A comparison of activity, toxicity, and conformation of tritrpticin and two TOAC-labeled analogues. Effects on the mechanism of action

Abstract

A strategy that has been gaining increased application for the study of the conformation, dynamics, orientation, and physicochemical properties of peptides is labeling with the paramagnetic amino acid TOAC. This approach was used to gain a deeper understanding on the mechanism of action of the antimicrobial peptide tritrpticin (TRP3). TRP3 was labeled with TOAC at the N-terminus (prior to V¹, TOAC⁰-TRP3) or internally (replacing P⁵, TOAC⁵-TRP3). Functional studies showed that labeling led to peptides with higher activity against Gram-positive bacteria and lower hemolytic activity with respect to TRP3. Peptide-induced model membranes permeabilization and ion channel-like activity studies corroborated the functional assays qualitatively, showing higher activity of the peptides against negatively charged membranes, which had the purpose of mimicking bacterial membranes. TOAC presented a greater freedom of motion at the N-terminus than at the internal position, as evinced by EPR spectra. EPR and fluorescence spectra reported on the peptides conformational properties, showing acquisition of a more packed conformation in the presence of the secondary structure-inducing solvent, TFE. CD studies showed that TOAC⁰-TRP3 acquires a conformation similar to that of TRP3, both in aqueous solution and in TFE, while TOAC⁵-TRP3 presents a different conformation in all environments. While the mechanism of action of TRP3 was impacted to some extent by TOAC labeling at the N-terminus, it did change upon replacement of P⁵ by TOAC. The results demonstrated that TOAC-labeling could be used to modulate TRP3 activity and mechanism of action and, more importantly, the critical role of P⁵ for TRP3 pore formation.

Keywords: Antimicrobial peptides; Conformational properties; Membrane permeabilization; Structure-activity relationship; TOAC; Tritrpticin.