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.

Understanding the mechanism of action of antimicrobial peptides (AMPs) is a key step in developing new antimicrobial agents. This mechanism is often linked to the peptide’s orientation upon interaction with lipid membranes—whether it inserts into the membrane or remains surface-bound. Despite its importance, the literature lacks a systematic approach to predict peptide orientation based on physicochemical characteristics. This review highlights how peptide orientation at membranes can be qualitatively predicted by integrating sequence-level features, length/effective span, amphipathicity, charge distribution, and helix propensity, with membrane/environmental variables. The approach is illustrated with representative AMPs and it is discussed how external factors (pH, anionic fraction, cholesterol, ionic strength) bias surface-bound versus transmembrane regimes. The review is built upon theoretical observations and integrates structural, thermodynamic, and electrostatic parameters drawn from experimental data in the literature. While this study does not aim to provide a definitive classification, it offers a starting point that may guide experimental validation and future refinement.

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.

Antimicrobial peptides (AMPs) interact with lipid membranes through multistep processes that may include interfacial adsorption, partial insertion, membrane deformation, pore formation, and transmembrane organization. Although these processes have been extensively investigated, a unified operational framework connecting surface-bound, inserted, and intermediate peptide–membrane states remains underdeveloped. Here, we present a regime-based field-coupling framework that organizes peptide–membrane interactions according to the relative predominance of interfacial electrostatic coupling, hydrophobic-core coupling, and mixed coupling contributions. Surface-bound states are characterized primarily by interactions between cationic peptide residues and anionic lipid headgroups, whereas inserted states display stronger engagement of nonpolar peptide surfaces with lipid acyl chains. Mixed and pore-associated states retain both polar and hydrophobic interactions and may additionally involve water penetration, lipid-headgroup reorientation, membrane thinning, curvature, and peptide oligomerization. The operational use of the framework integrates peptide charge and charge distribution, hydrophobic moment, length and topology, membrane anionic charge, acyl-chain saturation, bilayer thickness, and peptide concentration. These variables are related to coordinated computational and experimental observables, including insertion depth, peptide–headgroup contacts, nonpolar residue–acyl-chain contacts, anionic lipid enrichment, peptide orientation, membrane order, water penetration, deformation, and peptide–peptide association. The framework generates conditional and falsifiable predictions for surface-bound, inserted/core, carpet-like, barrel-stave, and mixed/toroidal regimes. Its application to representative peptide–membrane systems illustrates how published observations can be organized within a common operational map. The proposed framework remains qualitative, and quantitative boundaries between coupling regimes require validation across peptides and membranes with distinct physicochemical properties. Nevertheless, it provides a structured basis for comparing mechanisms, designing computational and experimental tests, and interpreting transitions among peptide–membrane interaction states.

This work aims to highlight the importance of practical experience in the professional development of undergraduate students. The simulation of real-life scenarios allows students to cultivate both technical competencies and soft skills, better preparing them for the demands of the job market. This article presents a practical simulation involving the application of an agile methodology in software development. The example aims to support a teaching approach in which practice plays a central role in students' personal and professional growth. Furthermore, it illustrates how an interdisciplinary experience can contribute both technically and pedagogically to higher education.