Journal: bioRxiv

Article Title: Conserved architecture of Tc toxins from human and insect pathogenic bacteria

doi: 10.1101/596536

Figure Lengend Snippet: pH-induced pore formation of Xn-XptA1, Mm-TcdA4 and Yp-TcaATcaB. (a) Negative stain electron micrograph of the Xn-XptA1 prepore at pH 8 (left) and the pore reconstituted in nanodiscs at pH 11 in the presence of 3 mM CaCl2 (right). (b) Negative stain electron micrographs of the Mm-TcdA4 prepore at pH 8 (left) and the pore reconstituted in liposomes at pH 5 or in nanodisc at pH 11 and 5 mM CaCl2 (right). (c) Negative stain electron micrographs of the Yp-TcaATcaB prepore at pH 8 (left) and the pore reconstituted in nanodiscs at pH 4.7 or pH 10.5 (right). Particles in the pore state are highlighted with colored circles. One typical particle in the pore state is magnified for each protein.

Article Snippet: For Pl-TcdA1(WT) and the three alanine mutants (Pl-TcdA1-E158A-R1873A, Pl-TcdA1-D965A-R1971A and Pl-TcdA1-E1086A-R1166A), 50 nM toxin pentamer was incubated with 300 nM nanodiscs MSP1D1-⊗H5-His with POPC (Cube Biotech) and then dialyzed against 20 mM CAPS pH 11, 150 mM NaCl over a period of 72 h. For the mutants Pl-TcdA1-H1202A and Pl-TcdA1-Y1168F-Y1205F, 0.3 μM toxin and 2 μM nanodiscs (MSP2N2-His with POPC for Pl-TcdA1-H1202A and MSP1D1-⊗H5-His with POPC for Pl-TcdA1-Y1168F-Y1205F from Cube Biotech) were incubated and analyzed for 72 h against buffer at pH 11.

Techniques: Staining, Liposomes

Journal: bioRxiv

Article Title: Conserved architecture of Tc toxins from human and insect pathogenic bacteria

doi: 10.1101/596536

Figure Lengend Snippet: Alanine mutants of Pl-TcdA1 reveal differences in protein stability. (a-c) The left panels show an SDS-PAGE gel after Ni-NTA purification and gel filtration at pH 8 of Pl-TcdA1-D158A-R1873A (a), Pl-TcdA1D965A and R1971A (b), and Pl-TcdA1-D1086A-R1166A (c). M = marker, L = lysate, SN = supernatant, P = pellet, elutions at increasing imidazole concentrations ranging from 5 – 150 mM, GP = gel filtration peak fraction. The 300 kDa band corresponding to the TcA monomer is marked with an arrow. The right panels show negative stain electron micrographs of the peak fraction applied to the grid directly after purification at pH 8 or after reconstitution in nanodiscs at pH 11. Particles in the prepore and pore state are marked with solid and dashed circles, respectively. Due to the insufficient purity of the Pl-TcdA1-D158A-R1873A variant, the reconstitution step was omitted. (d) Table with measured melting temperatures Tm (nanoDSF) for all analyzed TcAs at pH 8. The measurements were performed as triplicates and the mean Tm as well as the standard deviation are shown.

Article Snippet: For Pl-TcdA1(WT) and the three alanine mutants (Pl-TcdA1-E158A-R1873A, Pl-TcdA1-D965A-R1971A and Pl-TcdA1-E1086A-R1166A), 50 nM toxin pentamer was incubated with 300 nM nanodiscs MSP1D1-⊗H5-His with POPC (Cube Biotech) and then dialyzed against 20 mM CAPS pH 11, 150 mM NaCl over a period of 72 h. For the mutants Pl-TcdA1-H1202A and Pl-TcdA1-Y1168F-Y1205F, 0.3 μM toxin and 2 μM nanodiscs (MSP2N2-His with POPC for Pl-TcdA1-H1202A and MSP1D1-⊗H5-His with POPC for Pl-TcdA1-Y1168F-Y1205F from Cube Biotech) were incubated and analyzed for 72 h against buffer at pH 11.

Techniques: SDS Page, Purification, Filtration, Marker, Staining, Variant Assay, Nano Differential Scanning Fluorimetry, Standard Deviation

Journal: bioRxiv

Article Title: Conserved architecture of Tc toxins from human and insect pathogenic bacteria

doi: 10.1101/596536

Figure Lengend Snippet: Mutational studies of Pl-TcdA1. (a) SDS-PAGE gel after expression of Pl-TcdA1 mutants Pl-TcdA1-E1086A, Pl-TcdA1-R1166A, and Pl-TcdA1-E1086A-R1166A. For all proteins, a sample of the lysate (L), supernatant (SN) and pellet after ultracentrifugation (P) was applied. M = marker. (b-c) Close-up view of a conserved interaction site in Pl-TcdA1 (b), and alignment of residues, which are involved in the interaction (c). The conserved residues in all four TcAs are highlighted in yellow and the ones only conserved in Pl-TcdA1, Xn-XptA1 and Mm-TcdA4 in green. (d-e) SDS-PAGE gel of gel filtration fractions (left) of Pl-TcdA1-H1202A (d) and Pl-TcdA1-Y1168F-Y1205F (e) as well as negatively stained complexes at pH 8 (middle) and pH 11 after reconstitution in nanodiscs (right). The ratio of toxin-nanodisc ratio was 1 : 10 in (d), leading to a background of empty nanodiscs. The star marks the band corresponding fraction used for electron microscopy. Particles in the prepore and pore state are marked with solid and dashed circles, respectively. Scale bars, 100 nm.

Article Snippet: For Pl-TcdA1(WT) and the three alanine mutants (Pl-TcdA1-E158A-R1873A, Pl-TcdA1-D965A-R1971A and Pl-TcdA1-E1086A-R1166A), 50 nM toxin pentamer was incubated with 300 nM nanodiscs MSP1D1-⊗H5-His with POPC (Cube Biotech) and then dialyzed against 20 mM CAPS pH 11, 150 mM NaCl over a period of 72 h. For the mutants Pl-TcdA1-H1202A and Pl-TcdA1-Y1168F-Y1205F, 0.3 μM toxin and 2 μM nanodiscs (MSP2N2-His with POPC for Pl-TcdA1-H1202A and MSP1D1-⊗H5-His with POPC for Pl-TcdA1-Y1168F-Y1205F from Cube Biotech) were incubated and analyzed for 72 h against buffer at pH 11.

Techniques: SDS Page, Expressing, Marker, Filtration, Staining, Electron Microscopy