Characterization of kinetic folding intermediates of recombinant canine milk lysozyme by stopped-flow circular dichroism

Masaharu Nakao, Kosuke Maki, Munehito Arai, Takumi Koshiba, Katsutoshi Nitta, Kunihiro Kuwajima

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

The intermediate in the equilibrium unfolding of canine milk lysozyme induced by a denaturant is known to be very stable with characteristics of the molten globule state. Furthermore, there are at least two kinetic intermediates during refolding of this protein: a burst-phase (first) intermediate formed within the dead time of stopped-flow measurements and a second intermediate that accumulates with a rate constant of 22 s-1. To clarify the relationships of these intermediates with the equilibrium intermediate, and also to characterize the structural changes of the protein during refolding, here we studied the kinetic refolding reactions using stopped-flow circular dichroism at 10 different wavelengths and obtained the circular dichroism spectra of the intermediates. Comparison of the circular dichroism spectra of the intermediates, as well as the absence of observed kinetics in the refolding from the fully unfolded state to the equilibrium intermediate, has demonstrated that the burst-phase intermediate is equivalent to the equilibrium intermediate. The difference circular dichroism spectrum that represented changes from the kinetic intermediate to the native state had characteristics of an exciton coupling band, indicating that specific packing of tryptophan residues in this protein occurred in this phase. From these findings, we propose a schematic model of the refolding of canine milk lysozyme that is consistent with the hierarchical mechanism of protein folding.

Original languageEnglish
Pages (from-to)6685-6692
Number of pages8
JournalBiochemistry
Volume44
Issue number17
DOIs
Publication statusPublished - May 3 2005

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Dichroism
Muramidase
Circular Dichroism
Canidae
Milk
Protein Refolding
Kinetics
Protein folding
Proteins
Protein Folding
Schematic diagrams
Flow measurement
Tryptophan
Molten materials
Rate constants
Wavelength

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Characterization of kinetic folding intermediates of recombinant canine milk lysozyme by stopped-flow circular dichroism. / Nakao, Masaharu; Maki, Kosuke; Arai, Munehito; Koshiba, Takumi; Nitta, Katsutoshi; Kuwajima, Kunihiro.

In: Biochemistry, Vol. 44, No. 17, 03.05.2005, p. 6685-6692.

Research output: Contribution to journalArticle

Nakao, Masaharu ; Maki, Kosuke ; Arai, Munehito ; Koshiba, Takumi ; Nitta, Katsutoshi ; Kuwajima, Kunihiro. / Characterization of kinetic folding intermediates of recombinant canine milk lysozyme by stopped-flow circular dichroism. In: Biochemistry. 2005 ; Vol. 44, No. 17. pp. 6685-6692.
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