Fig. 2

Characterisation of SiaT. a SiaT is able to rescue growth of E. coli ∆NanT on Neu5Ac as the sole carbon source. The growth lag observed for ΔNanT+pNanT and ΔNanT+pSiaT is due to IPTG induction of the T5 promoter on pNanT and pSiaT. Growth curves represent the mean of six experiments ± SEM. b Time course of Neu5Ac uptake into proteoliposomes reconstituted with SiaT. In (black circle, black square, white triangle, black triangle), valinomycin was added to facilitate K⁺ movement prior to transport. In (white circle), ethanol was added instead of valinomycin as a control. In (white circle, black square, black circle), 10 mM NaCl was added together with [³H]-Neu5Ac; in (white triangle) 10 mM KCl was used in place of NaCl; in (black triangle) no salts were used in the transport assay. In (black square), transport was measured in empty liposomes. On the left Y-axis, specific transport activity is reported; on the right Y-axis transport in empty liposomes. is reported. Uptake data were fitted in a first-order rate equation for time course plots. c The transport of [³H]-Neu5Ac in the presence of 10 mM NaCl was measured in proteoliposomes reconstituted with SiaT, with an imposed K⁺ diffusion membrane potential. Data were plotted using the Michaelis–Menten equation. d The kinetics of Neu5Ac transport by SiaT sialic acid binding site variants. The transport of [³H]-Neu5Ac with or without NaCl was measured in proteoliposomes reconstituted with wild type and mutated variants, with an imposed K⁺ diffusion membrane potential. All proteoliposome measurements (b–d) are presented as means ± SD from five independent experiments. e MST binding assay of Neu5Ac binding to SiaT. f Representative isothermal titration calorimetry raw data (top) and binding isotherm (bottom) of Neu5Ac binding with SiaT. g Chemical structures of N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc) and ketodeoxynononic acid (KDN). h, i MST binding assay of Neu5Gc (h) and KDN (i) binding to SiaT. j Determination of the SiaT Na⁺ Hill coefficient. Data were plotted using the Hill equation. The inset represents the same data plotted using a log-scale for the X-axis to increase the resolution of low concentration data points. MST (e, h, i) and ITC (f) experiments represent the mean of three independent experiments ± SEM; for each, data from one representative experiment is shown