Application Notes

1 Label-free Protein-Sugar Interaction Analysis Application Note NT-MO-028 Interaction of maltose binding protein (MBP) with maltose in a truly label-free assay Beate Kern 1 , Amit J. Gupta 1 , Tanja Bartoschik 1 1 NanoTemper Technologies GmbH, Munich, Germany Abstract Protein-sugar interactions are essential for nutrient transport and metabolism in all types of cells. Here, we investigate the interaction of maltose binding protein (MBP) and its substrate, maltose, drawing upon label-free MicroScale Thermophoresis (MST) as the ideal technique. We also show that sample viscosity did not affect the binding assay, and discuss how MST is not limited by sample viscosity. Introduction MicroScale Thermophoresis (MST) is a powerful technique to study biomolecular interactions in solution. MST makes use of thermophoresis, the phenomenon of molecules migrating in a solution when a temperature gradient is applied. Since thermophoretic behaviour is specific to the molecule in question, it will change upon the binding of a ligand, and this allows the extraction of binding affinity information from observations of a molecule's thermophoresis. For an MST experiment, a non-fluorescent ligand is titrated against a fluorescent target, and changes in thermophoresis of the target are monitored over a range of ligand concentrations [1, 2]. For experiments performed with the Monolith NT.LabelFree, fluorescence is detected via intrinsic tryptophan fluorescence. Thus, the label- free approach allows the investigation of proteins that are difficult or even impossible to label. A common example of interactions analyzed in this context are protein-sugar systems like the one presented here. Maltose binding protein (MBP) is part of the periplasmic transport system of Escherichia coli and involved in the transport of maltose into the bacterium. It binds the disaccharide once it has crossed the outer membrane, and then assists its translocation across the inner membrane [3]. Additionally, it is often used as a fusion tag for protein purification or solubilization [4]. The interaction between MBP and maltose is well- characterized [5, 6] and will serve as an example for protein-sugar interactions that can conveniently be measured label-free. Figure 1: Structural representation of maltose binding protein (MBP). MBP undergoes a conformational change upon binding maltose (colored in red), changing from the unbound (cyan) to the bound conformation (blue). (PDB 1OMP and 1ANF [7, 8]). Structural alignment and rendering was performed with Chimera.

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