Characterization of kinetic folding intermediates of recombinant canine milk lysozyme by stopped-flow circular dichroism.
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.
Nakao
Masaharu
M
Department of Physics, School of Science, University of Tokyo, Japan.
Maki
Kosuke
K
Arai
Munehito
M
Koshiba
Takumi
T
Nitta
Katsutoshi
K
Kuwajima
Kunihiro
K
D000818:Animals; D002942:Circular Dichroism / Q000379:methods*; D004285:Dogs; D019791:Guanidine; D006358:Hot Temperature; D007700:Kinetics; D008894:Milk Proteins / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry
D008894:Milk Proteins; D011994:Recombinant Proteins; D014364:Tryptophan; D009113:Muramidase; D019791:Guanidine