Many antimicrobial peptides act by disrupting cellular membranes, with therapeutic selectivity arising from their ability to discriminate between bacterial and mammalian cells, a distinction largely governed by membrane surface charge. Here, we investigate the interfacial organisation and membrane insertion of the cationic antimicrobial peptide magainin 2 using specular neutron reflectometry on lipid monolayers that mimic bacterial and mammalian membranes. Negatively charged dipalmitoylphosphatidylglycerol (DPPG) and zwitterionic dipalmitoylphosphatidylcholine (DPPC) were used as respective representative model systems. Experimental conditions were adjusted to compensate for the markedly different membrane association constants of magainin 2, enabling direct comparison of peptide behavior at the two interfaces.Despite similar peptide coverages, magainin 2 exhibits strikingly different interfacial behavior. At the DPPG interface, the magainin 2 α-helix inserts deeply and adopts an orientation parallel to the membrane surface, with hydrophobic residues facing the lipid chains and cationic residues interacting with the polar headgroups. In contrast, at the DPPC interface, the peptide displays limited interaction with the monolayer and a diffuse intensity distribution across the headgroup and acyl-chain regions, consistent with a largely disordered conformation.These results demonstrate that electrostatic interactions not only promote membrane binding but also dictate peptide insertion depth and interfacial organisation. By directly linking membrane charge to peptide penetration and structural arrangement, this work provides mechanistic insight into the selective antibacterial activity of magainin 2 and establishes a general framework for understanding the membrane-disruptive action of cationic antimicrobial peptides.

Background/Objectives: Antimicrobial peptides are promising agents for combating resistant infections. They exhibit bactericidal activity against a wide range of microbes, primarily by disrupting the permeability of the bacterial membrane and ultimately causing cell death. Effective bacterial killing requires a high number of membrane-bound peptide molecules. Therefore, it is conceivable that peptide accumulation on the membrane could also interfere with essential cellular processes by altering bilayer dynamics, a hypothesis referred to as the “sand-in-a-gearbox” model. Methods: We systematically investigated how membrane dynamics is affected by a set of well-characterized yet highly diverse peptides: the natural AMP magainin 2, the toxin melittin, the synthetic peptides LAH4 and Killer-FLIP, and small membrane-active peptidomimetics with bactericidal activity. These effects were examined using fluorescence spectroscopy techniques, by measuring anisotropy, generalized polarization, and excimer formation of specific probes inserted at different depths within the lipid bilayer. Results: The activity of all compounds extends beyond membrane permeabilization, and the perturbation of membrane dynamics, often associated with the so-called “sand-in-a-gearbox” mechanism, is a common feature among all systems analyzed. The membrane-active compounds induced a stiffening of the phospholipid bilayer by reducing lipid lateral mobility and decreasing water penetration, at least on the nanosecond timescale accessible to fluorescence measurements. Conclusions: The concentration range in which this behavior occurred was the same for all compounds studied. This threshold is, generally, higher than that required for membrane permeabilization and reflects near-complete coverage of the bilayer surface.

Traditionally, Nuclear Magnetic Resonance (NMR) infrastructures have relied on in-person access, requiring researchers to travel to centralized facilities to conduct experiments. However, recent advancements in remote access technologies, accelerated by the constraints imposed by the COVID-19 pandemic, have demonstrated the feasibility and strategic benefits of transitioning NMR operations toward remote accessibility. This review examines the key challenges and opportunities associated with remote access to NMR instrumentation, including standardized protocols for sample handling, secure authentication mechanisms, real-time instrument control, and data management. By establishing a unified framework for remote access, we aim to enhance the sustainability and accessibility of NMR facilities. Our findings highlight the necessity for collaborative efforts to develop best practices that ensure reproducibility, high-quality data acquisition, and equitable access to NMR infrastructure on a global scale.

