PH-DEPENDENT MODULATION OF PEPTIDE-MEMBRANE INTERACTIONS: A STUDY OF HISTIDINE-SUBSTITUTED MP1 ANALOGS

Electrostatic interactions play a fundamental role in the selectivity of lytic antimicrobial peptides (AMPs). Rich in cationic and non-polar residues, AMPs interact preferentially with the negatively charged outer leaflet of microbial membranes, without the need for specific receptors. Polybia-MP1 (MP1), derived from the venom of Polybia paulista, is a broad-spectrum AMP that exhibits antibacterial and antitumoral activity without causing hemolysis. Its amphipathic structure is stabilized by the proximity of acidic (Asp) and basic (Lys) residues, which modulate its net charge and membrane affinity depending on the solution pH. To explore the role of pH in regulating peptide activity, we synthesized H-MP1, a histidine-substituted analog of MP1, replacing all lysines with histidines. Given the pKa of histidine (~6.5), H-MP1 shows increased sensitivity to acidic environments, such as tumor extracellular microenvironments. Our results show that H-MP1 exhibits stronger binding and lytic activity at acidic pH, correlating with enhanced protonation of histidines and stronger electrostatic attraction to anionic membranes. Zeta potential measurements confirmed enhanced membrane association of H-MP1 at pH 5.5, while fluorescence spectroscopy revealed pH- dependent shifts in tryptophan emission, enabling us to determine partition constants and binding isotherms. Interestingly, despite H-MP1’s enhanced pH selectivity, its overall membrane affinity and lytic activity were lower than MP1, suggesting that individual lysine residues may contribute differently to peptide function. To investigate this, we designed three new analogs—[His4]MP1, [His5]MP1, and [His11]MP1—each with a single lysine replaced by histidine. These variants will be evaluated for their structural behavior, net charge, and lipid binding across physiological and acidic pH conditions, using zeta potential and tryptophan fluorescence. Model membranes mimicking healthy (PC:PE) and tumor (PC:PE:PS) cells will be used to assess the critical peptide concentration for lytic activity. Theoretical modeling based on Gouy– Chapman theory will support interpretation of electrostatic contributions.This study aims to elucidate the role of charged residues in modulating AMP-membrane interactions and to explore the potential of pH-responsive peptides as selective agents for targeting tumor cells.