Hi,
I have got CD results for my protein. However I do not know how to interpret the data for CD. I have uploaded my results in word format. Kindly download to take a look at the results.
I understand that each of the three structure of a protein gives a characteristic CD Spectrum. Based on my CD spectrum results (in Figure 1), what I can interpret is that my protein is predominantly in the form of alpha-helices (Correct me if I am wrong). However, what confuses me here is that the table (Table 1 in the word file ) that shows my protein consists of almost ~47% of Random coil?
Does the presence of high random coil indicate that that my protein is unfolded?
Awaiting for your help.
Thank you.
Biochimica et Biophysica Acta 1751 (2005) 119 – 139 is a good reference for most of your basic protein CD questions
Perhaps I misunderstand what you have reported. Something does not look right. You should double check your calculation for the ellipticity. The reported y-axis values do not seem realistic.
[theta] = (theta x 10^6)/(c x l x n)
Units: [theta] = mean residue ellipticity = degrees cm^2 decimol^-1
theta = instrument dichroism in millidegrees
c = protein concentration in micromolar
l = pathlength in mm
n = number of backbone peptide units
Dear Saranpal Chhabra,
how did you derive the values in the table? They do not seem to be correct. As you already stated, the CD spectrum looks like you have a lot of alpha helix and only little random coil.
Usually the programs which derive secondary structure from CD-data (like K2D3, http://k2d3.ogic.ca/) expect mean residue ellipticity (mdeg cm^2 dmol^-1) as an input (as Kurt Berndt discribed). You have to convert the ellipticity values from your instrument accordingly. Maybe you can also check the tutorial on the K2D3 web-page for detailed information.
I advice you to also run a sequence-based secondary structure prediction algorithm like jpred or psipred. They are quite reliable and you can compare the results with what you get from your CD-analysis. Predict protein (https://www.predictprotein.org) is a nice webserver which can give you valuable information on your protein based on the primary sequence.
Best regards,
Helge
i find very usefull the use of dicoprot
https://dicroprot-prabi.ibcp.fr/
a free available software that allow the integration and analisis of your spectra
Almost all have been said that you must know about the analysis of CD spectrum of a protein. I hope, by now, you would have been able to know the contents of secondary structure of your protein. (I hope you must have gone through the literature suggested to you.) I would like to add the following that one must consider, 1, Does your protein contain any prosthetic group that contributes to the observed CD of the protein? 2. Have you subtracted contribution of the blank solution from the observed CD of the protein? Is the signal to noise ratio at each wavelength in the recommended range?
In the end I must say that the far-UV CD is an excellent technique to monitor change in the overall peptide backbone orientation. However, it MAY NOT be that good in the qualitative analysis of secondary structure of protein due to obvious reasons.
Best wishes,
Faizan
Beyond the excellent tips that you received, please be aware that what you call random coil / unfolded protein could also be due to an intrinsically disordered region. The minimum at about 205 nm and the fact that the spectrum rises towards a maximum, over 190 nm make one think. An analysis of the composition of AA regions in the sequence will clarify this aspect.
Hi,
please help me to analyse my CD data. I want to see the effect of quencher binding to my protein. I have calculated the alpha helix value from MRE and i found the content of alpha helix decrease but at higher conc. of quencher i got the -ve value. So what -ve value actually say (from 23% to -3%).
Pritee, Before I give you any explanation for your observation, let me know how you calculate alpha helix. Secondly, it seems to me that you have measured the far-uv CD spectrum of your protein in the presence of different concentrations of the quencher. If it is so, plot CD222 as function of the quencher to see whether the plot is sigmoidal.
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