Sequential four-state folding/unfolding of goat α-lactalbumin and its N-terminal variants

Proteins
Katsuaki TomoyoriK Kuwajima

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 ...Continue Reading

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Citations

Sep 21, 2017·International Journal of Molecular Sciences·Reina Shinozaki, Michio Iwaoka
Nov 17, 2012·Protein Engineering, Design & Selection : PEDS·Koki MakabeKunihiro Kuwajima
Aug 23, 2020·Biomolecules·Eugene A Permyakov

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