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Abstract
During the folding of many proteins, collapsed globular states are formed prior to
the native structure. The role of these states for the folding process has been widely
discussed. Comparison with properties of synthetic homo and heteropolymers had suggested
that the initial collapse represented a shift of the ensemble of unfolded conformations
to more compact states without major energy barriers. We investigated the folding/unfolding
transition of a collapsed state, which transiently populates early in lysozyme folding.
This state forms within the dead-time of stopped-flow mixing and it has been shown
to be significantly more compact and globular than the denaturant-induced unfolded
state. We used the GdmCl-dependence of the dead-time signal change to characterize
the unfolding transition of the burst phase intermediate. Fluorescence and far-UV
CD give identical unfolding curves, arguing for a cooperative two-state folding/unfolding
transition between unfolded and collapsed lysozyme. These results show that collapse
leads to a distinct state in the folding process, which is separated from the ensemble
of unfolded molecules by a significant energy barrier. NMR, fluorescence and small
angle X-ray scattering data further show that some local interactions in unfolded
lysozyme exist at denaturant concentrations above the coil-collapse transition. These
interactions might play a crucial role in the kinetic partitioning between fast and
slow folding pathways.