Protein dissection experiments reveal key differences in the equilibrium folding of alpha-lactalbumin and the calcium binding lysozymes.

The alpha-lactalbumins and c-type lysozymes have virtually identical structure but exhibit very different folding behavior. All alpha-lactalbumins form a well populated molten globule state, while most of the lysozymes do not. alpha-Lactalbumin consists of two subdomains, and the alpha-subdomain is considerably more structured in the molten globule state than the beta-subdomain. Constructs derived from the alpha-subdomain of human alpha-lactalbumin containing the A, B, D, and 3(10) helices are known to form a molten globule state in the absence of the rest of the protein (Demarest, S. et al. (1999) J. Mol. Biol. 294, 213-221). Here we reported comparative studies of constructs derived from the same regions of canine and equine lysozymes. These proteins form two of the most stable molten globule states among all the lysozymes. A construct containing the A, B, D, and 3(10) helices of equine lysozyme is partially helical but is less structured than the corresponding human alpha-lactalbumin peptide. Addition of the C-helix leads to a construct that is still less structured and less stable than the alpha-lactalbumin construct. The corresponding construct from canine lysozyme is also less structured and less stable than the alpha-lactalbumin peptide. Thus, molten globule formation in human alpha-lactalbumin can be driven by the isolated alpha-subdomain, while more extensive interactions are required to generate a stable molten globule in the two lysozymes. The stability of the canine and equine lysozyme constructs is similar, indicating that the extraordinary stability of the canine lysozyme molten globule is not due to an unusually stable isolated alpha-subdomain.