Downhill folding is predicted to occur under conditions of extreme native bias, i.e. at low temperatures or in the absence of denaturants.
Free energy barriers in protein folding are predicted to be small because they arise as a result of compensation between large energetic and entropic terms.
However, the status of BBL as a downhill-folding protein, and by extension the existence of naturally occurring downhill folders, has been controversial.
[5][6][7] The current controversy arises from the fact that the only way a protein can be labeled as two-state or downhill is by analyzing the experimental data with models that explicitly deal with these two situations, i.e. by allowing the barrier heights to vary.
[8] Nevertheless, in some proteins such as the yeast phosphoglycerate kinase and a mutant human ubiquitin, non-exponential kinetics suggesting downhill folding have been observed.
The data analyzed with a structure-based statistical mechanical model resulted in a unimodal population distribution at all temperatures, indicating a structurally uncoupled continuous unfolding process.
For example, DSC gives information on the heat capacity changes (and hence enthalpy) associated with unfolding, fluorescence on the immediate environment of the fluorophore, FRET on the average dimensions of the molecule and CD on the secondary structure.
A more stringent test would involve following the chemical shifts of each and every atom in the molecule by nuclear magnetic resonance (NMR) as a function of temperature/denaturant.
This assumes even more importance in case of DSC experiments as the changes in heat capacity correspond to both fluctuations in the protein ensemble and exposure of hydrophobic residues upon unfolding.
[11] Instead of attempting a model-free inversion of the DSC profile to extract the underlying probability density function, they assumed a specific free energy functional with either one or two minima (similar to the Landau theory of phase transitions) thus enabling the extraction of free energy barrier heights.
[15] Coarse-grained simulations by Knott and Chan also support the experimental observation of global downhill folding in BBL.