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The observed wild-type ESBL equilibrium intermediate has decreased fluorescence but full secondary structure.
When solidification was complete, the other two equilibrium intermediate phases were detected by SEM and XRD.
In addition, using native-state hydrogen exchange-directed protein engineering, we created a mimic of the equilibrium intermediate.
We stabilize here with a single mutation the equilibrium intermediate of apoflavodoxin thermal unfolding and determine its solution structure by NMR.
However, a native-state hydrogen exchange experiment subsequently detected an equilibrium intermediate that only involves the formation of the C-terminal domain.
Global kinetic analysis revealed that the unfolded state partitions between an off-pathway refolding intermediate and the on-pathway equilibrium intermediate early in folding.
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These data suggest that equilibrium intermediates that can be formed through different means are likely to resemble a kinetic intermediate.
Because they sample different regions of the energy landscape, kinetic and equilibrium intermediates will not necessarily be similar.
In other proteins, this trend is less clear, with only a subset of the interactions of equilibrium intermediates seen in kinetic intermediates.
However, proteins displaying equilibrium intermediates under certain conditions abound and it is unclear whether the energetics of native/intermediate equilibria is well represented in current knowledge.
The red shifts of fluorescence spectra show a complex unfolding process involving multiple equilibrium intermediates indicating differential stability of the subdomains of the enzyme.
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