FOLDING AND STABILITY OF ENDOGLUCANASE-III, A SINGLE-DOMAIN CELLULASEFROM TRICHODERMA-REESEI

The reversible folding of an endoglucanase (EGIII) from the filamentou s fungus Trichoderma reesei was investigated by activity, tryptophan f luorescence, and peptide CD measurements. Equilibrium stability was de termined by urea denaturation at various pH and temperature values. Un folding and refolding rates were measured over a range of urea concent rations. The data from the equilibrium and kinetic studies fit a simpl e two-state model, except at lower urea concentrations, where the fold ing kinetics indicate a transient intermediate. Unfolding is very slow , with a half-life of about 2 h in 8 M urea at pH 5.5 and 25 degrees C . Comparison of the urea dependence of the folding kinetics and equili brium indicates the protein undergoes 93% of its total change in solve nt exposure on going from the unfolded state to the transition state. Thus, the transition state is quite compact. The presence of dithiothr eitol destabilized the protein by 7 kcal/mol, indicating the presence of an unusually strong disulfide linkage between the two cysteines in the molecule. Protein stability is dramatically reduced at alkaline pH values; this can be attributed to a titratable shift (pK(a) = 7.8) in the slope of the urea dependence of unfolding.