Biochimica et Biophysica Acta (BBA) - Biomembranes

Small-angle scattering can be used to derive structural information about membrane proteins reconstituted in suitable carrier systems enabling solubilization of the membrane proteins in question. Since the studies are done in solution, there is no need for crystallization or deposition on sample grids, and it is in principle possible to obtain structural information about intrinsically disordered regions which cannot be resolved by crystallography or the quantitative link to which is hard to establish using e.g. electron microscopy methods. In this study, tetramers of the gated spinach aquaporin SoPIP2;1 were reconstituted into nanodiscs and small-angle x-ray scattering data were recorded. From these data, we refine structural models of the entire nanodisc-membrane protein complex including the flexible regions using newly developed models based on Fast Debye sums. We introduce software for these computations available via online repositories and discuss the implications and limitations of these methods. Author summaryWhen it comes to investigating the structure and function of the proteins, a particular class of proteins are known to be cumbersome and problematic: membrane proteins that reside in the cell membrane and regulate and facilitate a number of critical biological processes. Such proteins can often not be studied by conventional means as they unravel and denature structurally or even precipitate in solution. To add insult to injury, such membrane proteins also often contain parts that are intrinsically disordered rendering them irresolvable by e.g. traditional crystallographic techniques and hard to describe structurally. Here, we present a combined computational and experimental approach (as well as the necessary software) to analyze and determine the structure of such proteins in close-to-native conditions in so-called nanodiscs, a biological carrier systems, using small-angle scattering and molecular simulations.

The physical properties and chemical composition of lipid droplets inside cells of the yeast Cryptococcus curvatus were investigated using quasi-elastic neutron scattering and mass spectrometry with complementary surface modeling using 3D microscopy. With temperature decrease from 310 to 280 K, their phase remained fluid, i.e., the droplets remained in the physiological state, unlike synthetic lipid membranes that transition to a gel phase. The lipid dynamics in the droplet was described by a model implying diffusion of the lipid and its hydrocarbon chains. The diffusion coefficient of the lipid chains (274x10-3 [A]2/ps at 310 K) was much higher than that observed in a synthetic lipid membrane because of the larger volume (up to 12 [A]) for the local dynamics in the droplet. These physical properties were correlated with the types of lipids composing the droplet. Based on that, the lipid packing and resulting energetic value of the yeast droplets are discussed in relation to their usefulness as biofuels.

Bicelles have been demonstrated to be a valuable tool for studying membrane protein interactions and structure in vitro. They are distinguished by a distinct lipid bilayer that mimics the plasma membrane of cells making it more native-like than its detergent micelle counter-part. Bicelles are typically comprised of a long-chain phospholipid such as dimyristoylphosphatidylcholine (DMPC) and a short-chain phospholipid such as dihexanoylphosphatidylcholine (DHPC). When mixed together in solution DMPC-DHPC bicelles assume a discoidal structure comprised of a heterogeneous arrangement where the short-chain lipids gather around the rim of the disk and the long-chain lipids form the flat, planar, bilayer region. In this study, the nonionic surfactant, C8E5, was used to prepare mixtures with DMPC to determine if it adopts properties similar to bicelles with a q [≥] 0.5. At q [≥] 0.5, DMPC-DHPC bicelles are bilayered and DMPC is sequestered from the detergent micelle-like DHPC. Mixtures of DMPC and C8E5 were prepared at various q values, a parameter used to describe the mole ratio of DMPC to DHPC in the preparation of bicelles. Employing biophysical methods like dynamic light scattering, 31P-NMR and analytical ultracentrifugation, properties of these lipid-detergent complexes are described. Interestingly they adopted a spherical-shaped micellar structure morphology and did not assume a discoidal shape typical of bicelles at q [≥] 0.5. However, they appear to retain bilayer-like properties that may prove beneficial for in vitro biophysical studies of membrane proteins.

