Isolation of a Highly Reactive β‑Sheet-Rich
Intermediate of Lysozyme in a Solvent-Free Liquid Phase
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Abstract
The
thermal denaturation of solvent-free liquid lysozyme at temperatures
in excess of 200 °C was studied by synchrotron radiation circular
dichroism spectroscopy. Temperature-dependent changes in the secondary
structure were used to map the equilibrium denaturation pathway and
characterize a reactive β-sheet-rich unfolding intermediate
that was stable in the solvent-free liquid phase under anhydrous conditions
but which underwent irreversible aggregation in the presence of water.
The unfolding intermediate had a transition temperature of 78 °C
and was extremely stable to temperature, eventually reaching the fully
denatured state at 178 °C. We propose that the three-stage denaturation
pathway arises from the decreased stability of the native state due
to the absence of any appreciable hydrophobic effect, along with an
entropically derived stabilization of the reactive intermediate associated
with molecular crowding in the solvent-free liquid