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 alpha-lactalbumin.
The molten globule state of alpha-lactalbumin (alpha LA) 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 3(10) helices (residues 101-120) of alpha LA 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 alpha LA make to the structure and stability of the alpha LA 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 long-range, mutually stabilizing interactions within the molten globule state of the protein. Proteins 2001;42:237-242.
Demarest
S J
SJ
Department of Chemistry, State University of New York at Stony Brook, Stony Brook, New York 11794-3400, USA.
Horng
J C
JC
Raleigh
D P
DP
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D006801:Humans; D007768:Lactalbumin / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding
D010455:Peptides; D007768:Lactalbumin