Membrane mimetics such as lipid bicelles and nanodiscs have become indispensable platforms for high-resolution structural, dynamical, and functional studies of membrane-associated systems by NMR spectroscopy, cryo-electron microscopy, and X-ray crystallography. In particular, magnetically aligned bicelles and nanodiscs uniquely enable the measurement of anisotropic NMR interactions, providing direct access to membrane geometry, lipid order, thickness, and molecular dynamics. However, the quantitative interpretation of such anisotropic NMR spectra has been hindered by the absence of physically rigorous dynamic models that properly account for the coupled effects of molecular diffusion, orientational distribution, and membrane deformation. Here, we present a comprehensive theoretical framework for the dynamic simulation of 31P chemical shift anisotropy and 14N quadrupolar NMR lineshapes in bicelles and nanodiscs. The model explicitly incorporates lipid diffusion, orientational distributions on curved membrane geometries, and membrane thinning, enabling physically consistent and quantitatively accurate reproduction of experimentally observed anisotropic lineshapes. Using this framework, we simulate dynamic 31P and 14N NMR spectra of DMPC/DHPC bicelles and nanodiscs and demonstrate how membrane thinning and lipid diffusion govern the apparent reduction of anisotropic interactions commonly observed upon peptide or protein association. This approach establishes a general physical basis for interpreting anisotropic NMR spectra of aligned membrane mimetics and provides a unified platform for quantitative investigation of membrane structure, dynamics, and membrane-active biomolecular interactions.

Lipids in all biological membranes are distributed heterogeneously across the bilayer. A particularly striking example for this asymmetry is the yeast plasma membrane (PM), which exhibits a high concentration of very long-chain sphingolipids (SL) in its outer leaflet. Experimental observations indicate the existence of highly ordered gel-like PM domains that are enriched in SL but depleted in the major yeast sterol ergosterol. For a better mechanistic understanding of these unusual domains we have performed coarse-grained molecular dynamics simulations with membranes containing varying concentrations of very long-chain lipids. In agreement with experimental results we observed formation of a gel phase, with high order parameter of the acyl chains and hexagonal arrangement of lipid tails, at higher concentrations of very long-chain lipids. Our simulations also show that ergosterol is excluded from these gel regions and that, even when embedded into a liquid disordered phase, gels remain stable on the simulation time scale.

The study focuses on investigating the interaction of SARS-CoV-2 fusion peptide fragment with model membranes of various lipid composition to elucidate the molecular mechanisms of peptide-derived membrane fusion. The work utilized the short fragment of SARS-CoV-2 fusion peptide which is homologous to 816-827 region of the native SARS-CoV-2 FP (FP816-827) and contains the highly conserved LLF motif responsible for membrane fusion, and its ineffective analogue (mFP816-827), where LLF motif was replaced for AAA. Using fluorescence fusion assay, it was demonstrated that the LLF motif plays a key role in inducing liposome fusion, whereas its replacement completely abolishes this capability. The fusogenic activity of the peptide strictly depended on the vesicle lipid composition. It was potentiated by phosphatidylethanolamine and inhibited by phosphatidylserine. Molecular dynamics revealed that both peptides predominantly adopt an -helical conformation; however, the native peptide interacts more strongly with the hydrophobic core of the membrane by increasing peptide-lipid hydrophobic contacts, while the mutant version exhibits a more superficial localization. Differential scanning microcalorimetry data indicated that the ability of FP816- 827 to disturb lipid packing increased with decreasing membrane lipid tail length. The molecular mechanisms underlying the fusogenic activity of the SARS-CoV-2 fusion peptide were identified, specifically its ability to cluster phospholipid head groups in its own vicinity. As a result, local regions with positive spontaneous curvature are formed in the outer monolayer, facilitating membrane fusion. The findings highlight the role of membrane composition and lipid architecture in the mechanism of viral fusion with host cells.

