We are working with a protein to induce fibrillation forms. Our FTIR (deep peak at 1624 qnd 1695) results and AFM images (twisted long fiberils) confirmed fibril formation. In spite of these results, CD study indicated a sharp negative peak at 224 versus 218. I cannot find any report for beta sheet CD spectum in this region. Nobody knows why it was happening.
Hi Dina,
I subscribe Karim and Jogender. ThT and Congo Red are good additional tests. Have you tested the kinetics of your sample aggregation.
I am sure that you have fibers, with beta sheet, as AFM and FTIR suggest. And they will certainly deposit. In FTIR ATR (I am not sure if that is what you are doing), all this will give a signal, as in AFM. Actually, the bigger and heavier and more deposited material that really sinks and sticks toi the bottom will give a good signal there,
In CD you may indeed be able to see some other structural details as Karim suggested. And scattering can be a issue as well (you will also see your signal very noisy in that case). I would add that in Cd you are most likely seeing the more soluble material that keeps floating. These would be smaller more soluble fibrils and protofibrils and perhaps intermediate structures and oligomers. These intermediate oligomers may pass through a stage rich in alpha-helical structure (see work by dobson CM for example and many others). If you have two mixed populations you may let it age longer and/or, centrifuge to see the supernatant in FTIR/CD/AFM. You may see a decrease of signal in all techniques but you may be able to better capture the alpha-helical structure of those intermediates.
Alternatively, from the concentration of your sample and number of residues, you can calculate how much signal in mear residue molar elipticity you ought to more or less expect from your samples in CDE. If you are having a lot less it is possible that it is most of it sinking. Maybe you can agitate while/before doing the CD measurement?
Light scattering studies of the kinetics of your sample aggregation might also be very informative.
Kind regards,
Ivo
Hi Dina,
it might be that you see a shift in the spectrum (i.e. your neg. peak) due to a superhelical structure of your beta-sheets and as the spectrum results out of a superposition of multiple spectra it might interfere with the reading, which is something you probably won't see in an FTIR spectrum. Also the CD spectra for alpha-helical structures have a negative peak at ~ 224 nm, which might be an indication for a helical structure.
You might also want to consider using Thioflavin T as an additional characterisation method of your fibrill formation, which is a nice fluorescent read out method as well.
Hope that helps.
Cheers,
Karim
Thank you for your response. We have tested ThT fluorescence assay . The intensity was high, and also red shifting in Congo red assay. Only thing that was unusual was Cd spectrum. I do not know about super helix in beta strand . Is it any reference about it?
Hi Dina,
below here are two references for superhelical fibril structures. I would also recommend the research of Roland Winter from the TU Dortmund University as he is an expert in fibril formation.
http://www.sciencedirect.com/science/article/pii/S0006349505732008
http://pubs.acs.org/doi/pdf/10.1021/ja000645zhttp://pubs.acs.org/doi/pdf/10.1021/ja000645z
Best wishes,
Karim
Hi Dina,
Do the fibrils form turbid solution? Are the fibrils insoluble?
If that is the case then there will be scattering of light in CD and the signal you will get would not be reliable. Also in that case the CD signal will be less.
Since all other probes show the presence of amyloid fibrils, the unusual CD spectrum is most likely because of the insolubility of the fibrils. Long twisted fibrils, as you observe by AFM, are generally insoluble.
Best,
Jogender
Hi,
I agree with Jogender, but I've done some CD experiments on fibril formation myself and didn't have any problems with insolubility. It is definitely worth looking into it but might not be the only reason.
Best,
Karim
Is your fibril formation n process completed, or is it possible that you may have some amount of native protein left in solution? Let's say 10% of monomer could really make clear impact on CD spectrum, while stay almost invisible in FTIR.
Hi Dina,
I subscribe Karim and Jogender. ThT and Congo Red are good additional tests. Have you tested the kinetics of your sample aggregation.
I am sure that you have fibers, with beta sheet, as AFM and FTIR suggest. And they will certainly deposit. In FTIR ATR (I am not sure if that is what you are doing), all this will give a signal, as in AFM. Actually, the bigger and heavier and more deposited material that really sinks and sticks toi the bottom will give a good signal there,
In CD you may indeed be able to see some other structural details as Karim suggested. And scattering can be a issue as well (you will also see your signal very noisy in that case). I would add that in Cd you are most likely seeing the more soluble material that keeps floating. These would be smaller more soluble fibrils and protofibrils and perhaps intermediate structures and oligomers. These intermediate oligomers may pass through a stage rich in alpha-helical structure (see work by dobson CM for example and many others). If you have two mixed populations you may let it age longer and/or, centrifuge to see the supernatant in FTIR/CD/AFM. You may see a decrease of signal in all techniques but you may be able to better capture the alpha-helical structure of those intermediates.
Alternatively, from the concentration of your sample and number of residues, you can calculate how much signal in mear residue molar elipticity you ought to more or less expect from your samples in CDE. If you are having a lot less it is possible that it is most of it sinking. Maybe you can agitate while/before doing the CD measurement?
Light scattering studies of the kinetics of your sample aggregation might also be very informative.
Kind regards,
Ivo
Thank you for your all helping comments. The CD spectrum of monomer also had just one negative peak at 215 nm. Yes, our sample was turbid but before CD assay, we centrifuged it, and the supernatant was examined. However, the signal was very strong and sharp with no more negative peak at 208 nm, just at 224nm. As I said before the AFM images showed thick fibrils. I had had also experiment with several known proteins such as ASN, Lys and Ins , and also assessed their CD after 1 week but none of their spectra were very far from 218 nm( Ins only a bit at lower wavelength 216 nm).
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