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. 2020 Sep 29;10:15938. doi: 10.1038/s41598-020-72854-2

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Investigation of the potential binding of UDP-GlcNAc to YvcJ by partial proteolysis and Thermal Shift Assay (TSA). (A) Coomassie-stained SDS-PAGE of YvcJ partial proteolysis profile. YvcJ was incubated with endoproteinase Glu-C (Promega) in the presence or in the absence of 1 mM UDP-GlcNAc for 0, 5, 10 or 20 min at 37 °C. The digestion profiles were assessed by electrophoresis in 12.5% SDS-PAGE. Full-length gel is presented in Fig. S8. (B) TSA in the presence of increasing concentrations of UDP-GlcNAc. YvcJ (top) and YvcJ(K22A) (bottom) melting profiles were monitored in the presence of increasing concentration of UDP-GlcNAc (0–1 mM). One curve corresponds to data obtained for one concentration of UDP-GlcNAc. The melting temperature of the protein (T_m) is obtained at the midpoint of each melting curve and corresponds to the minimum of the negative derivative curves. The T_m is an indicator of protein stability and is increased by the addition of UDP-GlcNAc. For the WT protein, T_m = 48 °C in the absence of UDP-GlcNAc and T_m = 58.5 °C in the presence of 1 mM UDP-GlcNAc. For YvcJ(K22A), T_m = 49.5 °C and is not increased by addition of UDP-GlcNAc. (C) TSA results for the binding of UDP-GlcNAc to YvcJ. Assays were performed with YvcJ and YvcJ(K22A) in the presence of increasing concentrations of ligands (0–0.7 mM). The difference of temperature (the shift of T_m induced by the presence of ligand) was plotted against the concentration of UDP-GlcNAc. Each experiment was reproduced at least in triplicate and the standard deviations are represented by the error bars. Curve fitting was performed by using Microcal Origin 5.0 software (Microcal software Inc).