Why do MST traces cross over?

In MST measurements, the change in fluorescence intensity of a fluorescent interaction partner is recorded as a function of temperature as well as concentration of its associated ligand. In a typical MST experiment, the fluorescence intensity of the labeled target molecule decreases upon local sample heating introduced by a precise IR-laser. Two effects, TRIC (Temperature Dependent Intensity Change) and thermophoresis, contribute to the overall fluorescence variation. See Figure 1.

Figure 1: In a typical MST/TRIC experiment, the fluorescence intensity decreases over time due to sample heating with an IR-laser. The extent of fluorescence change is different for the bound and unbound state of the monitored target molecule.

TRIC and MST can result in either the bound or unbound state having a greater F_norm response, i.e. your experiment may look inverted compared to Figure 1. It is also possible to have an increase in F_norm in response to the temperature increase. In these cases, it is possible to see the cross-over events described in this FAQ.

Specific cases of MST trace cross-over

In some cases, prolonged heating of the sample (especially at high MST-Power) may lead to structural destabilization of biomolecules, thus altering the chemical environment of the fluorophore. Ultimately, this can affect the temperature-dependent variation of the fluorescence intensity, or in other words the MST trace. Binding of a ligand to the fluorescent target may stabilize or destabilize the target molecule and thus directly influence the “shape” of the MST traces. In the given example, the fluorescence signal of the bound target molecule starts to raise after the initial fluorescence decrease, potentially indicating a destabilization of the bound target molecule resulting in an altered chemical environment of the attached dye. See Figure 2.

Figure 2: In some cases, the fluorescence intensity can raise again after the initial decrease, due to structural destabilization of the target molecule and thus changes to the local microenvironment of the attached dye.

In other examples, MST traces of bound and unbound target molecule initially behave similar, but the traces start to separate after prolonged heating (initially the fluorescence signal drops but after some time the fluorescence intensity increases again). See Figure 3.

Figure 3: In some cases, the fluorescence decrease of bound and unbound target molecule can behave very similarly during the first seconds of the measurement. Structural destabilization of the target molecule by prolonged heating can lead to an increasing fluorescence signal due to changes to the local microenvironment of the fluorescent dye.

Finally, it is also possible that MST traces cross during an experiment, e.g. if the bound state of the target shows a stronger initial temperature dependency than the unbound state (faster initial decrease in fluorescence intensity). Due to structural changes in the bound target molecule, induced by prolonged sample heating, the microenvironment of the fluorophore may change during the measurement and thereby affect the fluorescence signal in an opposite direction. See Figure 4.

Figure 4: MST traces may cross if one state of the target molecule (bound or unbound) shows a stronger initial fluorescence decrease which is counteracted by structural changes upon prolonged heating.

Generally, MST experiments should be evaluated after the shortest possible MST on-time yielding a sufficient signal-to-noise ratio to evaluate the binding event. Still, in rare cases thermal destabilization of the target molecule might be necessary to monitor binding events. MO.Control software automatically computes the best evaluation time for your MST experiments.