Compositional asymmetry is a hallmark of biological membranes and plays a crucial role in pore formation. However, the molecular mechanism by which leaflet asymmetry influences pore formation remains unclear. Here, we employed molecular dynamics simulations of asymmetric lipid bilayers composed of zwitterionic phosphatidylcholine (POPC) and anionic phosphatidylserine (POPS) to elucidate this process. The calculated free-energy barrier for pore nucleation in asymmetric bilayers was higher than that in symmetric ones. In the asymmetric system, water defects initiating pore formation preferentially appeared on the POPC leaflet, whereas the subsequent pore growth involved rearrangements in the POPS leaflet. During this process, hydrogen bonds between POPS molecules were disrupted and partially replaced by hydrogen bonds between POPS and water molecules, accompanied by increased headgroup cohesion and structural ordering within POPS-rich regions, thereby increasing the energetic cost of pore nucleation. In contrast, POPC molecules lacked intermolecular hydrogen bonding, resulting in weaker headgroup cohesion. We then used a continuum-based theoretical model to rationalize the preferential water entry on one leaflet observed in asymmetric membranes. Finally, using an erythrocyte-mimetic multicomponent membrane model, we show that leaflet-biased water entry and the accompanying reorganization of headgroup interaction networks persist in a multicomponent asymmetric membrane. Overall, leaflet-resolved headgroup chemistry and membrane mechanics jointly shape the free-energy landscape of pore nucleation in asymmetric membranes. Lipid-lipid headgroup hydrogen bonding, together with membrane thickness and elastic response, can modulate pore nucleation and early pore stabilization, and may be relevant to asymmetry-dependent processes such as apoptosis and transmembrane signaling.
We present a robust and easy-to-use methodology for determining the nanoscopic miscibility transition temperature, nm-Tmix, of lipid bilayer mixtures from FRET measurements. The method relies on the use of freely diffusing fluorescent donor and acceptor lipids that partition non-uniformly between coexisting phases. When this condition is met, changes in lipid clustering that occur as the sample passes through the transition result in abrupt changes in the spatial distribution of probes and consequently, abrupt changes in the FRET signal. FRET vs. temperature data can then be modeled with a phenomenological piecewise function that describes how the signal changes above and below nm-Tmix. Using lattice simulations, we show that the transition between these regimes occurs when the size of lipid clusters surpasses a critical threshold that is approximately equal to twice the Förster distance of the donor/acceptor pair, or about 10 nm. Because other, temperature-dependent factors unrelated to lateral organization-such as changes in lipid molecular area and donor photophysics-can also influence the FRET signal, we also fit the data using a simpler model of uniform mixing. An information theory-based test comparing the fit quality of the uniform and phase separated models provides a straightforward and robust criterion for objectively assessing whether a given sample undergoes a nanoscopic miscibility transition within a temperature range of interest. We highlight the distinction between nm-Tmix determined by FRET (or methods with comparable spatial resolution) and the micron-scale transition temperature, μm-Tmix, determined from diffraction-limited optical techniques. The analysis software is freely available from an online repository.
The present study extends our previous experimental investigation of liposome-amyloid composite nanocarriers for doxorubicin (DOX) delivery by providing a mechanistic analysis of how experimentally observed loading and release parameters influence drug depot behavior and systemic exposure. In our earlier work, the encapsulation capacity and release kinetics of DOX were characterized for phosphatidylcholine (PC) liposomes, cardiolipin (CL)-containing liposomes, lysozyme and insulin amyloid fibrils (LzF and InsF), and their corresponding composite systems. Here, the experimentally determined retained drug fractions and apparent release constants were used as input parameters for computational analysis of drug release, depot depletion, and systemic pharmacokinetic behavior. Using a first-order kinetic framework combined with a two-compartment pharmacokinetic model, theoretical release profiles, depot persistence, and plasma concentration-time curves were reconstructed to compare the investigated formulations under identical conditions. The results demonstrate that formulation composition strongly affects depot size and release dynamics, which subsequently reshape systemic exposure profiles. Systems with higher retained drug fractions were shown to form larger depots and produce delayed and reduced peak concentrations, whereas low-loading formulations allow rapid systemic drug availability. Whereas our previous experimental study identified PC+InsF composites as the most promising systems based on encapsulation efficiency and fluorescence-derived characteristics, incorporation of release kinetics and pharmacokinetic modeling in the present work indicates that PC+LzF systems offer a more advantageous balance between depot persistence and systemic exposure. However, this finding requires experimental validation under physiological conditions before solid translational conclusions can be drawn. Overall, the study demonstrates how computational pharmacokinetic modeling can extend experimental characterization and support the rational design of hybrid protein-lipid nanocarriers for controlled drug delivery.
