Hi,
I am currently analyzing a thermal denaturation of protein by CD, from the spectra, there are two peaks at around 222nm and 208nm. I know that for a helix, it should have a negative value near 222nm and 208nm and for b-sheet, it has a peak near 217nm.
So I collected data from 208nm and 222nm while increasing temperature from 20 degrees to 90 degrees. For most papers, they use the value at 220nm as CD signal(is it a good estimation for the total content of a-helix and b-sheet?)
When I analyze the curve, the value at 208nm shows a standard two-state model and value is close to 0 above 60 degrees. But for value at 222nm, it's still around -10(the initial value is -34) at 60 degrees and decreases very slowly.
I have used a fluorescence method to test the stability of my protein, the curve, and melting temperature are very similar to the curve at 208nm by CD.
Does that suggest that the protein lose a-helix while still has some b-sheet structure at 60 degrees? Thanks!
Hello Xinji Zhu, our group works with a class of protein that exhibits the same behaviour you observed: protein denaturation with loss of alpha-helical and "gain" of beta-sheet content. We relate this profile to the formation of Amyloid-fibrils due to the aggregation process on the proteins.
As Wieslaw Swietnicki and Rob Keller said, you can monitor the full spectrum of ellipticity during the denaturing process to ensure alpha/beta content changes through specific peaks and use chemical denaturation to access other states of this thermodynamic process.
If you want to fully understand the process of secondary structure changes using CD, I recommend you to use SR-CD (synchrotron-radiation CD) which can enhance the signal-to-noise ratio in the region below 200 nm, that is essential for characterizing the secondary structure content, allowing you to do PCA analysis, e.g. This technique also allows you to overcome buffer restrictions imposed by the CD technique.
In addition, I advise you to look at Thioflavin T assays, where you can monitor through a fluorimeter the interaction of this fluorophore to amyloid structures during the denaturation process. The formation of these amyloid structures is related to the presence of beta-sheet content and may be related to the secondary structure profile that you observed. There are several topics in the literature and here at Research Gate about this technique. We recently published a paper about this and this is available in the following link if you want to check:Article Correct partner makes the difference: Septin G-interface pla...
Good Luck!
You could check the shape of the (complete) curve at say 60 oC to confirm your assumption. In other words at 60 oC you should see a curve with higher beta-sheet content.
It is often shown that proteins/peptides tend to aggregate at higher temperature. So indeed it is possible that the protein loses alpha-helix and increase in beta-sheet content.
PS. Good thing to check, what you want to find out, by using two different approaches.
Hi Xinji,
Try doing it the right way. Plot the values and look at the plateau. The other option is to use chemical denaturation with GuHCl and monitoring 222 nm signal. You should know when the protein is unfolded fully.
The signal at 208 is too low to have a meaningful reading as in the case of 222 nm readouts. You may increase protein concentration and decrease cell thickness to reduce background but I doubt you will get a meaningful transition curve that way.
Hello Xinji Zhu, our group works with a class of protein that exhibits the same behaviour you observed: protein denaturation with loss of alpha-helical and "gain" of beta-sheet content. We relate this profile to the formation of Amyloid-fibrils due to the aggregation process on the proteins.
As Wieslaw Swietnicki and Rob Keller said, you can monitor the full spectrum of ellipticity during the denaturing process to ensure alpha/beta content changes through specific peaks and use chemical denaturation to access other states of this thermodynamic process.
If you want to fully understand the process of secondary structure changes using CD, I recommend you to use SR-CD (synchrotron-radiation CD) which can enhance the signal-to-noise ratio in the region below 200 nm, that is essential for characterizing the secondary structure content, allowing you to do PCA analysis, e.g. This technique also allows you to overcome buffer restrictions imposed by the CD technique.
In addition, I advise you to look at Thioflavin T assays, where you can monitor through a fluorimeter the interaction of this fluorophore to amyloid structures during the denaturation process. The formation of these amyloid structures is related to the presence of beta-sheet content and may be related to the secondary structure profile that you observed. There are several topics in the literature and here at Research Gate about this technique. We recently published a paper about this and this is available in the following link if you want to check:Article Correct partner makes the difference: Septin G-interface pla...
Good Luck!
Hello Xinji Zhu, Whether you have checked denatured states induced by heat and Chemicals (GdmCl or Urea) is same? In some cases denatured states contains significant amount of residual structure and that also cooperatively denatured on further unfolding. Also at 208 nm there is contribution from both alpha-helix and random colis but at 222 nm only from alpha-helix.
Also whether you normalise the concentration and check that mre value ( at 222 nm) close to reported value for denatured proteins.
Yes you are right.
At 222 nm, the dichroic extinction coefficients of the alpha helix are 2–3 times greater than that of the beta structure and an order of magnitude greater than that of the irregular structure. Therefore, the CD signal at 220 nm always has a negative sign.
At 208 nm, only the beta structure is characterized by a positive sign. If at 208 nm the CD signal is close to 0, this means almost complete destruction of the alpha helix and preservation of the beta structure аnd irregular structure. A positive beta signal is compensated for by a negative signal from an irregular structure.
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