Unfolding mechanism of a hyperthermophilic protein O6-methylguanine-DNA methyltransferase

Abstract

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.

Energetics of Protein Stability at Extreme Environmental Temperatures in Bacterial Trigger Factors

“…Denaturant concentrations were determined from refractive index measurements using a R5000 Atago hand refractometer . Reversible equilibrium unfolding curves of Ph TF were analyzed on the basis of a two-state model as described previously, − whereas Ec TF and Tm TF were analyzed on the basis of a three-state model: − y obs = [ y normalN + p [ D ] + exp false( a false) y I + exp false( a + b false) false( y normalU + q [ D ] false) ] / [ 1 + exp false( a false) + exp false( a + b false) ] .25ex2ex with .25em a = prefix− [ Δ G ° ( H 2 O ) NI + m NI false[ normalD false] ] / ( R T ) and .25em b = prefix− [ Δ G ° …”