Fig. 6: TMcin oligomerizes to form β-barrel pores.
a TMcin-G1905 mediated leakage of carboxyfluorescein-loaded liposomes (7:3 DMPG:cardiolipin molar ratio) relative to Triton X-100 (100%). (n = 3 biological replicates, mean ± standard error; ***, p < 0.001). b A representative current trace through planar lipid bilayer formed from DPhPC in 150 mM KCl, pH 7.4 after addition of 11.5 nM TMcin-G1905 to the cis compartment. Blue line indicates current after equal-volume addition of the mock-purified control to the cis compartment. Inset (top-left) shows individual stepwise current increase events marked by red horizontal lines. c Histogram of stepwise conductance events (n = 332). d Current-voltage (I/V) curves obtained for 4 different conductance levels (G) induced by TMcin-G1905 in planar membranes made in 150 mM (cis) / 730 mM (trans) KCl gradient. The reversal potential (Ψrev) was calculated as the intercept of the linear regression fits for each conductance level. e Top-view of a 21-mer TMcin pore model colored by pLDDT (for scale see Fig. 1i). f Lateral view and cross-section (right, 11 protomers visible) of the 21-mer pore model colored by chain (top), pLDDT (middle), and molecular lipophilicity potential (bottom). g Ribbon model showing backbone hydrogen bonding (left) and a salt bridge (right, arrow, Lys 32 and Glu 49) between β-strands of neighboring protomers. h–l MD simulation analysis. h, i Overlays of representative structures spaced uniformly throughout the trajectories of the monomeric TMcin peptide in solution (h) and pore complex in a bilayer (i), colored by secondary structure. j Cα RMSF values for the monomer (black) and monomeric units of the pore complex (red). k Cα RMSD values for the monomer (black) and monomeric units of the pore complex (red). j, k n = 3 independent simulations, mean (line) ± standard deviation (shade). l Renderings of the pore complex system after 0.5 µs (replica 1), showing the TMcin pore, lipids (DMPG and cardiolipin in grey and green, respectively), water (transparent blue), and ions (K and Cl in silver and gold, respectively). m Circular dichroism analysis. Far-UV mean residue ellipticity of 2 µM TMcin-G1905 in phosphate buffer and 5% acetonitrile (black) fitted with the closet match of a linear combination of reference proteins to estimate secondary structural components (blue). Source data are provided as a Source Data file.
