Abstract: The equilibrium unfolding and the kinetics of unfolding and refolding of equine lysozyme, a Ca2+-binding protein, were studied by means of circular dichroism spectra in the far and near-ultraviolet regions. The transition curves of the guanidine hydrochloride-induced unfolding measured at 230 nm and 292.5 nm, and for the apo and holo forms of the protein have shown that the unfolding is well represented by a three-state mechanism in which the molten globule state is populated as a stable intermediate. The molten globule state of this protein is more stable and more native-like than that of α-lactalbumin, a homologous protein of equine lysozyme. The kinetic unfolding and refolding of the protein were induced by concentration jumps of the denaturant and measured by stopped-flow circular dichroism. The observed unfolding and refolding curves both agreed well with a single-exponential function. However, in the kinetic refolding reactions below 3 M guanidine hydrochloride, a burst-phase change in the circular dichroism was present, and the burst-phase intermediate in the kinetic refolding is shown to be identical with the molten globule state observed in the equilibrium unfolding. Under a strongly native condition, virtually all the molecules of equine lysozyme transform the structure from the unfolded state into the molten globule, and the subsequent refolding takes place from the molten globule state. The transition state of folding, which may exist between the molten globule and the native states, was characterized by investigating the guanidine hydrochloride concentration-dependence of the rate constants of refolding and unfolding. More than 80% of the hydrophobic surface of the protein is buried in the transition state, so that it is much closer to the native state than to the molten globule in which only 36% of the surface is buried in the interior of the molecule. It is concluded that all the present results are best explained by a sequential model of protein folding, in which the molten globule state is an obligatory folding intermediate on the pathway of folding.

Abstract: The pressures of developing and maintaining intricate social relationships may have led to the evolution of enhanced cognitive abilities in many nonhuman primates. Knowledge of the dominance ranks and social relationships of other individuals, in particular, is important in evaluating one's position in the rank hierarchy and affiliative networks. Triadic interactions offer an excellent opportunity to examine whether decisions are taken by individuals on the basis of such knowledge. Allogrooming supplants among wild female bonnet macaques (macaca radiata) usually involved the subordinate female of a grooming dyad retreating at the approach of a female dominant to both members of the dyad. In a few exceptional cases, however, the dominant member of the dyad retreated; simple non-cognitive hypotheses involving dyadic rank differences and agonistic relationships failed to explain this phenomenon. Instead, retreat by the dominant individual was positively correlated with the social attractiveness of her subordinate companion (as measured by the duration of grooming received by the latter from other females in the troop). This suggests that not only does an individual evaluate relationships among other females, but does so on the basis of the amount of grooming received by them. Similarly, the frequency of approaches received by any female was correlated with her social attractiveness when she was the dominant member of the dyad, but not when she was the subordinate. This indicated that approaching females might be aware of the relative dominance ranks of the two allogrooming individuals. In logistic regression analyses, the probability of any individual retreating was found to be influenced more by her knowledge of her rank difference with both the other interactants, rather than by their absolute ranks. Moreover, information about social attractiveness appeared to be used in terms of correlated dominance ranks. The nature of knowledge acquired by bonnet macaque females may thus be egotistical in that other individuals are evaluated relative to oneself, integrative in that information about all other interactants is used simultaneously, and hierarchical in the ability to preferentially use certain categories of knowledge for the storage of related information from other domains.