Venlafaxine (VLX) and its active metabolite O-desmethylvenlafaxine (ODV) must partition into lipid membranes before reaching their membrane-embedded transporter targets. The principal active metabolite, ODV, differs from VLX by the removal of a methyl group and is itself marketed as an antidepressant. Here, we combined long-timescale all-atom molecular dynamics simulations with experimental membrane partitioning measurements to investigate how demethylation, stereochemistry, and drug concentration influence their interactions with phosphatidylcholine bilayers. All drug species spontaneously partitioned into the lipid-water interface and stabilized at ∼1.4 nm from the bilayer center while maintaining a conserved amphipathic orientation. Free-energy calculations revealed similar interfacial free-energy minima of approximately -20 kJ mol-1 for both compounds, whereas the energetic penalty for translocation toward the bilayer core exceeded 40 kJ mol-1. Demethylation of VLX lowered the energetic barrier for membrane insertion without altering the thermodynamically favored interfacial state, indicating that ODV accesses the membrane interface more efficiently while preserving comparable equilibrium membrane affinity. Experimental second-derivative UV-Vis spectroscopy further confirmed similar membrane partitioning for VLX and ODV. At low drug concentrations, membrane structure remained largely unperturbed following insertion. In contrast, elevated drug loading induced cooperative membrane perturbation characterized by lateral bilayer expansion, membrane thinning, and reduced acyl-chain order, with these effects consistently more pronounced for ODV. Together, these findings identify the lipid bilayer under studied conditions as a dynamically adaptive kinetic interface rather than a passive equilibrium reservoir, suggesting that functional differences between VLX and ODV arise primarily from differences in interfacial accessibility and subsequent protein-specific interactions rather than nonspecific membrane affinity alone.
Membrane shape is commonly described by curvature, area, and thickness, which underpin bilayer mechanics and protein-membrane interactions. In molecular dynamics (MD) simulations, these quantities must be inferred from discrete coordinates, and common grid-based smoothing approaches can introduce resolution-dependent artefacts and ambiguities, especially under periodic boundary conditions. Here we present CATpie, a Python framework for a unified geometric analysis of simulated membranes represented as smooth leaflet height fields over a periodic domain (from user-defined leaflet reference atoms or beads). CATpie fits real Fourier series to the upper and lower leaflets, constructs the mid-surface, and evaluates mean curvature, Gaussian curvature, and real area from analytic expressions of the fitted surfaces. It further defines a normal-line thickness as the distance between the intersections of each mid-surface normal with the two leaflets, providing a geometrically controlled measure of leaflet separation that complements the commonly used vertical separation Dzz. Validation on analytic benchmark surfaces, including a sinusoidal surface with zero Gaussian curvature and a separable cosine surface with non-zero Gaussian curvature, shows high-accuracy recovery of curvature fields, real areas, and leaflet separation. Application to a mixed POPS/POPC bilayer demonstrates that mid-surface curvature and normal-line thickness quantify local deformations and thickness modulations, while the ratio Tnorm/Dzz highlights regions where vertical separation overestimates normal-line spacing due to membrane tilt. CATpie supports atomistic and coarse-grained simulations of planar and undulated bilayers that remain representable as single-valued leaflet height fields z=Z(x,y) over a periodic domain (without overhangs or topological changes), enabling systematic geometric characterisation of membrane simulations.
