It is shown that, in contrast to hen egg white lysozyme in which the α and β 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 Ca 2+. The calcium binding β-domain unfolds at a lower temperature and with more extensive heat absorption than the α-domain. Binding of Ca 2+increases the stability of the β-domain, but even in the holo form it is less stable than the α-domain. The thermodynamic characteristics of Ca 2+binding have been determined, and indicate that it is an entropically driven process. The unfolding of equine lysozyme largely resembles the unfolding of α-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 α-lactalbumin, being almost of the same magnitude as the enthalpy of egg white lysozyme unfolding, which proceeds as a single two-state transition. Analyses of the unfolding enthalpy function of various lysozymes, which bind or do not bind Ca 2+, and unfold in one or two stages, have led us to the conclusion that the main reason for the loss of interdomain cooperatively 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 α and β domains.