Hi! So i attempted to understand a novel protein behavior towards heat application by analyzing its secondary structure change. I subjected the protein to a thermal denaturation analysis using circular dichroism and now I have a problem in interpreting the result. As the heat is applied, progressively most of my alpha helix and coil structure shifted to beta sheet. Is this possible? Why does my coil structure resorted to a more stable structure when heat is applied? Is it not supposed to be the other way around? I could relate it to the fact that my active site is potentially in the coil form hence the structure refolded to beta sheet is actually a bad thing. But i am confuse on its possibility. Hopefully someone can provide me with some insight on why does this happen. Fyi my protein is not thermophilic, theoretically and proven in conducted lab test.
Dear Fatin Safiudin This is not unusual. See for example
Article Effect of Heat Treatment on the Property, Structure, and Agg...
Upon denaturation the protein (tertiary) structure becomes more open and, in some cases, (when present) hydrophobic (possibly helical) regions become surface exposed. These hydrophobic regions ‘stick’ together, in other words aggregate, and this often is shown in terms of beta-sheet formation.Best regards.
PS. There is a fairly easy method to check whether your protein contains hydrophobic regions: Article New User-Friendly Approach to Obtain an Eisenberg Plot and I...
How did you performed thermal denaturation? Did you use one specific wavelength to do CD? Higher temperature leads to denaturation, loss of secondary structures and if aggregated, they are mostly Beta-sheets.
I think Zeyaul is on the right track. Isn't CD more of a qualitative technique? In other words, you can't tell the amount of beta sheets in your sample only the percentage. So, it might be what you are observing is your relatively less heat stable helices unfolding and only beta sheets remaining.
Good luck!
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