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. 2017 Jun 23;7:4139. doi: 10.1038/s41598-017-04064-2

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© The Author(s) 2017

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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YvcK binds to UDP, UDP-Glc and UDP-GlcNAc. (A) Coomassie-stained SDS-PAGE of YvcK partial proteolysis profile. YvcK was incubated with Trypsin (Promega) in the absence or in the presence 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 Supplementary data. (B) Thermal Shift Assay (TSA) in the presence of increasing concentrations of UDP-GlcNAc. YvcK melting profiles were monitored in the presence of increasing concentration of UDP-GlcNAc (0 to 4 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 the each melting curve and corresponds to the minimum of the negative derivative curves (see the arrow that indicates the T_m of YvcK in the absence of UDP-GlcNAc). The T_m is an indicator of protein stability and is increased by the addition of UDP-GlcNAc (T_m = 42 °C in the absence of UDP-GlcNAc and T_m = 50.5 °C in the presence of 4 mM UDP-GlcNAc). (C) TSA results for the binding of UDP, UDP-Glc, UDP-GlcNAc and Glc-NAc. Assays were performed in the presence of increasing concentrations of ligands (0 to 4 mM). The difference of temperature (the shift of T_m induced by the presence of ligand) was plotted against the concentration of UDP (black circle), UDP-Glc (grey triangle), UDP-GlcNAc (black square) and Glc-NAc (white triangle). All the curves correspond to the average of data from at least 3 independent experiments and the standard deviations are representated by the error bars. Curve fitting was performed by using Microcal Origin 5.0 software.