Fig. 4

Biochemical and Biophysical characterisation of Van2. a. PcaV and Van2 analysis in solution by size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS). PcaV and Van2 traces are shown as monodispersed (blue and red peaks respectively) and the molecular weight at 34.0 kDa and 33.5 kDa (blue and red strong lines inside the peak) indicating formation of a homodimer. b. Surface Plasmon Resonance (SPR) of PcaV and Van2 interaction with the biotinylated DNA palindromic probe PV Bio. Concentrations of PcaV from 0.94 nM to 60 nM and Van2 ranging 9.4 nM to 600 nM were tested. The signal was plotted and a curve was traced to calculate the affinity constants (Kd). PcaV showed experimental affinity to the PV Bio probe with Kd of 5.05 nM. Van2 showed a Kd of 86.7 nM. c to e Electrophoretic Mobility Shift Assays (EMSA) of PcaV and Van2 with the 5′-infrared probe PV Bio. c. PcaV - PV probe complex is disrupted in the presence of 5 mM of PCA, but not with vanillin or vanillic acid at the same concentrations. d. Van2 forms complex with PV, but not with the Rnd probe containing the same nucleotides completely randomized. e. EMSA titrations with increasing concentrations of vanillin and PCA showed similar pattern of Van2-PV complex disruption