NMR structural study of two-disulfide variant of hen lysozyme: 2SS[6-127, 30-115]--a disulfide intermediate with a partly unfolded structure.

The 15N-labeled recombinant hen lysozyme and two species of two-disulfide variants, denoted as 2SS[6-127, 30-115] and 2SS[64-80, 76-94], were studied by means of NMR spectroscopy. The former variant contains two disulfide bridges in the alpha-domain, while the latter has one disulfide bridge in the beta-domain and the other one at the interface between two domains. Resonance assignments were performed using 3D TOCSY-HSQC and NOESY-HSQC spectra. The 15N-1H-HSQC spectrum of 2SS[6-127, 30-115] was similar to that of recombinant lysozyme as a whole, although a number of cross-peaks disappeared. On the other hand, the HSQC spectrum of 2SS[64-80, 76-94] was characteristic of unfolded proteins. The structure of 2SS[6-127, 30-115] was thoroughly examined on the basis of NOE contacts determined by NMR spectroscopy. The structure of the alpha-domain was quite similar to that of authentic lysozyme, while the beta-domain was largely unstructured. However, NMR data clearly demonstrated that some residual structures exist in the beta-domain. The beta1 and beta2 strands were maintained stably as an antiparallel beta-sheet. In addition, the residues 55 and 56 were located in the vicinity of the end of the B-helix. Further, the C-helix was properly set with side-chains of I88, V92, K96, and V99 facing toward the hydrophobic core in the alpha-domain. These residual structures inherent in the amino acid sequence were evaluated concerning the folding process of lysozyme. Our experiments imply that the establishment of the backbone conformation ranging from residues 76-99 plays a key role in attaining the cooperativity between two domains required for the folding transition.