Application Notes

3 experiments. Thus, the control experiments confirm that the change in MBP thermophoresis with increasing concentrations of maltose is indeed caused by the interaction of the two molecules. Figure 5: High-concentration titration of maltose against lysozyme (blue). Even for maltose concentrations as high as 500 mM, no viscosity artefacts were observed in the thermophoresis of lysozyme. Thermophoretic traces are shown in the inlet. Thermophoresis and dose-response of MBP are shown as a reference (red, see Figure 3). Conclusions In this study, we show that label-free MST is the perfect method to analyze protein-sugar interactions. Intrinsic tryptophan fluorescence eliminates the need to use a fluorescent label, while non-fluorescent sugars are ideally suited as ligands for label-free MST experiments. To illustrate this, we investigated the well- characterized interaction of maltose binding protein (MBP) with its substrate maltose and derived an affinity of 3.3 µM, in good agreement with orthogonal techniques. The conformational change of MBP upon maltose binding leads to a large MST signal since it contributes significantly to the protein's thermophoretic behavior, even though the increase in mass upon binding is neglectable. Interactions without conformational changes can of course also be analyzed by MST. Any binding will induce alterations in the target molecule's surface area, charge, or hydration shell, which can readily be detected by MST. In addition, by performing appropriate negative control experiments, we could confirm that viscosity even at high maltose concentrations did not influence the binding assay. Larger carbohydrates, for example polysaccharides like dextran or cellulose, would have a more pronounced effect on sample viscosity. High viscosity in and of itself is not a problem for MST; any sample can be measured that can be loaded into capillaries, as long as the viscosity is constant in all capillaries. This includes very viscous samples like protein solutions high in glycerol. In case a ligand titration is causing a viscosity gradient, one of the following two control experiments can be applied to control for viscosity effects. Performing an experiment with a non- binding target, as done in this study, can verify specificity of the signal. Alternatively, a second, non-interacting ligand can be counter-titrated to keep viscosity constant in all capillaries. MST is therefore very well-suited to deal with sample viscosity, facilitating also the study of low-affinity interactions, where very high ligand concentrations are used. To sum up, we showed the sensitivity and convenience of MicroScale Thermophoresis when it comes to truly label-free experiments in a close- to-native environment. Additionally, MST is not limited by sample viscosity, further proving its versatility.

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