058 | Interaction between cyclic peptides and lipids: spectroscopic, calorimetric and structural analysis

Anna Laura La Monaca, Gabriele Cavalieri, Erik Laurini, Sabrina Pricl | Molecular Biology and Nanotechnology Laboratory MolBNL@UniTs, Department of Engineerig and Architecture DEA, University of Trieste, Italy.

This study investigates the interaction between cyclic peptides and model lipid membranes, with the aim of elucidating the mechanisms underlying their ability to interact with complex biological systems. In particular, the cyclic peptides βA-RYFFDMWY (RYF) and βA-GRLRWLRV (GRL), previously reported to exhibit biological activity, were examined using liposomes composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) as a simplified model of the bacterial membrane. The cyclic peptide RYF, also known as G4CP2, was originally identified for its ability to bind the transcription factor GAL4 and downregulate its activity in Saccharomyces cerevisiae. Owing to its target specificity, cell permeability, and enhanced resistance to proteolytic degradation, RYF represents a promising scaffold not only for studying galactose metabolism but also for potential biotechnological and pharmacological applications. Structurally, G4CP2 is cyclized through a lactam linkage between β-alanine and the eighth residue, conferring conformational rigidity, increased stability, and prolonged lifetime in biological environments, while exposing aromatic and hydrophobic residues critical for high-affinity target recognition. Peptide–lipid interactions were characterized using a multidisciplinary approach combining calorimetric, spectroscopic, and structural techniques. Isothermal Titration Calorimetry (ITC) was employed to determine the thermodynamic parameters of binding, while fluorescence spectroscopy (steady-state, synchronous, and three-dimensional) and UV–Vis spectroscopy were used to monitor changes in the microenvironment of aromatic residues and interaction-induced conformational effects. Circular dichroism spectroscopy was applied to assess alterations in peptide secondary structure, and dynamic light scattering provided information on liposome size, stability, and morphology. The results indicate that both peptides interact with the lipid component of the membrane, suggesting that direct lipid binding may contribute to their biological mechanism of action. Integrated analysis of spectroscopic, calorimetric, and structural data reveals that GRL and RYF interact with DOPC-based model membranes with overall weak affinity, yet through markedly different molecular mechanisms. GRL exhibits a predominantly surface-associated interaction, characterized by a weakly exothermic and moderately entropy-driven thermodynamic profile, consistent with adsorption mediated by electrostatic interactions and hydrogen bonding with lipid headgroups, and minimal membrane perturbation. In contrast, RYF displays a strongly entropy-driven and endothermic binding behavior, indicative of desolvation and hydrophobic partitioning processes. Spectroscopic changes and liposome size variations support a deeper interfacial insertion involving aromatic residues and inducing a more pronounced structural impact on the membrane, while preserving the native peptide conformation. Overall, these findings demonstrate that amino acid composition governs not only binding affinity but, more importantly, the mode and depth of peptide–membrane interaction, clearly distinguishing between superficial adsorption and interfacial insertion.