Fig. 4: Testing the regulatory properties of full-length CdaA.

Proteoliposomes with 200 mM KCl inside and 50 mM KCl outside were used to simulate a hypotonic stress, and proteoliposomes with 50 mM KCl inside and 200 mM KCl outside simulate hypertonic stress. We emphasize that these vesicles do not maintain much of an osmotic pressure difference, and the hypertonicity will result in shape changes and invagination of the vesicles (30). We also compared these osmotic conditions with vesicles with equimolar concentrations of KCl inside and outside, either at 50 mM or 200 mM. All assays were performed using 1 μM CdaA. All error bars represent the SEM for ≥ 3 biologically independent replicates. A Ionic strength-dependent activity of CdaA and CdaA-DAC. Assays were performed using CdaA-containing nanodiscs composed of 38 mol% DOPG, 12 mol% DOPC plus 50 mol% DOPE in 50 mM HEPES-NaOH pH 8.0 containing 1 mM MnCl₂ plus the indicated concentration of KCl. For CdaA n = 3 and for CdaA-DAC n = 2 biologically independent samples. B DOPG dependence of CdaA in nanodiscs. Nanodisc composition is the indicated DOPG mol% plus the required mol% DOPC, and the buffer composition was 50 mM HEPES-NaOH pH 8.0 containing 1 mM MnCl₂ plus 200 mM KCl. n = 3 biologically independent samples. C DOPE dependence of CdaA in nanodiscs. Nanodisc composition is the indicated DOPE mol% plus the required mol% DOPC and the buffer composition was the same as for (B). n = 3 biologically independent samples. D Testing of osmotic effect on activity of CdaA-containing vesicles. The liposome assays only measures CdaA oriented inside-out, as shown in the pop-out where the grey circle represents the lipid vesicle. n = 3 biologically independent samples. E Testing of effect of membrane potential on activity of CdaA-containing vesicles. Membrane potential was generated using valinomycin-mediated K⁺ diffusion potential. For an inside-negative/outside-positive potential the vesicles were loaded with 200 mM KCl in buffer and suspended in buffer containing 200 mM NaCl, whereas for an inside-positive/outside-negative potential the liposomes were loaded with 200 mM NaCl and then suspended in buffer with 200 mM KCl. n = 3 biologically independent samples.