Antimicrobial peptides (AMPs) represent a current strategy to develop new antibiotics against multi-resistant pathogens. The potential antibiotic activity of AMPs is related to their amphipathic properties and the presence of positively charged residues, which may interact with the negatively charged bacterial membranes. In contrast, they exhibit lower interaction with the eukaryotic, neutrally charged membranes. This is the primary reason AMPs can distinguish between eukaryotic and prokaryotic membranes. AMPs are usually modified or designed de novo, and their properties can be changed by inserting specific amino acid residues into their sequence. To assist in the rational design of AMPs, it is helpful to explore the biophysical changes they may induce in target cell membranes. Therefore, bacterial and eukaryotic model lipid membranes have been extensively used for this purpose. Parameters such as selective binding, lipid membrane interactions, membrane packing, permeability, hydration, and restructuring facilitate the exploration of peptide regions of interest. These parameters can be studied using various physicochemical techniques, including differential scanning calorimetry, X-ray diffraction, nuclear magnetic resonance, and fluorescence spectroscopy. This review aims to provide a practical guide to the main biophysical techniques used to explore the potential antibiotic activity of AMPs using model membranes, and to examine lipid-peptide interactions in order to define the mechanisms of action of these antimicrobial peptides. These techniques determine whether the peptide interacts specifically with bacterial membranes, the preferred bacterial target of a given AMP, the binding affinities of AMPs, potential pore formation and its geometry, and the impact of these interactions on both bacterial and eukaryotic membranes.
Cell-penetrating peptides (CPPs) such as penetratin are known to traverse lipid membranes, yet the nanoscale structural consequences of their membrane interactions remain incompletely understood. Using atomic force microscopy (AFM), we visualized penetratin-induced remodeling in supported lipid bilayers (SLBs), focusing on discrete POPC bilayer patches whose exposed edges sensitively report early structural changes. In POPC patches, penetratin first accumulated at patch boundaries, forming elevated peripheral rings, and at higher concentrations generated shallow nanoscale pits across the patch interior. Continuous POPC bilayers exhibited a closely parallel pathway-elevated protrusions at 1 μM penetratin and widespread nanoscale pore-like depressions at 2-4 μM-indicating that similar peptide-lipid structures form even without membrane edges. Bilayers containing anionic POPS showed greatly enhanced susceptibility, progressing from peripheral depressions and aggregates to full fragmentation into nanoscale lipid-peptide particles, whereas cholesterol-containing bilayers remained largely resistant, developing only a few isolated deep defects. Our findings reveal an array of penetratin-induced remodeling events shaped by membrane composition and geometry, providing new mechanistic insight into how penetratin modulates membrane structure at the nanoscale.
Cellular lipids are wonders of biomolecular self-organization whose structure and dynamics are intimately connected with their functionality. Here we review the development and use of NMR spectroscopy in the study of lipid membranes. For liquid-crystalline bilayers, the structure is described by orientational order parameters, while the dynamics entail fluctuations about the mean geometry. Addressing the information gap between molecular structure, dynamics, and function involves magnetic resonance spectroscopy combined with X-ray and neutron scattering approaches. Cholesterol gives a crucial test in liquid-ordered (lo) membranes, where the bending rigidity oppositely affects solid-state NMR observables-the order parameters increase yet the relaxation rates decrease. By contrast, nonionic surfactants in the liquid-disordered (ld) state soften the bilayer and decrease the order parameters, thereby enhancing the spin relaxation. This enigma is explained by a model-free power-law that combines the mean-squared amplitudes and fluctuation rates. Collective modes appear on the mesoscale of the bilayer thickness and less, indicating how membrane elasticity emerges from atomistic-level interactions that drive the response to external forces. The unified power-law scaling shows how the bilayer fluidity corresponds to a hydrocarbon liquid of similar chain length. Magnetic resonance spectroscopy thus yields insights into properties that underlie bilayer phase transitions, curvature, and protein-lipid interactions.
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.
The hydrophobic tail structure of lipids or amphiphiles plays a critical role in governing the self-assembly, structure, dynamics, and function. In the present work, β-alaninol-based amphiphiles bearing cholesterol, saturated, unsaturated, and branched fatty acid-based tails were synthesized in N-acyl and N-cholesteroyl forms and systematically investigated to elucidate how tail architecture controls physicochemical properties. Results revealed that variations in amphiphile tail structure influenced pronounced differences in phase transition behavior and supramolecular packing. Amphiphiles bearing saturated, unsaturated, or branched tails favored more tilted or less-ordered bilayer arrangements than the cholesterol-based amphiphile, which lead to lower phase transition temperatures of fatty acid-based amphiphiles than the cholesterol-based amphiphile. Thermal analysis studies revealed that N-acylation substantially enhanced thermal stability, with decomposition temperatures increased by 50-70 °C compared to the corresponding parent fatty acids; however, the cholesteroyl-based amphiphile exhibited lower enhancement than cholesterol. Laurdan and ANS (8-anilinonapthalene-1-sulphonic acid) fluorescence studies revealed a strong correlation between tail structure, membrane packing and interfacial hydration, in which unsaturation in tail lowers the gel-to-liquid-crystalline transition temperature and enhances ANS accessibility. Dynamic light scattering and field-emission scanning electron microscopy confirmed the formation of unilamellar vesicles, where tail architecture significantly influences vesicle size, polydispersity, and zeta potential. Comparison of bilayer-related properties between matched-chain saturated ethanolamine- and β-alaninol-based amphiphiles revealed only moderate effects arising from the headgroup substitution. Overall, these findings establish amphiphile tail design as a key structural parameter for controlling amphiphile packing, phase behavior, and hydration, offering molecular-level insights for tuning properties of lipid-based assemblies for various applications.
