Abstract
A molten globule‐like state of hen egg‐white lysozyme has been characterized in 25% aqueous hexafluoroacetone hydrate (HFA) by CD, fluorescence, NMR, and H/D exchange experiments. The far UV CD spectra of lysozyme in 25% HFA supports retention of native‐like secondary structure while the loss of near UV CD bands are indicative of the overall collapse of the tertiary structure. The intermediate state in 25% HFA exhibits an enhanced affinity towards the hydrophobic dye, ANS, and a native‐like tryptophan fluorescence quenching. 1‐D NMR spectra indicates loss of native‐like tertiary fold as evident from the absence of ring current‐shifted ^1^H resonances. CD, fluorescence, and NMR suggest that the transition from the native state to a molten globule state in 25% HFA is a cooperative process. A second structural transition from this compact molten globule‐like state to an “open” helical state is observed at higher concentrations of HFA (≥50%). This transition is characterized by a dramatic loss of ANS binding with a concomitant increase in far UV CD bands. The thermal unfolding of the molten globule state in 25% HFA is sharply cooperative, indicating a predominant role of side‐chain‐side‐chain interactions in the stability of the partially folded state. H/D exchange experiments yield higher protection factors for many of the backbone amide protons from the four α‐helices along with the C‐terminal 3~10~ helix, whereas little or no protection is observed for most of the amide protons from the triple‐stranded antiparallel β‐sheet domain. This equilibrium molten globule‐like state of lysozyme in 25% HFA is remarkably similar to the molten globule state observed for α‐lactalbumin and also with the molten globule state transiently observed in the kinetic refolding experiments of hen lysozyme. These results suggest that HFA may prove generally useful as a structure modifier in proteins.