Cooperative folding of the isolated α-helical domain of hen egg-white lysozyme

Proteins in the α-lactalbumin and c-type lysozyme family have been studied extensively as model systems in protein folding. Early formation of the α-helical domain is observed in both α-lactalbumin and c-type lysozyme; however, the details of the kinetic folding pathways are significantly different. The major folding intermediate of hen egg-white lysozyme has a cooperatively formed tertiary structure, whereas the intermediate of α-lactalbumin exhibits the characteristics of a molten globule. In this study, we have designed and constructed an isolated α-helical domain of hen egg-white lysozyme, called Lyso-α, as a model of the lysozyme folding intermediate that is stable at equilibrium. Disulfide-exchange studies show that under native conditions, the cysteine residues in Lyso-α prefer to form the same set of disulfide bonds as in the α-helical domain of full-length lysozyme. Under denaturing conditions, formation of the nearest-neighbor disulfide bonds is strongly preferred. In contrast to the isolated α-helical domain of α-lactalbumin, Lyso-α with two native disulfide bonds exhibits a well-defined tertiary structure, as indicated by cooperative thermal unfolding and a well-dispersed NMR spectrum. Thus, the determinants for formation of the cooperative side-chain interactions are located mainly in the α-helical domain. Our studies suggest that the difference in kinetic folding pathways between α-lactalbumin and lysozyme can be explained by the difference in packing density between secondary structural elements and support the hypothesis that the structured regions in a protein folding intermediate may correspond to regions that can fold independently.