A protein dissection study demonstrates that two specific hydrophobic clusters play a key role in stabilizing the core structure of the molten globule state of human α-lactalbumin

The molten globule state of α-lactalbumin (αLA) has served as a paradigm for understanding the role of these partially folded states in protein folding. We previously showed that a peptide construct consisting of the A and B helices (residues 1-38) cross-linked to the D- and C-terminal 3₁₀ helices (residues 101-120) of αLA is capable of folding to a stable molten globule-like state. Here, we report the study of three peptide constructs that are designed to investigate the contribution two short hydrophobic sequences located near the C-terminus of αLA make to the structure and stability of the αLA molten globule state. These regions of the protein have been shown to form stable non-native structures in isolation. The three peptide constructs contain residues 1-38 cross-linked to three separate C-terminal peptides via the native 28-111 disulfide bond. The C-terminal peptides consist of residues 101-114, 106-120, and 106-114. The results of CD, fluorescence, ANS binding, and urea denaturation experiments indicate that constructs that lack either of the hydrophobic sequences (residues 101-105 and 115-120) are significantly less structured. These results highlight the importance of longrange, mutually stabilizing interactions within the molten globule state of the protein.