Antimicrobial peptides are promising agents for combating resistant infections. They exhibit bactericidal activity against a wide range of microbes, primarily by disrupting the permeability of the bacterial membrane and ultimately causing cell death. Effective bacterial killing requires a high number of membrane-bound peptide molecules. Therefore, it is conceivable that peptide accumulation on the membrane could also interfere with essential cellular processes by altering bilayer dynamics, a hypothesis referred to as the “sand in the gearbox” model. In this work, we systematically investigated how membrane dynamics is affected by a set of well-characterized yet highly diverse peptides: the natural AMP magainin 2, the toxin melittin, the synthetic peptides LAH4 and Killer-FLIP, and small membrane-active peptidomimetics with bactericidal activity. These effects were examined using fluorescence spectroscopy techniques, by measuring anisotropy, generalized polarization, and excimer formation of specific probes inserted at different depths within the lipid bilayer. Our results show that the activity of all compounds extends beyond membrane permeabilization, and that perturbation of membrane dynamics is a common feature among all systems analyzed. The membrane-active compounds induced a stiffening of the phospholipid bilayer by reducing lipid lateral mobility and decreasing water penetration, at least on the nanosecond timescale accessible to fluorescence measurements. Interestingly, when accounting for differences in the resulting membrane surface area coverage, the concentration range in which this behavior occurred was the same for all compounds studied. This threshold is, generally, higher than that required for membrane permeabilization and reflects near-complete coverage of the bilayer surface.

Antimicrobial peptides (AMPs) are hoped to complement classical antibiotics in view of increasing microbial resistance. We investigated two members of a family of designed cyclic AMPs with different activity and selectivity. The cyclic hexapeptides are rich in arginine and tryptophan and have been shown previously to target cell membranes and to affect model membranes differently depending on the lipid membrane composition. To better understand their mechanisms of membrane perturbation, we investigated the interactions of cyclic RRRWWW and cyclic RWRWRW with various model membranes containing lipids commonly found in either bacterial or eukaryotic membranes. Using 31P and 2H solid-state NMR methods, we systematically analyzed the interactions between the peptides and lipid membranes at the molecular level. When POPE/POPG model membranes are investigated the two peptides exhibit distinct interactions with the PE and PG components, reflected in a different decrease in lipid chain order parameters. This decrease in deuterium order parameters and deformation of the vesicle shapes indicate disturbance of the lipid membrane structure by the peptides. Our study provides new insights into the molecular mechanisms of AMP-membrane interactions and can contribute to the understanding and development of novel antimicrobial agents.

Here we present studies of the structure and membrane interactions of ecPis-4 s, a new antimicrobial peptide from the piscidin family, which shows a wide-range of potential biotechnological applications. In order to understand the mode of action ecPis-4 s, the peptide was chemically synthesized and structural investigations in the presence of anionic POPC:POPG (3:1, mol:mol) membrane and SDS micelles were performed. CD spectroscopy demonstrated that ecPis-4 s has a high content of helical structure in both membrane mimetic media, which is in line with solution NMR spectroscopy that revealed an amphipathic helical conformation throughout the entire peptide chain. Solid-state NMR experiments of ecPis-4 s selectively labeled with 15N/2H and reconstituted into uniaxially oriented POPC:POPG membranes revealed an ideal partition of hydrophilic and hydrophobic residues within the bilayer interface. The peptide aligns in parallel to the membrane surface, a topology stabilized by aromatic side-chain interactions of the Phe-1, Phe-2 and Trp-9 with the phospholipids. 2H NMR experiments using deuterated lipids revealed that anionic lipid accumulates in the vicinity of the cationic peptide upon peptide-membrane binding.

Collagen VI is an extracellular matrix protein forming complex microfibrillar networks in connective tissues. Specifically, we focused on its role in innate immunity, in particular on cationic sequence motifs from the α3(VI)-chain, which exhibit strong antibacterial properties against both Gram-positive and Gram-negative bacteria in vitro and in vivo. Cytotoxicity assays revealed minimal to no adverse effects, even at concentrations effective against bacterial pathogens. This favorable safety profile suggests that these antimicrobial peptides selectively target bacterial membranes while sparing host cells, making them promising candidates for therapeutic development. The membrane structure and interactions of two antimicrobial peptides were investigated in quantitative detail using solid-state NMR, CD and fluorescence spectroscopies. Whereas calcein release was somewhat more pronounced from POPE/POPG 3/1 vesicles when compared to POPC/30 % cholesterol, this activity is about two orders of magnitude increased when POPC/POPG 3/1 liposomes are investigated. This pronounced lipid dependence was reproduced with magainin 2, a well-known linear cationic AMP. In lipid titration experiments both collagen-derived peptides showed a transition from predominantly random coil to helical conformations. Quantitative evaluation of membrane association required the presence of PEG-lipids which are known to prevent the agglutination of POPE/POPG 3/1 liposomes. A dissociation constant in the 260 μM range was observed for GVR28 while the binding isotherms reveal an intermediate state when SFV33 associates with bacterial membranes. 2H solid-state NMR reveals considerable membrane disorder of the deuterated PG palmitoyl chain in POPE/POPG membranes. The ensemble of biophysical data suggests two distinct modes of action for the collagen derived peptides.

