Oligomerisation of tryptocidine C, a Trp-rich cyclodecapeptide from the antimicrobial tyrothricin complex

Tryptocidine C (TpcC, cyclo[D-Phe¹-Pro²-Trp³-D-Trp⁴-Asn⁵-Gln⁶-Trp⁷-Val⁸-Orn⁹-Leu¹⁰]) is a broad-spectrum antimicrobial peptide in the tyrothricin complex produced by a soil bacterium, Brevibacillus parabrevis. Electrospray mass spectrometric studies reveal the oligomerisation of TpcC into dimers and higher oligomers, analogous to tyrocidine C (TrcC, Trp⁷ replaced by Tyr⁷). Ion mobility mass spectrometry (IMMS) further confirms the formation of stable peptide dimers and tetramers with diameters of 2.7 nm and 3.3 nm, respectively, calculated from collisional cross section (CCS). Molecular dynamic simulations and docking studies support the formation of amphipathic dimers, with a diameter of 2.5 ± 0.07 nm calculated from low energy model CCS. Circular dichroism and IMMS studies point towards dynamic hydrogen-bonded conformational changes up to 28-33 μM after which the structures become more static (or in equilibrium). Fluorescence studies indicate aromatic stacking of Trp residues with a CMC of 18 μM in aqueous solutions. The concentration and time dependent interaction of Trp in oligomers indicate cooperativity in the TpcC oligomerisation that leads to the formation of higher order microscopic structures. Scanning electron microscopy studies unequivocally shows that TpcC forms nanospheres with a mean diameter of 25 nm. Repeated smaller oligomeric units, possibly dimers and tetramers, self-assemble to form these nanospheres.