Cellular organelles are uniquely specialized membrane-bound structures that enable cells to organize and coordinate biochemical processes. Specifically, mitochondria are essential organelles for cellular metabolism, coordinating energy production, and connecting signaling networks for cellular homeostasis. 99% of mitochondrial proteins are encoded by nuclear genes that require precise and efficient translation and import into mitochondria for biological processes. This process is mediated by coordinated pathways involving the mitochondrial specific translocation complexes, chaperones, and specialized targeting routes. Tight regulation of these import mechanisms allows for proper protein localization, folding, and assembly. Disruptions in the mitochondrial protein import pathway compromise organelle homeostasis and activate proteostatic stress and quality control pathways. Such defects have been observed in a wide range of pathophysiological conditions, including cardiovascular disease, neurodegeneration, and cancer. The import defects destabilizing mitochondrial proteins can impair oxidative phosphorylation and metabolic signaling. In sum, defects to mitochondrial function can highlight a central role of mitochondrial protein import beyond maintaining cellular function and how defects at distinct stages of import contribute to disease, underscoring opportunities for therapeutic intervention targeting mitochondrial proteostasis.
Antimicrobial peptides (AMPs) are promising alternatives to conventional antibiotics due to their broad-spectrum activity and low tendency to induce resistance. Based on their modes of action, AMPs are generally classified as membrane-active or intracellular-targeting peptides. Rational design of next-generation AMPs with enhanced efficacy and reduced cytotoxicity requires a detailed understanding of their antimicrobial mechanisms. Solid-state nuclear magnetic resonance (ssNMR) has emerged as a powerful tool for probing AMP-target interactions, providing high-resolution structural and dynamic information under native-like membrane conditions. This review summarizes recent advances in ssNMR methodologies applicable in model membranes and intact cells and highlights their contributions to elucidating AMP antimicrobial mechanisms. Representative investigations reveal that the membrane-active peptide protegrin-1 disrupts lipid bilayers via a toroidal pore mechanism, whereas lipid II-targeting AMPs inhibit cell wall biosynthesis by immobilizing lipid II through supramolecular assembly.
One of the major obstacles in treating diseases that affect the central nervous system is delivering drugs across the blood-brain-barrier (BBB). Cell-penetrating peptides (CPPs) can be used as delivery vectors, but their translocation mechanism is still poorly understood, in part due to the simplistic membrane models applied to their interpretation. Here we investigate the translocation mechanism of two CPPs, R9 and MPG, using molecular dynamics and enhanced sampling techniques on a realistic membrane model of human brain microvascular endothelial cells. The results suggest that R9 induces greater membrane disruption compared to MPG, yet that both face a significant free energy barrier to translocation. In both peptides the first interactions were initiated by the N-terminus and prominently involved arginine residues even for MPG. The crucial role of the plasticity of both partners (BBB bending, partial CPP unfolding) on the translocation energetics was also explored by sampling ad hoc collective variables, revealing the important role of long polyunsaturated acyl chain lipids. Together, these findings provide mechanistic insight into CPP-mediated transport and offer guidelines for rational design.
Parkinson's disease (PD) is a progressive neurodegenerative disorder lacking reliable, early-stage diagnostic biomarkers. This study utilizes Fourier Transform Infrared (FTIR) spectroscopy to identify biochemical alterations in plasma-derived small extracellular vesicles (PsEVs) from PD patients. PsEVs were isolated and characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and immunoblotting for canonical markers (TSG101, CD63, and CD9). FTIR spectra were recorded in the mid-infrared region (900-3100 cm-1) to probe molecular signatures of lipids and proteins. Significant spectral differences were observed between PD and AM-C sEVs, in the lipid-associated regions- I2960/I2929 for CH₃/CH₂ asymmetric stretch (AUC = 0.991; Sn = 100%, Sp = 86.67%), I3040/I2929 for olefinic/saturated lipid ratios (AUC = 0.973; Sn = 96.67%, Sp = 86.67%), and protein-associated amide-I region, using Erik Goormaghtigh's MATLAB Kinetics and demonstrated a strong discriminatory power. Multivariate PCA and hierarchical clustering analyses also confirmed clear segregation. FTIR revealed lipid-based spectral biomarkers in PsEVs, highlighting their potential for early diagnostic tools for PD screening and monitoring.