Using antimicrobial peptides as a template, triazolium-based peptoids were designed with strong and selective antibacterial activities. To probe their mechanism, eight distinct peptoids were investigated using biophysical methods with lipid bilayers modeling bacterial or eukaryotic membranes. Calcein leakage experiments closely parallel antibacterial assays testing activities against Gram-negative or Gram-positive bacteria and toxicity for human red blood cells. This excellent correlation shows that the membrane-association underlies these peptoids' biological activities. While circular dichroism spectroscopy confirms their designed PPI (polyproline I) helical fold, fluorescence assays quantitatively evaluate membrane association and indicate localization at the membrane interface. In the presence of peptoids, a significant reduction in lipid order parameters is observed by solid-state NMR spectroscopy. Collectively, these findings support a membrane-mediated mechanism of action for the triazolium-based peptoids similar to that for linear cationic antimicrobial peptides. Furthermore, the physicochemical and structural features of the peptoids explain their different degrees of biological activities.

Vectofusin (VF) is a histidine-rich amphipathic peptide designed to enhance lentiviral transduction for gene therapeutic applications, where its assembly into fibrils requires polyvalent anions. In this study, we used solid-state NMR, transmission electron microscopy, and titration experiments to investigate the peptide's phosphate-driven supramolecular assembly. A VF variant lacking two lysines (V2K) was used to further assess the role of charge in these assemblies. Our results show that VF-pyrophosphate self-assembles into ordered, raft-like sheet structures. NMR confirmed that VF-pyrophosphate aggregates maintain an α-helical conformation, with distinct phosphate populations, one of which closely interacts with lysine residues. In contrast, the V2K variant showed weaker pyrophosphate interactions, highlighting the importance of electrostatic contacts in assembly. Based on these findings, we propose a model in which phosphates act as electrostatic glue, linking peptides via their lysine side chains. These insights support the design of new self-assembling biomaterials and improve understanding of phosphate-polypeptide interactions in biological processes.

Cathelicidin-BF (CatBF) is a LL-37 homologous antimicrobial peptide (AMP) isolated from Bungarus fasciatus with an exceptional portfolio of antimicrobial, antiviral, antifungal, and anticancer activities. Contrary to many AMPs, it showed a good pharmacological profile with a half-life of at least 1 h in serum and efficacy against bacterial infections in mice. To evaluate its potential against resistant nosocomial infections, we assessed its activity against 81 clinically relevant resistant bacterial isolates. CatBF exhibited minimum inhibitory concentrations (MICs) as low as 0.5 μM against carbapenem-resistant Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli. Its wide-ranging activity, unaffected by resistance mechanisms or Gram phenotype, prompted us to investigate its molecular mode of action. NMR spectroscopy, paramagnetic probes, and molecular dynamics (MD) simulations were employed to define its structure, penetration depth, and orientation in various membrane models, including micelles, bicelles, oriented bilayers, and vesicles. We found that CatBF's potent activity relies on its strong charge, allowing membrane neutralization at low peptide/lipid ratios and selective recruitment of charged phospholipids. At higher concentrations, a change in peptide orientation reveals membrane invagination and the formation of transient pores possibly leading to bacterial death. Our findings highlight the potential of CatBF as a model for developing resistance-independent agents to combat multidrug-resistant (MDR) bacterial infections.

