Acceleration of the folding of hen lysozyme by trifluoroethanol.

The refolding of a partially structured state of hen lysozyme formed in 60% (v/v) 2,2,2-trifluoroethanol (TFE) has been studied using hydrogen exchange pulse labelling monitored by 2D 1H NMR, and by stopped flow fluorescence and CD measurements. The results are compared with similar studies of the refolding of the protein denatured in 6 M guanidine hydrochloride (GuHCl). Two conclusions have emerged from these studies. First, provided that the refolding conditions are identical, the two denatured states fold with very similar kinetics, despite the fact the extensive secondary structure is present in the TFE-denatured state but not in the protein denatured in 6 M GuHCl. This arises because of the rapid equilibration of structure in the species formed in the initial stage of folding. Second, whilst addition of GuHCl to the refolding buffer decreases the rate of folding, low concentrations of TFE increase the rate of folding. The result is consistent with slow steps in the refolding of lysozyme being associated primarily with the reorganisation of hydrophobic interactions rather than of hydrogen bonded structure.