I noticed only one broad peak around 222 for the Far-UV spectra of my protein. It containes mostly alpha-helics but the 208 peak is missing. I have tested CD for both MBP-fusion protein and the 80%-purified protein. But still the same pattern. When I tested MBP alone I got a normal alpha-helics spectra though!
Well, proteins with well-defined antiparallel β-pleated sheets (β-helices) have negative bands at 218 nm (you said around 222 so could be 218?) and positive bands at 195 nm, Is it the case? may be not alpha helices then. I recommend you to read:
Greenfield N, Fasman GD. Computed circular dichroism spectra for the evaluation of protein conformation. Biochemistry. 1969;8:4108–4116. and
Greenfield N, Nat Protoc. 2006; 1(6): 2876–2890.
Thanks for the answer Brigitte! Yes, it can be 218 also since the peak is really broad, I can not see any positive band at 195 though. The thing is I am quite sure about the alpha-helical structure of my protein and I have references showing the far-uv spectra for this protein with a normal alpha helics pattern! So I doubt if the signal belongs to beta structures!
Is ur protein is aggregated?
I have found similar observation in my own study. We speculate that it could be a cross bita-sheet signals.
Without looking at the spectrum I cant be sure, but I agree with Javed and Brigette that the program is likely aggregated and a mixture of random coil/beta sheet. A conformational change is not that uncommon during aggregation. Is there any visible sediment if you spin the protein down? Do you have access to a size exclusion column or or dynamic light scattering instrument? Is there an enzymatic activity for the protein? A DOSY NMR experiment can also tell you the exact size.
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