Folding-unfolding equilibrium and kinetics of equine beta-lactoglobulin: equivalence between the equilibrium molten globule state and a burst-phase folding intermediate.
The denaturant-induced equilibrium unfolding transition of equine beta-lactoglobulin was investigated by ultraviolet absorption, fluorescence, and circular dichroism (CD) spectra. An equilibrium intermediate populates at moderate denaturant concentrations, and its CD spectrum is similar to that of the molten globule state previously observed for this protein at acid pH [Ikeguchi, M., Kato, S., Shimizu, A., and Sugai, S. (1997) Proteins: Struct., Funct., Genet. 27, 567-575]. The unfolding and refolding kinetics were also investigated by the stopped-flow CD and fluorescence. A significant change in the CD intensity was observed within the dead time of measurements (25 ms) when the refolding reaction was initiated by diluting the urea-unfolded protein solution, indicating the transient accumulation of the folding intermediate. The CD spectrum of this burst-phase intermediate agrees well with that of the molten globule state at acid pH. The stability of the burst-phase intermediate was also estimated from the urea-concentration dependence of the burst-phase amplitude, and it shows a fair agreement with that of the equilibrium intermediate. These results indicate that the molten globule state of equine beta-lactoglobulin populates at moderate urea concentration as well as at acid pH and it is equivalent with the kinetic folding intermediate.
Fujiwara
K
K
Department of Bioengineering, Faculty of Engineering, Soka University, Tokyo, Japan.
Arai
M
M
Shimizu
A
A
Ikeguchi
M
M
Kuwajima
K
K
Sugai
S
S
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D019791:Guanidine; D006736:Horses; D007700:Kinetics; D007782:Lactoglobulins / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence / Q000379:methods; D014508:Urea
D007782:Lactoglobulins; D014508:Urea; D019791:Guanidine