Hexafluoroacetone hydrate as a structure modifier in proteins: Characterization of a molten globule state of hen egg‐white lysozyme

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 a f f i n i t y 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 '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 (250%). This transition is characterized by a dramatic loss of A N S 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. HID exchange experiments yield higher protection factors for many of the backbone amide protons from the four a-helices along with the C-terminal 3'0 helix, whereas little or no protection is observed for most of the amide protons from the triple-stranded antiparallel @-sheet domain. This equilibrium molten globule-like state of lysozyme in 25% HFA is remarkably similar to the molten globule state observed for a-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.

The compact and expanded denatured conformations of apomyoglobin in the methanol‐water solvent

“…The compact denatured state of b-lactoglobulin was reported not only in methanol but also in several types of organic solvents including ethanol, isopropanol, dimethylformamide, and dioxane~Uversky et al, 1997!. The compact denatured state in hexafluoroacetone hydrate was also reported for lysozymẽ Bhattacharjya & Balaram, 1997!. There results also indicate the general existence of the MG state in alcohol-water solvents.…”

Thermodynamics of thermal denaturation of ribonuclease A and cytochrome c in the presence of 1,1,1,3,3,3-hexafluoro-2-propanol

“…(9,31,32) Hexafluoroacetone hydrate (HFA) is an amphiphilic molecule possessing a highly hydrophobic fluorocarbon face and a hydrophobic hydrogen bond donating hydrated face. Balaram and Bhattacharya (33) have observed a molten globule-like state of hen egg-white lysozyme in 0.25 mass fraction aqueous HFA. Figure 6 shows the far u.v.…”

Interactions between single-walled carbon nanotubes and lysozyme

“…Because of the dispersion turbidity, a quartz cuvette with optical path length of 0.20 cm was used. Data reported here refer to the k range 190-260 nm, where lysozyme shows typical absorbing bands centered at 208 and 222 nm, respectively [28]. The measuring temperature, 25.0°C, is controlled to 0.1°C by an external water circulation bath.…”