Hi everyone,
I am working on CD thermal melts for a protein high in alpha-helical content. At high temperatures, I see the loss of one alpha-helical dip at 208 nm (image attached). I would like to know whether this indicates protein unfolding, and I see the CD signal of a random coil somehow overlapped with that of an alpha helix, or whether I see some structural rearrangement where protein 'gains beta sheet content' ?
I would also like to know the significance of the 2 alpha helical dips at 208 nm and 222 nm, and how the pi to pi* and n to pi* transitions can be explained in terms of loss of protein structure.
Thanks in advance!
Dear Radha Somarathne ,
Have a look at https://bitesizebio.com/59809/circular-dichroism-sample-preparation/ I would plot your data as indicated in this link in Figure 2C as well. It looks to me that your protein is remarkably temperature resistant (till round 60 oC there is hardly any change).
Like in the above link (with the example given in Figure 2) I tend to say that you are looking at a (slow) unfolding of your protein from a primarily helical towards a (partly) random coil transition. I would suggest (if not already known) to perform a secondary structure prediction analysis. There are numerous programs for this, see for some suggestions (and how to perform this) for example:Article The role and significance of potential lipid-binding regions...
Be careful with trying to quantify data based on circular dichroism. There are numerous discussions here on RG to what extent deconvolute circular dichroism is accurate, see for example: https://www.researchgate.net/post/Circular_dichroism_deconcolution_problemhttps://www.researchgate.net/post/Can-anyone-suggest-some-software-to-calculate-the-percentage-of-alpha-helix-and-beta-sheet-from-circular-dichroism-data
But it is safer and more significant to use circular dichroism in qualitive terms.
Best regards.
PS. Though the link I provided shows a simplified image in Figure 1 it is a bit outdated. The random coil is nowadays interpreted differently and looks somewhat different, see for example:
Chapter Characterization of helical structures in gelatin networks a...
Chapter Characterization of helical junction zones in gelatin networ...
Thank you Rob Keller for your very informative and helpful answer!
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