PEPTIDE MODELS OF LOCAL AND LONG-RANGE INTERACTIONS IN THE MOLTEN GLOBULE STATE OF HUMAN ALPHA-LACTALBUMIN

alpha-lactalbumin, a small calcium-binding protein, forms an equilibri um molten globule state under a variety of conditions. A set of four p eptides designed to probe the role of local interactions and the role of potential long-range interactions in stabilizing the molten globule of alpha-lactalbumin has been prepared. The first peptide consists of residues 20 through 36 of human alpha-lactalbumin and includes the en tire B-helix. This peptide is unstructured in solution as judged by CD . The second peptide is derived from residues 101 through 120 and cont ains both the D and 3(10) helices. When this peptide is crosslinked vi a the native 28 to 111 disulfide to the B-helix peptide, a dramatic in crease in helicity is observed. The cross-linked peptide is monomeric, as judged by analytical ultracentrifugation. The peptide binds 1-anil inonaphthalene-8-sulphonate (ANS) and the fluorescence emission maximu m of the construct is consistent with partial solvent exposure of the tryptophan residues. The peptide corresponding to residues 101 to 120 adopts significant non-random structure in aqueous solution at low pH. Two hydrophobic clusters, one involving residues 101 through 104 and the other residues 115 through 119 have been identified and characteri zed by NMR. The hydrophobic cluster formed by residues 101 through 104 is still present in a smaller peptide containing only residues 101 to 111 of alpha-lactalbumin. The cluster also persists in 6 M urea. A no n-native, pH-dependent interaction between the Y103 and H107 side-chai ns that was previously identified in the acid-denatured molten globule state was examined. This interaction was found to be more prevalent a t low pH and may therefore be an example of a local interaction that s tabilizes preferentially the acid-induced molten globule state. (C) 19 98 Academic Press.