I want to study conformational / structural change in the protein after ATP binding ( I am working with a yeast protein that shows ATPase activity) so I want to see and compare the binding effect of ATP with wt protein and its mutant. ( I want to do FAR-UV CD)
Any suggestion or experience in this kind of experiment, please share..?
Far UV CD will reveal changes in secondary structure. Although such changes are possible, in most cases, any conformational changes caused by binding of ATP may not include secondary structure changes, and may therefore cause no change in the far UV CD spectrum. You should also try near UV CD, which can reveal tertiary structure changes as a result of the altered environment of aromatic amino acid residues, especially tryptophan. Another experiment along the same lines is tryptophan fluorescence spectroscopy.
Since ATP itself has UV absorbance, you will have to be careful not to cause too high an absorbance for the CD spectrometer.
Dear Shapiro
Thanks for your answer.
I have done the Far UV CD studies of Wt and two mutants (M1 & M2). I am attaching here a snap shot, Lower panel (only protein). upper panel protein bound to ATP.
Please suggest what is happening here?
Thanks for your time.
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