The binding of local anesthetics (LAs) to cell membranes is required for LAs to reach the target ion channels, but lipid interactions may also play a role in a purely membrane-mediated mode of activity. Here, we used solid-state NMR and further biophysical techniques to characterize the effect of six LAs covering a wide range of structures and properties (benzocaine, bupivacaine, mepivacaine, lidocaine, procaine, QX-314) on membranes. Membrane partitioning log D values (between 2.1 and 3.7) varied little with pH, in contrast to octanol partitioning. Membrane thinning was induced by most LAs, except for benzocaine. A conformational change in the lipid headgroup was observed, with a pronounced dependence on the protonation state, indicating the importance of the positive charge that is maintained by most membrane-bound LAs. We found stabilization of negative membrane curvature in the case of benzocaine, and of positive curvature in the case of bupivacaine, procaine, mepivacaine and, most pronouncedly, for QX-314. Comparing the LAs with respect of their influence on membranes as observed in the different experiments, benzocaine and QX-314 were always found at either extreme of the scale, with bupivacaine and lidocaine closer in their effect to benzocaine. This order of influence correlates with the depth of membrane insertion and with the protonation state, both of which were identified as key factors for LA behavior. Finally, we found indications that LAs are able to alter the activity of bacterial mechanosensitive channels without any expected LA binding sites, thus supporting a membrane-mediated activity of LAs.
The structural and dynamic properties of lipid membranes vary with bilayer size and hydration. While Molecular Dynamics (MD) simulations are a powerful tool for studying cellular membranes, results can be sensitive to the analysis workflow and software. In this study, all-atom MD simulations were conducted on 128, 256, 512, and 1024 POPC lipids at 40, 80, and 160 water molecules per lipid. A two-fold study was performed: (1) to assess the convergence of structural and dynamic properties of POPC bilayers as a function of membrane size and hydration level, including area per lipid (APL), bilayer thickness, order parameter, headgroup orientation, and lateral diffusion, and (2) to compare the performance of four software packages: CPPTRAJ (CPP), GROMACS (GRO), MDAnalysis (MDA), and LiPyphilic (LiP). For the first objective, the average APL, bilayer thickness, order parameter, and headgroup orientation were largely independent of size and hydration, whereas the lateral diffusion coefficient was sensitive to both. Notably, 128-lipid systems were susceptible to artificially inflated lateral diffusion due to finite-size artifacts at higher hydration levels. Increasing system size primarily decreased the statistical variance of APL and thickness. For the second objective, all four packages produced consistent results for APL and thickness, with the main discrepancy being a known artifact of the gmx order tool applied to unsaturated carbons. CPP was the fastest serial tool, whereas parallelization benefited MDA and LiP for some metrics. These findings demonstrate that moderately sized systems (e.g., 256L), combined with CPP, offer an efficient workflow for membrane structural property analysis.
Biological membranes are considered as laterally heterogeneous, containing disordered and ordered regions ranging in size from a few nanometers to micrometers. Pulsed dipolar EPR spectroscopy (PDS) is specifically designed to determine distances between spin-labeled molecules in the range from 1.5 to typically 8 nm. A non-steroidal anti-inflammatory drug ibuprofen, due to its amphiphilic nature, is integrated into the cell membrane. In this study, we investigated spin-labeled ibuprofen (ibuprofen-SL) in a lipid raft-mimicking bilayer formed by a mixture of dioleoyl-glycero-phosphocholine (DOPC), dipalmitoyl-glycero-phosphocholine (DPPC) and cholesterol (35:35:30 mol%). Two variants of PDS were used: double electron-electron resonance (DEER, also known as PELDOR) and the simple 2-pulse electron spin echo (2p ESE) method. Analysis of PDS data in the time and frequency domains shows that ibuprofen-SL molecules at a threshold concentration of about 1 mol% form 6-membered clusters in the membrane. The clusters apparently have a prismatic structure oriented along the normal to the membrane, with a maximum diagonal of about 4 nm, and are most likely located in cavities formed between nanodomains approximately 30 nm in diameter.
