Physicochemical Evidence that Francisella FupA and FupB Proteins Are Porins
2. Results and Discussion
2.1. Structural Modelling of Fup Proteins Predicted Their Folding as Porins
2.2. Fluorescence Assays on Proteoliposomes Containing FupA/B Suggested Porin Activity
- The intensity of the FupA/B light signal emitted by the PL dot was quantified as being 73% of that emitted by the dot corresponding to the starting material (FupA/B-MBP-His tagged protein at 100 μg/mL) (Figure 2B). Given that the sucrose gradient fractions were of 1 mL, we estimated that the whole PL fraction contained 73 μg of FupA/B protein. Knowing that the molar mass of MBP-FupA/B is 100 kDa (Reference  and Figure 2A), we estimated that there were 73 × 10−11 moles of FupA/B, i.e., 4.38 × 1014 molecules, of FupA/B inserted in the total number of PLs.
- We calculated the number of lipid molecules per PL by dividing the whole surface occupied by lipids in one PL by the mean surface of one phospholipid. Dynamic Light Scattering (DLS) measured the mean radius of PLs as being 63.5 ± 3.6 nm (mean ± SD; n = 3). Given that PLs contain 2 layers of lipids, we calculated the whole surface occupied by lipids in one PL as 2 × (4π R2), thus as 1.01 × 105 nm2. Knowing that the mean surface of a phospholipid is estimated to be 0.65 nm2 , we estimated the number of lipid molecules per PL as being 1.01 × 105/0.65 = 1.55 × 105.
- To calculate the number of formed PLs, we divided the total number of lipid molecules found in all the PLs by the number of lipid molecules per PL. The liposomes were prepared with 2 mg, thus 2.85 µM, i.e., 1.71 × 1018 molecules of lipids. Assuming that there was no loss of lipids between the liposome formation step and that of PLs, the number of formed PLs was thus estimated to be 1.71 × 1018/1.55 × 105 = 1.03 × 1013.
- Finally, the number of FupA/B monomers per PL was calculated as the total number of FupA/B proteins in the whole number of PLs, divided by the estimated number of PLs, thus 4.38 × 1014/1.03 × 1013 = 39.7.
- Knowing that the PLs’ radius estimated by DLS was 63.5 ± 3.6 nm, we calculated the volume of any given PL as 4/3 × π × R3 = 1.072 × 10−21 m3 = 1.072 × 10−18 L.
- Knowing that 2 mg of lipids had been used for the experiment, with the production of 1.013 × 1013 PLs, the total volume occupied by the formed PLs in the 1 mL working fraction was calculated as 1.013 × 1013 × 1.072 × 10−18 = 1.04 × 10−5 L = 11.04 μL. The total volume of PLs thus represented 1.10% of the 1 mL working fraction.
- Given that PLs were incubated in a 20 mM ANTS solution and that the total volume of PLs represented 1.10% of the total volume (1 mL), we reasoned that the maximum concentration of ANTS reached inside the PLs would have been 20 × 103 × 1.10% = 220 μM if their membrane had been permeable to the dye. In contrast, we obtained a concentration of 3.70 ± 0.01 μM ANTS within the PLs (Figure 2C), which represented only 1.7% of the ANTS molecules that would have diffused passively through a permeable membrane.
- Given the ANTS concentration calculated within the PLs (3.70 ± 0.01 µM) and the calculated volume of one PL (1.072 × 10−18 L), we calculated that each PL would contain 4.02 × 10−24 moles of ANTS, i.e., 2.42 molecules of ANTS, while it would have contained 142 molecules if the PL membrane had been permeable to ANTS.
2.3. Impedance Spectroscopy Experiments Using Tethered Lipid Bilayer Membranes Confirmed the Porin Activity of FupA/B
3. Material and Methods
3.1. Protein Purification
3.2. Protein Insertion in Liposomes
3.3. Porin Activity by Fluorescent Uptake Assay in Proteo-Liposomes
3.4. Electrophysiological Measurement of Porin Ion Conductance in Tethered Lipid Bilayer Membranes
3.5. Evaluation of the Amount of Protein Inserted in the Tethered Lipid Bilayer Membrane
3.6. Statistical Analyses
Conflicts of Interest
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Siebert, C.; Mercier, C.; Martin, D.K.; Renesto, P.; Schaack, B. Physicochemical Evidence that Francisella FupA and FupB Proteins Are Porins. Int. J. Mol. Sci. 2020, 21, 5496. https://doi.org/10.3390/ijms21155496
Siebert C, Mercier C, Martin DK, Renesto P, Schaack B. Physicochemical Evidence that Francisella FupA and FupB Proteins Are Porins. International Journal of Molecular Sciences. 2020; 21(15):5496. https://doi.org/10.3390/ijms21155496Chicago/Turabian Style
Siebert, Claire, Corinne Mercier, Donald K. Martin, Patricia Renesto, and Beatrice Schaack. 2020. "Physicochemical Evidence that Francisella FupA and FupB Proteins Are Porins" International Journal of Molecular Sciences 21, no. 15: 5496. https://doi.org/10.3390/ijms21155496