Hi! I´m trying to analyze results from a competition binding assay to assess the possible heterodimerization of GPCRs. As far as I know, the best way to fit the data is using the two-state dimer receptor model equation. But there are some variables in it that I'm doubting, like Rt (total amount of dimers) and KDab (hybrid dissociation constants when B is bound to a dimer semi-occupied by A). Does anybody know how to obtain these values?
Yes, quite familiar.
Using non-linear regression Rt is obtained. The program requires a starting value that would be half the estimation of Bmax.
Concerning the KDab I would like to ask whether you have already fitted data using only A in a saturation experiment to obtain KDa1 and KDa2.
I would suggest that you do some reading on the subject, here are two papers of which the first is about GPCRs:
54. Bywater RP, Sørensen A, Røgen P, Hjorth PG. J Theor Biol. 2002 Sep 21;218(2):139-47. Erratum in: J Theor Biol. 2003 Apr 21;221(4):669. Construction of the simplest model to explain complex receptor activation kinetics.
73. Bywater RP. (2013) Protein folding: a problem with multiple solutions. J Biomol Struct Dyn 31(4) 351-362.
If competing compound is B the parameters from fitting data to the two-state-dimer-derived equation are: Rt, KDB1, KDB2 and KDab. You may try to fit first assuming no "hybrid" effects, i.e. Kdab may be fixed to 2 x Kdb1
(Kdab= 2 Kdb1)
Kda1 and Kda2 shold be known from a previous experiment. Ideally A binding should be "simple" monophasic non-cooperative
Yes, the saturation experiments are already done with a non-cooperative behaviour neither in the saturation nor in the competitive binding experiments. So taking this into account, can i assume value of 1 for KDa2? Asuming that this variable represents a second binding site in a receptor like the dopamine ones.
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