Dear all,
I am working on thermal stability of proteins. I have done CD analysis of protein samples at varying temperatures within 190-240nm wavelength and recorded the CD data as molar elliptical values. I wish to find out the individual melting temperatures of each protein samples, subsequently, I wish to plot melting curves vs temperature.
Is there any online software to analyse the CD spectra data and calculate the melting temperature of the individual proteins?
Thanks
Hi Gaurav
Fraction of unfolded (denatured) protein can calculated, using the following equation
FU=(FO-F)/(UF-F)
where FU is fraction unfolded, Fo is the value of ellipticity (θ) at 222 or 208 nm at F is the average of first value or average of few values of ellipticity where protein is folded, UF is the average of last value or average of last few values of ellipticity where protein is unfolded. The graph was plotted for fraction unfolded (FU) against temperature. Tm can be calculated by fittig the value in Boltzmann sigmoid equation, which assumes a two state model. origin or prism software can be used for fitting. or if you wish you can just plot raw values of ellipticity or molar ellipticity vs temperature and use tha same fitting. both will give you same melting temperature. Hope this will help you
Thanks Mrityunjay. How do we determine the FU, Fo, F and UF from the molar ellipticity values? How do we know the folding state of proteins from CD spectra graph?
Hello Gaurav,
Easiest way to do it is to plot the molar ellipticy per residue at 222nm, which you can obtain from each spectrum, as function of temperature in Kelvin degrees (you can reconvert to celcius degrees later on for ilustration purposes). That way you can obtain the melting temperature and associated enthalpy change assuming a 2-state transition model.
Here you can find the equations and procedure:
https://www.researchgate.net/publication/266625837_Thermodynamics_of_Membrane_Insertion_and_Refolding_of_the_Diphtheria_Toxin_T-Domain
Article Thermodynamics of Membrane Insertion and Refolding of the Di...
Hi Gaurav,
U can take ellipticity values as it is. The ellipticity value where protein is folded that is at low temperature is taken as F and Fo is the value for ellipticity at each temperature. Uf is the value at which protein is unfolded that is at highest temperature. When you put this values in the formula i mentioned above you will get Fu. Please find attached excell with values and graph plotted. Hope this will help you.
I am facing same problem with the calculations can you kindly share an excel sheet for calculations .
Hello Kandluri Geethika and Gaurav Kumar I am facing the same problems and would appreciate some tips.
You can use CDPal software for plotting data. this is very fast and reliable. Please inbox me if you need its setup. I used this software in my PhD work
Mrityunjay Kumar Singh Would you mind sharing an excel sheet? I have a similar problem with fitting variable temperature CD data
Hi Cynthia,
I have attached the excel with this message. Hope that will help you.
Cynthia Bessem Nkemeto
Hi Mrityunjay Kumar Singh thanks a lot for your reply and for the excel sheet. This would help a lot. I just have some observations:
1) In the column "J", you have additional Fu (fraction unfolded) values, and the formular seems to be wrong (as there are no entries for column "H"). Could this simply be an error? Or did you want to normalize the Fu values?
2) After obtaining the Fraction unfolded, next step will be to analyze the equil constant and subsequently delta G and deltaH. The problem is, some K values will be negative, and so it is not possible to calculate ln(-K).
Furthermore, deltaG = lnK RT. Since K is a negative value, lnK can not be determined, and subsequently delta G. Please, How did you solve this issue? How did you determine deltaH as well?
Best,
Cynthia
Hi Cynthia Bessem Nkemeto, ya there was a mistake in pasting, as it is just a copy paste of column G. Fraction unfolded is in G column. you can plot this vs absolute temperature and can calculate delta G.
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