Unfolding mechanism of a hyperthermophilic protein O(6)-methylguanine-DNA methyltransferase.
Unfolding intermediates have been found only rarely in earlier studies, and how a protein unfolds is therefore poorly understood. In this paper, we show experimental evidence for multiple pathways and multiple intermediates during unfolding reaction of O(6)-methylguanine-DNA methyltransferase from hyperthermophile Thermococcus kodakaraensis (Tk-MGMT). The unfolding profiles monitored by far-UV CD and tryptophan fluorescence were both biphasic, and unfolding monitored by fluorescence was faster than that monitored by CD. GdnHCl-induced titration curves indicate that the intermediates with significant alpha-helical structure accumulate during unfolding. Dependence of kinetic phases on initial GdnHCl concentrations and cysteine reactivity of Tk-MGMT were investigated, suggesting that the heterogeneity of native conformations and parallel unfolding pathways.
Nishikori
Shingo
S
School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Ishikawa 923-1292, Japan.
Shiraki
Kentaro
K
Fujiwara
Shinsuke
S
Imanaka
Tadayuki
T
Takagi
Masahiro
M
D003545:Cysteine / Q000737:chemistry; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry*; D017510:Protein Folding; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology; D013816:Thermodynamics
D004228:Dithionitrobenzoic Acid; D019853:O(6)-Methylguanine-DNA Methyltransferase; D003545:Cysteine