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

Bhattacharjya, Surajit and Balaram, Padmanabhan (1997) Hexafluoroacetone hydrate as a structure modifier in proteins: Characterization of a molten globule state of hen egg-white lysozyme. In: Protein Science, 6 (5). pp. 1065-1073.


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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 ‘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 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. 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 beta-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.

Item Type: Journal Article
Publication: Protein Science
Publisher: Cold Spring Harbor Laboratory Press
Additional Information: Copyright for this article belongs to Cold Spring Harbor Laboratory Press.
Keywords: CD;fluorescence;hexafluoroacetone;lysozyme;molten globule states;NMR;protein folding
Department/Centre: Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited: 25 Aug 2008
Last Modified: 19 Sep 2010 04:17
URI: http://eprints.iisc.ac.in/id/eprint/2202

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