Unfolding-refolding kinetics of the tryptophan synthase alpha subunit by CD and fluorescence measurements.
To elucidate the folding mechanism of the tryptophan synthase alpha subunit from Escherichia coli, the kinetics of the unfolding-refolding were studied by peptidyl circular dichroism (CD) and aromatic fluorescence measurement at pH 7 and 25 degrees C. The reactions were induced by concentration jumps of guanidine hydrochloride (GuHCl). The results can be summarized as follows. (1) The kinetic properties of the unfolding-refolding monitored by CD at 222 nm and aromatic fluorescence coincided with each other, indicating that the changes in the secondary and tertiary structures proceed simultaneously. (2) The unfolding kinetics showed two phases in the range of final GuHCl concentration above 1.8 M. The total amplitudes in the unfolding kinetics accounted for about 100% of the total change. (3) The refolding kinetics also showed two phases in the native condition. The total amplitudes observed in the two phases accounted for only 41% of the total change in maximum, indicating the presence of an undetectable early folding intermediate in the folding process. (4) The fast phases in both the unfolding and refolding were major phases as judged by the magnitudes of the amplitudes. (5) The amplitudes in terms of the CD values at 222 nm for the undetectable early folding intermediate in the refolding kinetics showed little dependence on final GuHCl concentration in the native condition, but depended on final GuHCl concentration in the transition zone, resulting in a similar equilibrium GuHCl unfolding curve. (6) The CD spectrum in the far-UV region for the early folding intermediate was similar to that for the equilibrium unfolding intermediate. (7) It is concluded that the early folding intermediate of the alpha subunit is equivalent to the equilibrium unfolding intermediate, which is assumed to be a molten globule.
Ogasahara
K
K
Institute for Protein Research, Osaka University, Japan.
Yutani
K
K
D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology; D019791:Guanidine; D006146:Guanidines; D007700:Kinetics; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D014367:Tryptophan Synthase / Q000737:chemistry*
D006146:Guanidines; D014367:Tryptophan Synthase; D019791:Guanidine