In the case of some interactions a ligand of interest, A, could have more than one binding site to its target, B. In our NanoTemper Technologies software, designed to analyze Monolith data, we provide two fit models that could be used to describe the interaction – the Kd model from the law mass action and EC₅₀ model from the Hill equation (Kd vs EC50 – NanoTemper Technologies).

If several molecules of ligand A bind to target B but all binding sites have equal affinities and are independent of one another, then the Kd fit model from the law of mass action should be applied. For a situation where multiple ligands (A) interact with target (B) but their interaction is dependent (cooperative) the Hill model should be applied.

Some binding interactions also yield two transitions, or a biphasic binding curve. This biphasic behavior suggests binding sites with two different affinities with the resulting curve being a superposition of the individual curves. Various shapes are possible, depending on the shape and direction of the individual curves, an example of such curve is shown in Figure 1.

Figure 1 Example of the biphasic binding curve

One possible cause of biphasic binding curves is the target having more than one binding site for the ligand. In this case, at low ligand concentrations, the higher affinity binding site is occupied first, and at higher ligand concentrations, the lower affinity binding site follows. Both binding events change the MST signal of the monitored target and will show up separately in the data. Another possibility is ligand dimerization. The exact outcome of the situation depends on the dimerization affinity and the affinity of the interaction under investigation. It is possible that at low ligand concentrations, the ligand is present as a monomer. The ligand binding to the target will of course change the target’s MST signal and manifest as a binding event in the binding curve. At higher concentrations the ligand may then dimerize, causing another change in the MST signal of the monitored target which is again reflected in the binding curve.

In both cases, the way to proceed is the same. If the affinities of the different binding events are very close together, it can be difficult to analyze them individually. If they are separated by approximately two orders of magnitude, they can usually be discriminately analyzed. In order to do this, treat them as two separate datasets by removing all points that do not belong to the binding event of interest and fit a curve. Repeat this for each binding event. Since this can mean having only a few data points per binding event, it can be helpful to extend the curve by repeating the experiment and extending the ligand dilution series to lower and/or higher ligand concentrations for full coverage. 

It is important to note that such a biphasic binding response should be verified to ensure specificity of all events. On rare instances, small initial fluorescence changes (below 20%) together with Kd rapidly adapting to the temperature changes (highly enthalpic interactions) can lead to a transition of the bound phase during the measurement. The result of such action is also a biphasic curve, also demonstrated in Figure 1.

As a solution to such cases, we recommend adapting the labeling strategy. Both factors – change of the initial fluorescence as well as Kd shift - need to occur together for the biphasic curve to appear. As the environment of the dye is largely affected by its fluorescence properties, changing the position of the label on the target or switching the type of a dye could prevent biphasic behavior of the binding response. Also, inverting the labeling by coupling the label to the ligand instead of the target provides an alternative solution. NanoTemper Technologies offers protein labeling kits with different coupling chemistries (Target labeling tutorial – NanoTemper Technologies).

In cases where changing the labeling strategy is not possible but a clear phase transition can be observed throughout the different MST-on times, analysis by selecting very early MST-on time and lower MST power can be sufficient to prevent the biphasic behavior.