Hello all,
I am interested in studying the interaction between a peptide that forms a trimer and a target protein by ITC. The peptide protein interaction is dependent on the peptide being a trimer. I am looking for some precedence in the literature and cannot find any. I am concerned that there are two competing interactions 1. the equilibrium between monomeric and trimeric peptide. 2. The interaction between trimer and target protein. I am wondering if anyone has heard of or is familiar with studying this type of interaction by ITC? I am relatively new at ITC and am wondering if anyone thinks this kind of assay would work?
What is the value for the monomer-trimer equilibrium constant for the peptide?
You just have to perform the experiment and see what happens. In an ITC experiment there are many potential factors affecting the output.
If you do not care about the monomer-trimer equilibrium for the peptide, you will obtain "apparent" binding parameters. If you know about the equilibrium constant for the trimerization of the peptide (and the associated enthalpy), you may refine the data analysis by coupling that equilibrium with the peptide-protein interaction, in order to obtain "intrinsic" binding parameters.
Beware that if you do a direct (peptide into protein) or a reverse (protein into peptide) titration, the influence of the peptide monomer-trimer equilibrium will be different.
In addition, you can perform titrations along reactants concentration ranges, so you will shift the equilibrium towards the trimer or the monomer.
I did not use the trimer before but I used DNA tandom. In my opinion, because ITC measurement does not have limitation of molecules size, so the trimer or monomer does not mater as long as it is soluble and stable during your experiment. However it may have many binding site so you just look for proper fitting model, then everything is ok. If possible, please look at this paper to compare your protein size with reported protein . "Falconer, R.J., Penkova, A., Jelesarov, I., & Collins, B.M. ‘Survey of the year 2008: applications of isothermal titration calorimetry’ J. Mol. Rec. 23: 395-413 (2010)"
There is no principal limitation. You may have to consider what other equilibria are there and the data may be more complex (due to coupled reactions).
This article describes a similar scenario, however, TNF alpha homotrimers are very stable.
Article Recognition of human tumor necrosis factor α (TNF-α) by ther...
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