The HIV-1 encoded protein Vpu forms an oligomeric ion channel/pore in membranes and interacts with multiple host proteins to support virus lifecycle. However, Vpu molecular mechanisms are currently not well understood. The structures of full-length Vpu in its monomeric and oligomeric forms are unknown, although both the monomer and oligomer are deemed important. Here, we report on the diversity of Vpu oligomeric structures and how the environment affects the Vpu oligomer formation. We produced a uniquely designed MBP-Vpu chimera protein in E. coli in soluble form. We subjected this protein to analytical size exclusion chromatography (SEC) and negative staining electron microscopy (nsEM). Strikingly, we found that MBP-Vpu forms stable oligomers in solution, presumably driven by Vpu transmembrane domain self-association. Our coarse modeling suggests that these oligomers are pentamers, in agreement with the pentameric membrane-bound Vpu. To the best of our knowledge, this is the first observation of Vpu self-association out of lipid membrane environment. We further found that MBP-Vpu oligomer stability decreases when the protein was reconstituted in lipid membrane mimetics, such as {beta}-DDM, and mixtures of lyso PC/PG or DHPC/DHPG--In these cases significant oligomer heterogeneity was observed with oligomeric order lesser than that of MBP-Vpu oligomer in solution, but larger oligomers were observed as well. Importantly, we found that in lyso PC/PG, above certain protein concentration, MBP-Vpu forms linear array-like structures, which is also novel. Thus, our studies provide unique information about Vpu protein quaternary organization by capturing multiple Vpu oligomeric structures, which we believe are physiologically relevant.

One way to mitigate the ongoing antimicrobial resistance crisis is to discover and develop new classes of antibiotics. As all antibiotics at some point needs to either cross or interact with the bacterial membrane, there is a need for representative models of bacterial membranes and efficient methods to characterize the interactions to novel antimicrobials - both to generate new knowledge and to screen compound libraries. Since the bacterial cell envelope is a complex assembly of lipids, lipopolysaccharides, membrane proteins and other components, constructing realistic synthetic liposome-based models of the membrane is both difficult and expensive. We here propose to let the bacteria do the hard work for us. Outer membrane vesicles (OMVs) are naturally secreted by Gram-negative bacteria, playing a role in communication between bacteria, as virulence factors, molecular transport or being a part of the antimicrobial resistance mechanism. OMVs consist of the bacterial outer membrane and thus inherit many components and properties of the native outer cell envelope. In this work we have isolated and characterized OMVs from E. coli mutant strains and clinical isolates of the ESKAPE members Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa. The OMVs were shown to be representative models for the bacterial membrane in terms of lipid composition with strain specific variations. The OMVs were further used to probe the interactions between OMV and antimicrobial peptides (AMPs) as model compounds by Surface Plasmon Resonance (SPR) and provide proof-of-principle that OMVs can be used as an easily accessible and highly realistic model for the bacterial surface in interaction studies. This further enables direct monitoring of the effect of induction by antibiotics, or the response to host-pathogen interactions.

The HIV-1 assembly occurs at the plasma membrane, where the GAG polyprotein plays a crucial role. The GAG-membrane association is directed by the matrix domain (MA), which is myristoylated and has a highly basic region that interacts with the anionic lipids. Several evidence suggests that the presence of phosphatidylinositol-(4,5)-bisphosphate (PIP2) highly influence this binding. In addition, MA also interacts with nucleic acids, which is proposed to be important for the specificity of GAG for PIP2-containing membranes. It is proposed that RNA could have a chaperone function when interacting with the MA domain, preventing GAG from associating with unspecific lipid interfaces. Here, we study the interaction of MA with monolayer and bilayer membrane systems, focused on the specificity for PIP2 and on the possible effects of a GAG N-terminal peptide to impair the binding for either RNA or membrane. We found that RNA decreases the kinetics of the protein association with lipid monolayers but without any effect on the selectivity for PIP2. Interestingly, for bilayer systems, this selectivity increases in presence of both the peptide and RNA, even for highly negative charged compositions, where MA by itself doesnt discriminate between membranes with or without PIP2. Therefore, we propose that the specificity of MA for PIP2-membranes might be related to the electrostatic properties of both membrane and protein local environments, rather than a simple difference in molecular affinities. This scenario gives a new understanding of the regulation mechanism with a macromolecular view instead of considering molecular interactions within a ligand-receptor model. ImportanceHIV-1 virions are formed at the PM of infected cells through a direct interaction of the viral GAG protein with lipids. This is a finely regulated process governed by the GAG N-terminal matrix domain, MA. Here, we obtained compelling evidence on how this process depends on the local dielectric environments of both, the membrane and MA. Using bio-membrane mimicking systems, we found how MA myristoylation is involved in the interfacial absorption and anchoring of the protein, where the interaction with RNA negatively regulates this process but in a lesser extent when traces of the PIP2 lipid are present. Additionally, an N-terminal GAG-derived peptide competes with MA for the nucleic acid binding and impair the protein-membrane interaction when PIP2 is absent. All these data allowed us to propose a model for MA association with lipid interfaces and how it depends on oligonucleotide binding, lipid composition and competing peptide presence.

