I am having trouble calculating free energy of folding, enthalpy and heat capacity of my peptide samples. I performed a thermal unfolding of the peptides, and obtained the ellipticity in mdeg.
Next, I converted the mdeg to mean residue ellipticity using the equation:
m.r.e. = mdeg / [path length, mm * Molar conc. * Number of residues].
Is this correct?
With Origin software, I used a Boltzmann's fit to determine the Tm of the peptides. But how can I calculate the enthalpy, heat capacity and free energy of unfolding assuming a 2-state model?
Which software permits me to do this? And how can I actually use equations to compute the mathematical simulations?
All suggestions would be appreciated.
Using the Boltzmann function is not a good way to analyze protein unfolding assays. This curve has an appropriate shape, but its parameters (inflection point location, difference between asymptotes, asymptotes) may not have any relation with the thermodynamic parameters you would get from the correct analysis (Tm, unfolding enthalpy, unfolding heat capacity) from which you can compute the stability profile (Gibbs energy of unfolding as a function of temperature). Even the temperature for maximal slope (center of the transition) may not correspond with the actual unfolding transition temperature (Tm). Therefore, the Boltzmann fit is an example of a curve that may go through the experimental points, but gives no information at all about the stability of the protein.
You should analyze the unfolding experiment using the two-state or multiple-state (non-two-state) unfolding model. You can use any data analysis software (Origin, Excel, KaleidaGraph, SigmaPlot, DataFit, LabPlot...), because all these packages will allow you defining user-defined fitting functions.
But, first, you should read articles, book chapters, and protocols where the two-state or the non-two-state models are described or employed.
As Adrian suggests, a good starting point is the following article by Norma Greenfield:
Article Using circular dichroism collected as a function of temperat...
Hello Cynthia,
I have used 2-state and 3-state models to calculate free energy and enthalpy for my protein folding system. Please let me know if you would like to collaborate.
My email id is [email protected]
Hello Adrian, Conceicao and Rohit. Thanks a lot for your answers.
Adrian Velazquez-Campoy and Conceicao Minetti i have actually read lots of literature on this, and some seem to be conflicting. Also, I am aware I have to use a 2-state model to calculate the thermodynamic properties of my peptides.
I have my mean residue ellipticity and temperature values. And would like to know which user-friendly software would enable me model a 2-state unfolding transition and determine parameters automatically. Some softwares which I tried to use for these (e.g. CDToolX, CDPal, etc) are not so user-friendly.
My only option now is actually using the equations manually to calculate the thermodynamic properties. And this is where I have lots of problems.
For example, at high temperatures, I have negative ellipticity values. This in turn leads to negative K values, and I can't calculate natural log of a negative value. So I am stuck. Also I am not able to program the various equations of a 2-state model in Origin or Excel.
Adrian Velazquez-Campoy Despite several efforts, I have not made any progress with calculating Gibb's free energy of unfolding and had to put this project on a hold. I tried using Excel and Origin to model 2-state thermal unfolding transition, But I could not compute the mathematical equations. My ultimate goal is to determine dG of a native state vs a mutated state, and then calculate the ddG values between the two states. Please could you help me out here?
Using the Jasco thermal denaturation analysis software, I could determine Tm, dH and dS. Each of these parameters had their respective standard deviation values. Normally, at T=Tm, then dG=0. But after directly entering my determined Tm, dH and dS values into the equation (dG = dH-TdS), I did not get a zero value for dG. I think this is because I have to consider their standard error values too? Could someone please give me a recommendation on how to solve this problem? I would be glad to receive some tips.
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