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Unread 02-14-2012, 03:00 AM
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Default HSQC perturbation

HSQC perturbation

Dear All,

I am trying to determine the interaction between 2 proteins. Is there any reference or literature which report the determination of binding constant between 2 proteins in which intensity drop of HSQC cross peaks can be used as the measuring parameter. As when I titrate these 2 proteins only change which I observe is intensity drop. I tried other biophysical techniques to determine binding constant, but results are not satisfactory.

Any suggestion would be useful

Regards

Arun



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  • I am assuming you tried ITC, but were unable to acquire satisfactory results?
  • If so, when you are titrating your unlabeled protein into the labeled sample, what molar concentration-fold do you typically see HSQC peak changes reach a maximum? That is, do you need 3-fold binding partner to elicit maximum decreases in HSQC peaks of the labeled protein? Or is binding tighter than that: sub-stoichiometric concentrations of unlabeled protein. That might give you a start/estimate of Kd...i.e. weak (mid to high uM) or tight binding in the low uM to high nM range.
  • I myself would be interested in literature that discusses quantitative binding constants derived from NMR titration experiments. Alas, I haven't seen any specific papers on the topic.
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