Staphylococcus aureus is a human opportunistic pathogen capable of causing multiple infections in both humans and animals. It secretes a group of exotoxins, known as hemolysins, which are released to enhance its pathogenicity. All of them exhibit cytolytic activity on a variety of host cell types, but -hemolysin stands out for being the most thoroughly studied variant. In this work, we show the production and purification of S. aureus -hemolysin following a straightforward protocol and in sufficient quantity to consider it as a potential procedure for future biotechnological approaches. Functional and structural characterization has indeed revealed that the protein is fully functional, confirming the key role of cholesterol in the necessary protein-lipid interaction. Furthermore, it has also been shown that the purified toxin can be assembled into single-particle individual pores within soluble lipid platforms in the form of cholesterol-containing nanodiscs.

Annexins (ANXs) are a family of peripheral membrane binding proteins which play a vital role in the maintenance and function of cellular membranes. These proteins are known to be part of the plasma membrane repair machinery where they are known to bind to negatively charged lipids in the cell membrane in a calcium dependent manner. The shape of the plasma membrane is known to be a regulator of protein density and thereby affects several biological functions of the cell such as exo-and endocytosis, cell motility, immune responses and also membrane repair. Membrane deformation and curvature sensing by proteins is a well described phenomenon which can assist in recruitment of specific proteins to certain regions in the cell and facilitate membrane bending. Following ruptures in the plasma membrane, calcium influx assist in the association of ANXs with the membrane around the ruptured area. Due to the expected increase in curvature at the damaged membrane site, it has been suggested that both membrane curvature and Ca2+ participate in the recruitment of ANXs. We have investigated the curvature sensing of ANXA4 in giant unilamellar vesicles (GUVs) by pulling high curvature membrane tethers from the vesicle surface using optical tweezers showing that ANXA4 recruitment increases with higher membrane curvature. We also describe an assay for determining protein density on the plasma membrane by utilizing ANXA4s property as a calcium dependent membrane binding protein. This new assay allows us to investigate the effect of protein density on curvature sensing.

Phase separation plays critical roles in several membrane functions, and reduction or disappearance of phase coexistence by action of membrane-interacting molecules have been implicated in membrane function impairment. Here, we applied differential scanning calorimetry, electron paramagnetic resonance (EPR), and non-linear least-squares (NLLS) spectral simulations to study the effects of nicotine, a parasympathomimetic drug, on the two-phase coexistence of dipalmitoyl phosphatidylcholine (DPPC) lipid membrane. The thermodynamic quantities describing the DPPC phase coexistence are temperature dependent, giving rise to non-linear vant Hoff behavior. Our results showed that nicotine preferentially binds to the fluid phase and modifies the enthalpy and entropy changes of the DPPC heat capacity profile, while marginally perturbing the homogeneous gel and fluid phases. An EPR/NLLS/vant Hoff analysis of the DPPC phase coexistence revealed that nicotine significantly modified the temperature dependence of the free energy change of the two-phase equilibrium from a cubic to a parabolic behavior, resulting in an alteration of the thermodynamical driving force and the balance of the non-covalent interactions of the lipids in equilibrium. The thermotropic behavior of the enthalpy, entropy, and heat capacity changes, as determined by EPR, indicated that nicotine modified the relative contributions of hydrogen-bonding, electrostatic interactions, and conformational entropy of the lipids to the thermodynamics of the phase coexistence. The predominantly entropically-driven gel-fluid transition in nicotine-free DPPC changes to a temperature-triggered entropically-driven or enthalpically-driven process in nicotine-bound DPPC. Further applications of this thermodynamic EPR/NLLS/vant Hoff analysis are discussed.

