Kelton Rodrigues de Souza

Background/Objectives: The increasing prevalence of multidrug-resistant bacteria presents a major global health challenge, prompting a search for innovative antimicrobial strategies. This study aimed to develop and evaluate a novel nanobiostructure combining alumina nanoparticles (NPs) with the antimicrobial peptide lunatin-1 (Lun-1), forming peptide-functionalized nanofilaments. The main objective was to investigate how the site of peptide functionalization (C-terminal vs. N-terminal) affects membrane interactions and antibacterial activity. Methods: NP–peptide conjugates were synthesized via covalent bonding between lun-1 and alumina NP and characterized using transmission electron microscopy (TEM), X-ray diffraction (XRD), zeta potential analysis, dynamic light scattering (DLS), Fourier-transform infrared (FTIR), and solid-state ¹³C NMR. Antibacterial activities were assessed against different Gram-positive and Gram-negative strains. Biophysical analyses, including circular dichroism (CD), isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and solid-state ²H NMR, were employed to evaluate peptide–membrane interactions in the presence of membrane-mimetic vesicles composed of POPC:POPG (3:1) and DMPC:DMPG (3:1). Results: Characterization confirmed the successful formation of NP–peptide nanofilaments. Functionalization at the N-terminal significantly influenced both antibacterial activity and peptide conformation compared to C-terminal attachment. Biophysical data demonstrated stronger membrane interaction and greater membrane disruption when lun-1 was conjugated at the N-terminal. Conclusions: The site of peptide conjugation plays a crucial role in modulating the biological and biophysical properties of NP–lunatin-1 conjugates. C-terminal attachment of lunatin-1 retains both membrane interaction and antibacterial efficacy, making it a promising strategy for the design of peptide-based nanotherapeutics targeting resistant pathogens.

Background/Objectives: This study investigates the structural and biophysical properties of the wild-type antimicrobial peptide LyeTx I, isolated from the venom of the spider Lycosa erythrognatha, and its analog LyeTx I-b, designed to enhance antibacterial activity, selectivity, and membrane interactions by the acetylation and increased amphipathicty. Methods: To understand the mechanisms behind these enhanced properties, comparative analyses of the structural, topological, biophysical, and thermodynamic aspects of the interactions between each peptide and phospholipid bilayers were evaluated. Both peptides were isotopically labeled with ²H₃-Ala and ¹⁵N-Leu to facilitate structural studies via NMR spectroscopy. Results: Circular dichroism and solid-state NMR analyses revealed that, while both peptides adopt α-helical conformations in membrane mimetic environments, LyeTx I-b exhibits a more amphipathic and extended helical structure, which correlates with its enhanced membrane interaction. The thermodynamic properties of the peptide–membrane interactions were quantitatively evaluated in the presence of phospholipid bilayers using ITC and DSC, highlighting a greater propensity of LyeTx I-b to disrupt lipid vesicles. Calcein release studies reveal that both peptides cause vesicle disruption, although DLS measurements and TEM imaging indicate distinct effects on phospholipid vesicle organization. While LyeTx I-b permeabilizes anionic membrane retaining the vesicle integrity, LyeTx I promotes significant vesicle agglutination. Furthermore, DSC and calcein release assays indicate that LyeTx I-b exhibits significantly lower cytotoxicity toward eukaryotic membranes compared to LyeTx I, suggesting greater selectivity for bacterial membranes. Conclusions: Our findings provide insights into the structural and functional modifications that enhance the antimicrobial and therapeutic potential of LyeTx I-b, offering valuable guidance for the design of novel peptides targeting resistant bacterial infections and cancer.