Native-like tertiary structure formation in the α-domain of a hen lysozyme two-disulfide variant

Structure formation in two species of the two-disulfide variant of hen lysozyme was investigated by means of CD spectroscopy, disulfide exchange measurement, and 1H-NMR spectroscopy. One species, 2SS [6–127, 30–115], which contained the two disulfide bonds found in the α-domain of authentic lysozyme, had amounts of secondary and tertiary structures, and bacteriolytic activity comparable to those of authentic lysozyme, and showed a cooperative thermal unfolding. By contrast, the other species, 2SS [64–80, 76–94], which contained the β-domain disulfide bond as well as the inter-domain one, had a limited amount of secondary structure and little tertiary structure. Disulfide-exchange did not occur for 2SS [6–127, 30–115], whereas it occurred for 2SS [64–80, 76–94], indicating that the protein main-chain fold coupled with the formation of two disulfide bonds is relatively stable for the former variant, while unstable for the latter. 1H-NMR spectra of 2SS [6–127, 30–115] showed that native-like local environment is present within the region that corresponds to the α-domain, while it is absent within the region that corresponds to the β or inter-domain. These results indicate that the α-domain of hen lysozyme can be an independent folding domain at equilibrium. Although the bipartite nature in the structure formation of hen lysozyme is similar to that reported for α-lactalbumin, differences exist between the disulfide-intermediates of the two proteins in terms of the structural domain that accomplishes tertiary structure.