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

Structure formation in two species of the two-disulfide variant of hen lyso zyme was investigated by means of CD spectroscopy, disulfide exchange measu rement, and H-1-NMR spectroscopy. One species, 2SS [6-127, 30-115], which c ontained the two disulfide bonds found in the a-domain of authentic lysozym e, had amounts of secondary and tertiary structures, and bacteriolytic acti vity comparable to those of authentic lysozyme, and showed a cooperative th ermal unfolding. By contrast, the other species, 2SS [64-80, 76-94], which contained the beta -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 occurr ed for 2SS [64-80, 76-94], indicating that the protein main-chain fold coup led with the formation of two disulfide bonds is relatively stable for the former variant, while unstable for the latter. H-1-NMR spectra of 2SS [6-12 7, 30-115] showed that nativelike local environment is present within the r egion that corresponds to the alpha -domain, while it is absent within the region that corresponds to the beta or inter-domain. These results indicate that the alpha -domain of hen lysozyme can be an independent folding domai n at equilibrium. Although the bipartite nature in the structure formation of hen lysozyme is similar to that reported for alpha -lactalbumin, differe nces exist between the disulfide-intermediates of the two proteins in terms of the structural domain that accomplishes tertiary structure. (C) 2001 Ac ademic Press.