Organic anion transporting polypeptide (OATP) 1B1 is crucial for hepatic drug uptake. Three N-glycosylation sites, Asn134, Asn503, and Asn516, have been identified, and substituting the amino acid at these sites with glutamine reduces the expression of the OATP1B1 protein on the plasma membrane. However, the effects of disrupted N-glycosylation on the transport kinetics of OATP1B1 molecule remained uninvestigated. This study quantitatively analyzed the impact of N-glycosylation modifications on the intrinsic transport activity of OATP1B1. We established artificial mutant OATP1B1-expressing HEK293 cell lines with substitution of asparagine at one or multiple N-glycosylation sites with glutamine. The change in molecular weight of OATP1B1 protein in the plasma membrane fraction was assessed by Western blotting. OATP1B1 transport activity was evaluated by measuring 2',7'-dichlorofluorescein (DCF) uptake to determine the Michaelis constant (Km) and maximum transport rate (Vmax). The Vmax value per OATP1B1 molecule (Vmax,c) was calculated by dividing Vmax by the expression level of OATP1B1 in the plasma membrane fraction quantified by LC-MS/MS. Western blotting revealed that all mutant OATP1B1 proteins showed lower molecular weights than the wild-type. Among mutation sites, Asn134 was confirmed to be the primary glycosylation site, as reported previously. Regarding transport activity, the Km value of the Asn134/503/516Gln mutant decreased to 0.3-fold that of the wild-type. However, its Vmax,c value decreased to 0.2-fold that of the wild-type. Thus, N-glycosylation of OATP1B1 is not only important for the plasma membrane localization but also for transport activity.
Staphylococcus aureus (S. aureus) is an opportunistic pathogen that is a global health concern for its ability to cause a wide spectrum of clinical infections ranging from minor skin abscesses to systemic conditions such as sepsis and pneumonia. Due to the emergence of resistance to commonly used antibiotics, there has been interest in exploring the use of antimicrobial peptides to treat S. aureus infections. However, changes in the lipid composition of the lipid bilayer membrane can alter the activity of peptides, and S. aureus is able to induce variations in lipid composition in response to environmental stress. Here, we explore how the main lipid components in S. aureus are altered when exposed to LL-37, a human cathelicidin involved in primary immune response, and ATRA-1, a short antimicrobial peptide derived from the snake Naja atra venom. A lipidomic study is conducted through HPLC-MS-MS (LC-ESI-MS/MS) to quantify phosphatidylglycerol, cardiolipin, lysyl-phosphatidylglycerol, monogalacto- and digalacto-diacylglycerol, and carotenoids. In addition, menaquinones, responsible for electron transport during oxidative phosphorylation, were also quantified. Biophysical properties such as membrane electric surface potential and lipid packing were assessed. We find that lipid adaptation is specific to the type of antimicrobial peptide, where ATRA-1 mainly induces changes in the electric surface potential through variations in Lysyl-PG, while exposure to LL-37 changes carotenoid levels, inducing an increase in membrane rigidity as measured by FTIR. In addition, both peptides induce a reduction in menaquinone and DGDG levels. These findings highlight the role of membrane lipid remodeling as a peptide-specific response mechanism in S. aureus, with implications for the development of AMP-based therapies.
Plants produce natural rubber in specialized organelles, termed rubber particles (RP). The structure of the RP consists of a polyisoprene (rubber) core surrounded by a shell layer composed of a lipid monolayer and proteins. Among the prominent RP-associated proteins is small rubber particle protein (SRPP), the second most abundant protein on the Hevea brasiliensis rubber particle monolayer membrane. In this study, a combination of protein modeling, biophysical analysis, biochemistry, and transmission electron microscopy have been utilized to further characterize Hevea SRPP1 (HbSRPP1) membrane architectural rearrangement capabilities. Results obtained reveal that HbSRPP1 possesses characteristics reminiscent of apolipoproteins, including the ability to reorganize aqueous phospholipid dispersions into stable, disk-shaped nanoparticles, termed nanodisks. This research provides the groundwork for further studies into the mechanism of SRPP's interaction with the RP membrane surface which likely involves conformational changes in RP topology.