Normally reversible unfolding refolding is assumed in a 2 state equilibrium process in protein denaturation to calculate protein stability or delta G. Membrane proteins often aggregate / are not following reversibility in refolding. Are delta G calculations therefore reliable, if we assume reversibility and ignore the fact that it's not?
Hi Sascha, as far as I know, the issue of reversibility is important for 2 state models because you assume that there is an equilibrium that is being stablished at each point of your curve, and that equilibrium is only between two species, Unfolded and Native. If there are more states than those, you cannot assume equilibrium, and therefore the dG value is only apparent as hysteresis may be driving your dG value higher than it should be.
An approach, I guess, is to recalculate the soluble protein concentration after denaturant incubation, so your measurement corresponds only to folded fraction, and the reweigh your signal (Abs, fluorescence, ellipticity, etc) by that protein concentration.
Since the equilibrium in a 2S model is stablished independently of the protein concentration, it should not affect the folded fraction for that denaturant concentration.
I hope it helps.
Good luck!
Delta G calculated in classic thermodynamics according to reversible and equlibrium conditions.
Maybe by lowering scan rate the results improve to a better condition.
As the above answers indicate, (dG) assumes reversibility. The initial step of protein denaturation is typically reversible. The problem is that there are subsequent steps that can occur once the initial structural change happens that aren't necessarily reversible (e.g., aggregation and precipitation). Proteins don't actually like aqueous solutions. Hydrophobicity is a primary driver in their tertiary structure. Once those hydrophobic residues are exposed in an unfolding event, they will find any other hydrophobic material to bond with to displace water away from them. You can slow this aggregation process down using dilute solutions, but it will always be there.
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