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

The α-lactalbumins and c-type lysozymes have virtually identical structure but exhibit very different folding behavior. All α-lactalbumins form a well populated molten globule state, while most of the lysozymes do not. α-Lactalbumin consists of two subdomains, and the α-subdomain is considerably more structured in the molten globule state than the β-subdomain. Constructs derived from the α-subdomain of human α-lactalbumin containing the A, B, D, and 3₁₀ 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 α-lactalbumin peptide. Addition of the C-helix leads to a construct that is still less structured and less stable than the α-lactalbumin construct. The corresponding construct from canine lysozyme is also less structured and less stable than the α-lactalbumin peptide. Thus, molten globule formation in human α-lactalbumin can be driven by the isolated α-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 α-subdomain.