title: Sequential four-state folding/unfolding of goat α-lactalbumin and its N-terminal variants.
abstract: Equilibria and kinetics of folding/unfolding of α-lactalbumin and its two N-terminal variants were studied by circular dichroism spectroscopy. The two variants were wild-type recombinant and Glu1-deletion (E1M) variants expressed in Escherichia coli. The presence of an extra methionine at the N terminus in recombinant α-lactalbumin destabilized the protein by 2 kcal/mol, while the stability was recovered in the E1M variant in which Glu1 was replaced by Met1. Kinetic folding/unfolding reactions of the proteins, induced by stopped-flow concentration jumps of guanidine hydrochloride, indicated the presence of a burst-phase in refolding, and gave chevron plots with significant curvatures in both the folding and unfolding limbs. The folding-limb curvature was interpreted in terms of accumulation of the burst-phase intermediate. However, there was no burst phase observed in the unfolding kinetics to interpret the unfolding-limb curvature. We thus assumed a sequential four-state mechanism, in which the folding from the burst-phase intermediate takes place via two transition states separated by a high-energy intermediate. We estimated changes in the free energies of the burst-phase intermediate and two transition states, caused by the N-terminal variations and also by the presence of stabilizing calcium ions. The Φ values at the N terminus and at the Ca(2+)-binding site thus obtained increased successively during folding, demonstrating the validity of the sequential mechanism. The stability and the folding behavior of the E1M variant were essentially identical to those of the authentic protein, allowing us to use this variant as a pseudo-wild-type α-lactalbumin in future studies.
authors.affiliation: Department of Physics, School of Science, University of Tokyo, Bunkyo-ku, Tokyo, Japan.
authors.forename: Katsuaki
authors.initials: K
authors.lastname: Tomoyori
authors.forename: Takashi
authors.initials: T
authors.lastname: Nakamura
authors.forename: Koki
authors.initials: K
authors.lastname: Makabe
authors.forename: Kosuke
authors.initials: K
authors.lastname: Maki
authors.forename: Kimiko
authors.initials: K
authors.lastname: Saeki
authors.forename: Kunihiro
authors.initials: K
authors.lastname: Kuwajima
mesh_terms: D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D006041:Goats; D019791:Guanidine; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D017510:Protein Folding; D055550:Protein Stability; D013816:Thermodynamics
chemical_list: D007768:Lactalbumin; D019791:Guanidine; D002118:Calcium