In this study, we present an atomic-level structural investigation of the magainin 2 antimicrobial peptide reconstituted in extended lipid bilayers that closely mimic the composition of bacterial membranes. Using state-of-the-art solid-state NMR spectroscopy, we show that within liquid-crystalline membranes the peptide exhibits site-specific motional regimes, which correlate with its amphipathic character. Peptide-lipid interactions are identified at the polar headgroup region consistent with an in-plane topology also observed by oriented 15N solid-state NMR spectroscopy. While 13C chemical shift analysis reveals α-helical conformations, the NMR line shapes indicate pronounced conformational heterogeneity, which can be explained by the existence of higher order arrangements along the membrane surface. A reduced degree of helicity is observed when the membrane is in the gel phase suggesting more superficial interactions of magainin 2. Notably, our NMR data show that membrane-associated magainin 2 can evolve into amyloid-like β-sheet structures, forming large peptide-lipid aggregates. This behavior occurs only in bacterial and not in mammalian membrane models, paving the way for a new understanding of the role of these supramolecular assemblies in host defense mechanisms, and highlighting a potential relationship between antimicrobial peptides and functional amyloid structures.

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.

RATIONALE:In this study, we applied cross-linking mass spectrometry (XL-MS) to characterize the oligomeric states of a PGLa/magainin 2 mixture and gain insight into the heterodimerization previously suggested in the literature. Both peptides have shown a synergistic enhancement of activity when tested in antimicrobial assays; however, the mechanism of action is still not well understood. METHODS:Peptides solutions were prepared in HEPES buffer in the presence of membrane-mimicking DDM detergent micelles or POPE:POPG 3:1 vesicles. Cross-linking experiments were performed using disuccinimidyl suberate (DSS) or disuccinimidyl glutarate (DSG), and MALDI-MS was used to follow the cross-linking performance. Nano liquid chromatography coupled to mass spectrometry was conducted on a Q Exactive Plus orbitrap to achieve linkage sites determination using pLink2 for data interpretation. Trypsin or pepsin digestion was performed for the characterization of intermolecular links. RESULTS:XL-MS performed in a DDM micelle environment provided direct evidence of a specific PGLa/magainin 2 heterodimer, but no other oligomeric states were detected. Monitoring the reaction using MALDI-MS allowed unambiguous characterization of the cross-linked stabilized oligomers and facilitated a rapid optimization of conditions to achieve the best balance between stabilizing complex formation and avoiding unspecific aggregation. Comparison of the cross-linked species in detergent micelles and lipidic POPE:POPG bilayers revealed different behaviors suggesting that interaction between the peptides might occur differently in both membrane-mimicking media. CONCLUSIONS:This study revealed that XL-MS was relevant at the peptidomic level. However, the cross-linking workflow had to be adjusted compared to its use in large-scale protein-protein interaction mapping in order to avoid technical bias arising from the rapid nature of the cross-linking reaction.

Apolipoprotein A-I (apoA-I) mimetic peptides, inspired by the principal protein component of high-density lipoprotein, self-assemble with lipids to form discoidal nanodiscs widely used in biomedical research and as versatile scaffolds for characterization of membrane proteins in structural biology. Here, we investigate the 14A apoA-I mimetic, quantifying its orientation around the lipid bilayer and identifying the interactions that are crucial for nanodisc stability and dynamics using all-atom molecular dynamics simulations. To assess model fidelity, we back-calculated solid-state NMR observables, namely 15N chemical shifts and 2H quadrupolar splittings from the trajectories and compared them with previously reported solid-state NMR data. The simulations support a dimeric, antiparallel, belt-like arrangement of 14A peptides around the discoidal bilayer, stabilized by π-π stacking between aromatic residues and by electrostatic and hydrophobic peptide-lipid interactions. These interactions yield structurally stable nanodiscs with pronounced heterogeneity in lipid ordering and bilayer thickness between the nanodisc center and rim. Collectively, our MD results provide atomistic evidence for previously hypothesized peptide-peptide and peptide-lipid interactions and clarify how amphipathic helices organize to form the rim of discoidal nanodiscs. These insights inform the rational design of apoA-I mimetics for biomedical applications and the optimization of nanodiscs as platforms for studying membrane proteins.

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.