A comparative study of peptide models of the α-domain of α-lactalbumin, lysozyme, and α-lactalbumin/lysozyme chimeras allows the elucidation of critical factors that contribute to the ability to form stable partially folded states

α-Lactalbumin (αLA) forms a well-populated equilibrium molten globule state, while the homologous protein hen lysozyme does not. αLA is a two-domain protein and the α-domain is more structured in the molten globule state than is the β-domain. Peptide models derived from the α-subdomain that contain the A, B, D, and 3₁₀ helices of αLA are capable of forming a molten globule state in the absence of the remainder of the protein. Here we report comparative studies of a peptide model derived from the same region of hen lysozyme and a set of chimeric α-lactalbumin-lysozyme constructs. Circular dichroism, dynamic light scattering, sedimentation equilibrium, and fluorescence experiments indicate that the lysozyme construct does not fold. Chimeric constructs were prepared to probe the origins of the difference in the ability of the two isolated subdomains to fold. The first consists of the A and B helices of αLA cross-linked to the D and C-terminal 3₁₀ helices of lysozyme. This construct is highly helical, while a second construct that contains the A and B helices of lysozyme cross-linked to the D and 3₁₀ helices of αLA does not fold. Furthermore, the disulfide cross-linked homodimer of the αLA AB peptide is helical, while the homodimer of the lysozyme AB peptide is unstructured. Thus, the AB helix region of αLA appears to have an intrinsic ability to form structure as long as some relatively nonspecific interactions can be made with other regions of the protein. Our studies show that the A and B helices plays a key role in the ability of the respective α-subdomains to fold.