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3 Peptide Sequence K d (MST) K d (Fluorescence anisotropy) Acceptor 5-FAM- GSDQNATF 0.42 +/- 0.17 µM 1.02 +/- 0.06 µM Inhibitor 5-FAM- GSDQ(Dab)ATF 3.80 +/- 0.2 µM 10.15 +/- 0.25 µM Table 1: Comparison of the K d values determined for the interactions of PglB with different peptide, determined by using MicroScale Thermophoresis and Fluorescence anisotropy. Material and Methods Labeled peptides: Synthetic peptides, fluorescently labeled with 5-Carboxyfluorescein, were reported by Gerber, Lizak [5]. Protein Preparation: PglB carrying a C-terminal decahistidine tag, was purified as previously described by Lizak, Gerber [2]. The protein was purified in desalting buffer (10 mM MES pH 6.5, 100 mM NaCl, 3 % glycerol, 0.016 % DDM) and concentrated to 35 µM in an Amicon Ultra-15 concentrator (Millipore) with a molecular mass cutoff of 100 kDa. Binding of labeled peptides to PglB: The concentrations of labeled peptide and MnCl 2 were kept constant at 50 nM and 10 mM respectively. For the interaction analysis of PglB with the peptides, PglB was titrated in 1:1 dilutions. The highest concentration of PglB was about 20-fold above the expected K d for the interaction PglB- acceptor peptide, which was previously determined as 1 µM by fluorescence anisotropy [3]. The samples were incubated for 10 min at 4 °C, and then filled into standard treated capillaries (Cat# K002, NanoTemper Technologies, Germany). Instrumentation: The measurements were performed in a NanoTemper Monolith NT.115 instrument, at 30 % LED power and 90 % MST power. References 1. Aebi, M., N-linked protein glycosylation in the ER. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2013. 1833(11): p. 2430-2437. 2. Lizak, C., et al., X-ray structure of a bacterial oligosaccharyltransferase. Nature, 2011. 474(7351): p. 350-5. 3. Lizak, C., et al., Unexpected reactivity and mechanism of carboxamide activation in bacterial N-linked protein glycosylation. Nat Commun, 2013. 4: p. 2627. 4. Bause, E., W. Breuer, and S. Peters, Investigation of the active site of oligosaccharyltransferase from pig liver using synthetic tripeptides as tools. Biochem J, 1995. 312 ( Pt 3): p. 979-85. 5. Gerber, S., et al., Mechanism of bacterial oligosaccharyltransferase: in vitro quantification of sequon binding and catalysis. J Biol Chem, 2013. 288(13): p. 8849-61. © 2014 NanoTemper Technologies GmbH