I am working with several compounds that are expected to have a Kd of ~ 20 uM. They are also poorly soluble in aqueous solutions so it is not possible to titrate high concentration of compound into a reasonable concentration of protein. The protein can be concentrated to 1 mM so it possible to perform a reverse titration. Would this set up be valid for low affinity compounds or it is always necessary to titrate concentrated compound into the protein?
Any other suggestions on how to deal with such scenarios would be greatly appreciated.
thanks!
I actually done this reverse, using Nanotemper to get my Kd. Thus my target protein became my ligand, and the ligand is now my target.
In the Nanotemper reverse analysis, I still got my sigmoid Kd curve, but "in reverse". What's more important is that you get your sigmoid plateaus saturated with high concentration of your ligand to produce an almost flat line, thus giving you an accurate Kd. Since Kd corresponds to the ligand molar concentration that is needed to occupy "half" of the target binding sites for the ligand, thus it doesn't matter if you get a reverse sigmoid curve, since you are looking for the half way point of either normal or reversed curve. I haven"t worked with ITC but I presume you should be able to do something similar.
Contrary to proteins having more than one ligand binding site, when a proteins have a single ligand binding site (1:1 stoichiometry), performing a direct titration or a reverse titration is equivalent, unless there are specific phenomena such as monomer-oligomer equilibrium, tendency to aggregate, low solubility, etc. in any of the reactants.
Very often, depending on the solubility and the availability of a given reactant, the experimental setup (direct or reverse titration) is designed according to that constraint. Molecular weight is not a required factor to be considered.
For 1:1 stoichiometry, the experimental binding isotherm obtained from the titration and the data analysis procedure is the same for direct and reverse titrations. For 1:n stoichiometry, the experimental binding isotherm may be quite different, and the data analysis procedure (mainly, the fitting function for the interaction model) is different, for direct and reverse titrations.
Anyway, I would not say that a Kd of 20 micromolar corresponds to low affinity; it would be moderate affinity. And the value of c can be modulated by using larger cell concentrations.
When dealing with low c-values in ITC, please check the published work from Bruce Turnbull and Joel Tellinghuisen.
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