Fluoroalcohol-induced stabilization of the α-helical intermediates of lentil lectin: implication for non-hierarchical lectin folding

An intermediate state of lentil lectin was characterized at pH 1 having low content of secondary as well as tertiary structure. Far- and near-UV CD spectroscopy showed loss of structure when pH was lowered from 7 to 0.8 but the structure loss was less than that of the protein in presence of 6 M GndHCl. Intrinsic tryptophan fluorescence studies, ANS binding, and acrylamide quenching experiments supported the existence of the intermediate at low pH. The unfolding process of lentil lectin at pH 1 was also studied by GndHCl denaturation monitored by intrinsic fluorescence spectroscopy. The non-cooperative unfolding at pH 1, in contrast to cooperative unfolding of the native protein further confirmed the presence of loose tertiary structure. The unfolded structure of the lectin at pH 1 was also shown by limited tryptic digestion studies. Further studies were performed on this intermediate state of lentil lectin obtained at low pH in presence of fluoroalcohols 2,2,2-trifluoroethanol (TFE) and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP). Lentil lectin is mainly a β-sheet protein, and both TFE and HFIP stabilized the acid unfolded structure by inducing α-helical contacts. Interestingly, it was observed that induction of the non-native structure resulted in regain of protein activity to some extent. At pH 1, loss in activity was found with both dextran and bromelain while the reported intermediate at the given pH was found to regain activity with bromelain in presence of HFIP and TFE. HFIP induced more structure as compared to TFE and hence a greater regain in activity of about 30% was observed with HFIP as compared to a 15% regain with TFE. Activity with dextran in presence of fluoroalcohols could not be determined as turbidity developed in the corresponding blank preparations. Our results presented here point out the possibility of the formation of a helical structure preceding the formation of the native β-sheet structure and thus support the non-hierarchical model of protein folding for lentil lectin.