Cationic peptides with amphiphilic characteristics were previously characterized for interactions with TPPS porphyrin molecules. Interaction with the peptides initiated formation of excitonically coupled J-aggregates (Caputo et. al. 2009). These peptide sequences were both amphiphilic and net cationic, reminiscent of antimicrobial peptides. As such, we conducted further investigations into the potential antimicrobial activity of these sequences. Three variants of the peptide were synthesized, differing only in the location of a single Trp. The primary use of the Trp was to act as a fluorescent reporter for any membrane binding interaction as well as interactions with TPPS. At a low micromolar efficacy, all variants exhibited similar degrees of antimicrobial activity when exposed to Gram+ and Gram- strains of bacteria. Biophysical characterization of the 3 peptides was carried out using fluorescence spectroscopy as well as quenching to explore the binding and membrane topography. All three peptides exhibited higher affinity for anionic bilayers (25% anionic lipid content) compared to that of zwitterionic bilayers, although there was little difference between the three peptide sequences. Again, only small differences were observed in acrylamide quenching experiments to determine Trp exposure when bound to the bilayer, indicating that the peptide is may not be adopting a uniform orientation since all three Trp variants exhibited similar quenching behavior. Using CD spectroscopy, all peptides adopted helical conformations when bound to the bilayers. The mechanism of antibacterial activity was investigated using bacterial membrane permeabilization assays. All peptides readily permeabilized the outer membrane of E.coli but none of the peptides exhibited substantial permeabilization of the E.coli inner membrane, indicative of localized activity to the outer membrane. Ongoing experiments are focused on new variants of the peptide with altered sequence composition (changing hydrophobic or cationic residue identities). Early results indicate that decreasing the hydrophobic content significantly impairs antimicrobial activity but not porphyrin binding.

Characterization of bifunctional peptides:  Porphyrin binding and antimicrobial activity

254th National Meeting (2017)

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