Dear all,
I have been trying to measure the least concentration of a nitroxide spin labelled peptide probe to use in paramagnetic relaxation enhancement based NMR experiment. In an PRE experiment where protein concentration was 100uM and the probe was 125uM and we saw lots of perturbation along with PRE effects (though disappered peaks in active spin state did not appear upon spin reduction; we exciuded them and measured peak intensity of other peaks). We tried a control experiment with the untagged vrsion of the spin-labelld peptide. There we had titrated the protein (30uM) with the untagged peptide and also saw 10 peaks completely broadened and some other 50% broadened. It seems intermediate exchange.
How I can design the control for such experiments?
From what you are observing it seems like your protein binds the PRE probe. Why don't you try to add the probe to wild-type protein and see what happens (I am assuming you mutated a residue to cys to attach the probe)? This bring up also another question, is the residue binding the probe on the surface of your protein? please get back if you need more info.
One thing I'd recommend doing first is continue adding your unlabeled peptide and see if the signals remain broadened. The exchange rate is modulated by the free ligand concentration, so if you continue increasing it you may go into the fast exchange regime and the peaks may return. You could also try changing the temperature of the experiment to modulate the exchange rate. Otherwise, run a control with the same unlabeled peptide concentration as you used for your labelled peptide in the PRE experiment and hope there are peaks that disappear in the PRE experiment that don't disappear in the control experiment.
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