title: Effects of the stabilization of the molten globule state on the folding mechanism of alpha-lactalbumin: a study of a chimera of bovine and human alpha-lactalbumin.
abstract: The N-terminal half of the alpha-domain (residues 1 to 34) is more important for the stability of the acid-induced molten globule state of alpha-lactalbumin than the C-terminal half (residues 86 to 123). The refolding and unfolding kinetics of a chimera, in which the amino acid sequence of residues 1 to 34 was from human alpha-lactalbumin and the remainder of the sequence from bovine alpha-lactalbumin, were studied by stopped-flow tryptophan fluorescence spectroscopy. The chimeric protein refolded and unfolded substantially faster than bovine alpha-lactalbumin. The stability of the molten globule state formed by the chimera was greater than that of bovine alpha-lactalbumin, and the hydrophobic surface area buried inside of the molecule in the molten globule state was increased by the substitution of residues 1 to 34. Peptide fragments corresponding to the A- and B-helix of the chimera showed higher helix propensity than those of the bovine protein, indicating the contribution of local interactions to the high stability of the molten globule state of the chimera. Moreover, the substitution of residues 1-34 decreased the free energy level of the transition state and increased hydrophobic surface area buried inside of the molecule in the transition state. Our results indicate that local interactions as well as hydrophobic interactions formed in the molten globule state are important in guiding the subsequent structural formation of alpha-lactalbumin.
authors.affiliation: Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama, Japan. mimeyuki@ms.toyama-mpu.ac.jp
authors.forename: Mineyuki
authors.initials: M
authors.lastname: Mizuguchi
authors.forename: Atsushi
authors.initials: A
authors.lastname: Matsuura
authors.forename: Yuko
authors.initials: Y
authors.lastname: Nabeshima
authors.forename: Kazuo
authors.initials: K
authors.lastname: Masaki
authors.forename: Masahiro
authors.initials: M
authors.lastname: Watanabe
authors.forename: Tomoyasu
authors.initials: T
authors.lastname: Aizawa
authors.forename: Makoto
authors.initials: M
authors.lastname: Demura
authors.forename: Katsutoshi
authors.initials: K
authors.lastname: Nitta
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Mori
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Shinoda
authors.forename: Keiichi
authors.initials: K
authors.lastname: Kawano
mesh_terms: D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates; D000818:Animals; D002417:Cattle; D005456:Fluorescent Dyes; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence
chemical_list: D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D010455:Peptides; D011993:Recombinant Fusion Proteins; C027132:1-anilino-8-naphthalenesulfonate; D007768:Lactalbumin; D019791:Guanidine