Analytical multi-technique approach to successfully tackle the complexity of metal-peptide interaction: the case of copper-microplusin

Investigating protein systems presents significant challenges, particularly due to the dynamic and environmentally dependent nature of metal-protein interactions. A comprehensive understanding of these systems requires extensive analyses under diverse experimental conditions, encompassing species distribution, ligand coordination modes, complex stoichiometry, and structural geometry. In recent years, an effective analytical method has emerged to study metal interactions in biological systems. The analytical approach aims to simplify the experimental investigation using model systems and in vitro simulations, applying a multi-technique approach for qualitative and functional characterizations [1, 2]. In this work we present how the use of suitable electroanalytical, thermodynamic, spectroscopic and spectrometric methods, combined with proper experimental conditions, can provide comprehensive information on the interaction between metal ions and peptide or protein systems, with a focus on their speciation in solution and thermodynamics of complex formation equilibria. The purpose of study is the antimicrobial peptide microplusin. Microplusin is a 10,204-Da peptide with antibacterial and antifungal activity, and such antimicrobial mechanism is primarily attributed to its ability to bind and sequester copper ions [3]. The complexity of metal-peptide interactions is tackled by different analytical techniques, including potentiometry, ultraviolet–visible absorption spectrophotometry, circular dichroism and high-resolution mass spectrometry.