Fluorescent anisotropy assay

Fluorescent anisotropy (FA) experiments were performed in a black 384-well plate (Greiner Bio-One Polystyrene Non-Binding Flat Bottom) in triplicates. Serial dilutions of the protein (PBP1bγ, FtsW, FtsW-PBP3 or MurJ) in the appropriate buffer and the NBD-lipid II probe was added at a 0.33 µM final concentration. FtsN was used as a negative control under the same conditions. Blank samples without proteins were also prepared for background fluorescence estimation. The mixtures (30 µL) were allowed to reach equilibrium for 20 min at room temperature with gentle shaking and the FA signals were recorded at 21 °C using an Infinite F Plex (Tecan, Männedorf, Switzerland) microplate reader equipped with polarization filters with excitation and emission wavelengths at 485 nm and 535 nm respectively. Fluorescence polarization (FP) and anisotropy (A) values were calculated using the equations FP = (I_|| − G·I_┴)/I_||+ G·I_┴) and A = 2· FP/3-FP respectively, where I_|| is the fluorescence intensity of emitted light parallel to excitation, I_┴ is the fluorescence intensity of emitted light perpendicular to excitation, and G is the correction factor that correct for instrument bias. The G factor is experimentally determined using the probe alone. FA in millianisotropy units (mA) is plotted as a function protein concentration in µM. For K_d determination, the fluorescence anisotropy data were analysed by nonlinear curve fitting to the Eq. (¹) using GraphPad Prism 6.0 software²⁷.

Where A, A_min, A_max are the observed, the minimum and the maximum anisotropy, respectively, and [R], [L] and [RL] are the equilibrium concentration of the protein, the probe; and the protein-probe complex, respectively; K_d is the dissociation constant between the protein and the probe.

The fluorescence intensities of bound and free species were evaluated to verify the influence of binding partners on the fluorescence intensity of the probe (Fig ^S6). Except from ramoplanin and compound 5b, the binding of the proteins or the compounds have no significant effect on the fluorescence intensities (within the experimental error). For these compounds, the anisotropy values were corrected (Ac) using Eq. (²) ²⁸:

where A is the observed anisotropy; A_f is the anisotropy of the free probe; A_b is the anisotropy of the probe in the bound state; Q_f is the intensity of free probe and Q_b is the intensity of the probe in the bound state.

Serial dilutions of each antibiotic in 10 mM Tris-HCl pH 7.5 and 3% DMSO were prepared in 384-well plates (30 µL) in triplicates. The probe NBD-lipid II was used at a 0.33 µM final concentration. Blank samples without antibiotic were also prepared for background fluorescence estimation. The mixtures (30 µL) were allowed to reach equilibrium for 20 min at room temperature with gentle shaking and the FA signals were recorded at 21 °C using an Infinite F Plex (Tecan, Männedorf, Switzerland) microplate reader equipped with polarization filters with excitation and emission wavelengths at 485 nm and 535 nm respectively. The data were analysed as described above with [R] and [RL] are, respectively, the equilibrium concentration of the compound and the compound-probe complex.

Fixed concentrations of NBD-lipid II (0.33 µM) and proteins (~1 µM, 50–80% FA saturation²⁹) were used. Serial dilutions of each antibiotic,unlabelled lipid II, or control compounds were prepared in 384-well plates in triplicates. Blank samples without proteins were also prepared for background fluorescence estimation. The mixtures (30 µL) were allowed to reach equilibrium for 20 min at room temperature with gentle shaking and the FA signals were recorded at 21 °C using an Infinite F Plex (Tecan, Männedorf, Switzerland) microplate reader equipped with polarization filters with excitation and emission wavelengths at 485 nm and 535 nm respectively. The data were plotted FA in millianisotropy units (mA) as a function of test compound concentration in µM. For the analysis of the completion data, fluorescence anisotropy values were fitted to the Eq. (³) using GraphPad Prism 6.0 software³⁰.

Where [RL] is concentration of the receptor-probe complex; [R] is the concentration of the protein; [L] is the concentration of the probe; [C] is the concentration of test compound; K_d is the dissociation constant between the receptor and the probe (determined above); K_i is dissociation constant between the test compound and its target.