I got two ΔG, one is from the equilibrium constant( ΔG=-RTlnK), the equilibrium constant(K) is from protein kinetic experiments (K=Kfold/Kunfold at H20). Another ΔG is from equilibrium folding experiments by denaturants (urea). I found that the two ΔG are very close. Is that a good justification that the folding of this protein follows a two-state model? Is there any reference that talks about that? I used to read a similar statement in one paper. Thanks!
Yes. It indicates that protein folding is two state. Also, analyse the data manually, if all the points in the transition can be represented as a linear combination of first and last point. 2ndly, if the delta G values are coming out same for folding paths using different experimental techniques(like fluorescence or CD)
You can analyze your fluorescence or CD data by fitting it to a two-state equation. From the curve you can determine the ΔG, m and Cm values for the folded to unfolded transition. For a two state model you will have a single ΔG or Cm value. Software that you can use: Origin.
Tushar Chakraborty I tried fitting my CD data in Origin, but was not able to determine ΔG. I used Boltzmann's fit in Origin and could calculate Tm. But I do not know how to proceed to determine ΔH, ΔS and ΔG. I have this problem for about 1 year now, and stuck since then to analyze my data. Please, could you have time to go through with me?
The most important thing is to quickly understand what kind of transition we are dealing with. The experimental result should be a sigmoidal transition curve with the instrumental observable (eg CD at 222 nm) in the ordinate and the concentration of the denaturant in the abscissa.
Now look at the slope of the transition. If the slope is very steep (the feet of the curve, top and bottom, should fall within a very narrow concentration range (about 0.5 conc. Units). The steeper the transition, the higher the probability of having a two-states transition.The more the slope decreases and the base of the transition widens, the greater the probability of having intermediates at equilibrium. A transition that falls within an interval of two or more conc. units is certainly multi-state.
It is a crude way, but it tells you immediately and with good reliability what you are dealing with. In this way you will then be able to better manage the analysis that you will do fitting experimental data to a mathematical model.
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