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4 Material and Methods MST assay conditions: The concentration of the intrinsically fluorescent protein was kept constant while the sugar was titrated. The final concentration of MBP in all capillaries was 250 nM, that of lysozyme was 1.25 µM. A 1:1 maltose dilution series was prepared, with the highest concentration being 2 mM (final maximum concentration in the experiment 1 mM) for the interaction experiment, and 1 M (final 500 mM) for the viscosity control experiment. Maltose was solved and the dilution series prepared in water. All dilutions of MBP were performed in MST-P buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 10 mM MgCl 2 , supplemented with 0.1 % Pluronic F-127). Lysozyme was diluted in an acetate buffer (20 mM sodium acetate pH 4.5). As a result, the final assay buffer was always a 1:1 mixture of water and protein buffer. For additional purification, a size exclusion chromatography column (Column B, NanoTemper Technologies Protein Labling Kit) was equilibrated with 9 ml protein buffer and protein was added to the column (total sample volume 500 µl). Elution was performed with 500 µl of protein buffer and protein concentration was determined spectroscopically. Instrumentation: All MST measurements were conducted on a NanoTemper Technologies Monolith NT.LabelFree instrument, at 20 % LED power and 40 % MST power. The capillaries used were Monolith NT.LabelFree Zero Background MST Premium Coated Capillaries (cat # MO-Z005) for MBP experiments and Monolith NT.LabelFree Zero Background Standard Treated Capillaries (cat # MO-Z002) for lysozyme experiments. Material: Maltose and (GlcNAc) 3 were purchased from Sigma-Aldrich (cat # M5885 and T2144 respectively). Lysozyme was taken from the Monolith NT.LabelFree Control Kit (cat # MO- C032), and the size exclusion Column B from NanoTemper Technologies protein labeling kits. Acknowledgement MBP was kindly provided by Susanna v. Gronau and Dr. Sabine Suppmann from the biochemistry core facility of the Max Planck Institute of Biochemistry, Martinsried. References 1. Seidel, S.A., et al., Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions. Methods, 2013. 59(3): p. 301-15. 2. Seidel, S.A., et al., Label-free microscale thermophoresis discriminates sites and affinity of protein-ligand binding. Angew Chem Int Ed Engl, 2012. 51(42): p. 10656- 9. 3. Nikaido, H., Maltose transport system of Escherichia coli: an ABC-type transporter. FEBS Lett, 1994. 346(1): p. 55-8. 4. Raran-Kurussi, S. and D.S. Waugh, The ability to enhance the solubility of its fusion partners is an intrinsic property of maltose- binding protein but their folding is either spontaneous or chaperone-mediated. PLoS One, 2012. 7(11): p. e49589. 5. Telmer, P.G. and B.H. Shilton, Insights into the conformational equilibria of maltose- binding protein by analysis of high affinity mutants. J Biol Chem, 2003. 278(36): p. 34555-67. 6. Vosyka, O., M. Molnar, and A.J. Gupta, Detection of binding-induced conformational changes by SAW. NanoTemper Technologies Application Note, 2015. NT-SE-001(NT-SE-001). 7. Quiocho, F.A., J.C. Spurlino, and L.E. Rodseth, Extensive features of tight oligosaccharide binding revealed in high- resolution structures of the maltodextrin transport/chemosensory receptor. Structure, 1997. 5(8): p. 997-1015. 8. Sharff, A.J., et al., Crystallographic evidence of a large ligand-induced hinge-twist motion between the two domains of the maltodextrin binding protein involved in active transport and chemotaxis. Biochemistry, 1992. 31(44): p. 10657-63. NanoTemper ® and Monolith ® are registered trademarks. © 2016 NanoTemper Technologies GmbH