Hi there,
I'm currentlty investigating a protein which upon binding to its target induces some sort of concormational changes to it.
Therefore I collected CD spectra of the single proteins and their respective mixtures. By summing up both single spectra I almost get the exact spectrum as for the determined complex but with one minor difference between 195 and 200 nm.
And here is my question:
Could anyone explain what kind of conformational change or shift in secondary structure elements could be deduced between 195 and 200?
I already looked up several things and used servers like K2D3 but I did not find a conclusive answer to my problem.
Thanks in advance!
Is this difference reproducible? Establish that before spending a lot of effort trying to explain it. It might just be noise.
Hi there, alpha helices and beta sheets contribute positively to CD signal between 195 and 200nm. Does the signal of the complex get bigger than the sum of the 2 individual spectra? If so it means the complex increases the global structuration of the 2 objects. If the opposite, it means some structure melting occurs in the complex.
Unfortunately was I unable to do a second run that day. But will do so any time soon. The thing is I did measurements against to targets of my protein with similar structures and a similar effect could be observed in both runs (see attachments, unit: mdeg). Maybe that will help or make it clearer.
The spectra don't look right.
The change at low wavelength is most like come from noise. What is optical length you used? Water can generate huge noise at this range. So the maximal path should not over 1 mm. You can find 0.1 mm covette that greatly reduce water signal.
Another possibility is the ligand. What is your ligand? Most compounds can interfere CD at this wavelength.
DMSO can also cause trouble even at very low concentration.
If it is not reproducible, then it might be just noise due to DMSO assuming you have this in your buffer...
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