The structural and functional characteristics of membrane proteins can be influenced by the composition of the membrane. Consequently, native membranes are most relevant for the study of receptors and other membrane proteins. In this study, we investigated two types of cell-derived vesicles: natively shed extracellular vesicles (EVs) and mechanically derived vesicles (MVs). To this end, we utilized the human breast cancer cell line SKBR3, which strongly overexpresses the receptor HER2. We designed a protocol based on designed ankyrin repeat proteins (DARPins) to purify EVs and MVs enriched in HER2, and to ensure the native orientation of the HER2 receptors within the vesicle. The isolated HER2-containing EVs and MVs were characterized by cryo-EM, cryo-electron tomography (cryo-ET) and mass spectrometry (MS), which revealed fundamental differences between the different vesicle types. Our study highlights the greater structural diversity of EVs over MVs. A single particle cryo-EM analysis and classification of all visible receptors on the vesicle surface yielded electron density consistent with HER2 at modest resolution. Taken together, our results suggest that MVs can serve better than EVs as a suitable platform for the structure determination of membrane proteins within their native membrane environments.

The transbilayer distribution of plasma membrane cholesterol remains uncertain despite repeated analysis. We propose a new mechanism driving cholesterol sidedness: sterols form simple stoichiometric associations with phospholipids. Our model postulates that the phospholipids in the plasma membrane bilayer are fully complexed with cholesterol. The cholesterol in each leaflet is then the product of the abundance of its phospholipid and its sterol stoichiometry. Notably, lipid affinities are not relevant. Applying literature values for the composition, abundance and sterol stoichiometry of the phospholipid in each leaflet, the model predicts that two-thirds of the cholesterol in the human erythrocyte membrane bilayer is located in its outer leaflet, an exofacial to endofacial ratio of 2:1. The model also predicts that the overall cholesterol content of the bilayer is [~]0.75 mole/mole phospholipid, in agreement with literature values. Furthermore, our analysis suggests that the areas of the two membrane leaflets are about the same. The concordance of prediction with observation validates the model and the values used for the parameters. The sterol in the exofacial leaflet of the plasma membrane of any cell is predicted to exceed that on its contralateral side when its phospholipids have a higher sterol stoichiometry and are fully complexed. SynopsisWe propose that the transbilayer distribution of cholesterol in the plasma membrane bilayer is determined by its complexation with the phospholipids in the two leaflets. Because the complexes are homeostatically filled to stoichiometric equivalence, leaflet cholesterol is given by the abundance of its phospholipids multiplied by its sterol stoichiometry. The model predicts that two-thirds of the cholesterol in the human erythrocyte membrane bilayer resides in the outer leaflet. It also predicts the cholesterol content of the bilayer as a whole.

Using the solvatochromic membrane probes Laurdan and Pro12A we investigated order-disorder phase transitions in cellular lipid membranes of HeLa cells. Due to internalization of Laurdan its fluorescence signal yields information about inner and outer cellular membranes while Pro12A exclusively stains the plasma membrane. The two different membrane-embedded dyes show an emission redshift upon increasing disorder of the surrounding lipids that can be quantified by the Generalized Polarization (GP). First, we compare the sensitivity of both probes to lipid phase transitions by analyzing GP of synthetic lipid vesicles as function of temperature. Second, we investigate the temperature dependent lipid order of HeLa cell membranes and find that the plasma membrane shows a partially reversible order-disorder transition regime at temperatures between T = 20 {degrees}C and T = 70 {degrees}C. Third, we calorimetrically detect an irreversible transition at T = 55 {degrees}C and conclude that the optically detected restructuring of the plasma membrane can be partially attributed to protein denaturation. At last, it is shown that the reversible plasma membrane transition temperature Tm shifts from Tm = 25 {degrees}C to Tm =-16 {degrees}C upon cholesterol depletion and sharpens from a half width of {Delta}TFWHM = 60 K to {Delta}TFWHM = 8 K. The reversibility and the sensitivity to cholesterol of this transition indicate a temperature-induced lipid melting within the plasma membrane of HeLa cells.

The rising challenge of antimicrobial resistance has accelerated the search for alternative therapeutics. Antimicrobial peptides (AMPs), a class of naturally occurring defense molecules found across diverse species, are promising candidates. Despite their potent membrane-disrupting activity, the atomic details of the pore formation process remain insufficiently understood. In this study, we employed all-atom molecular dynamics (MD) simulations to investigate the pore formation process of melittin under different initial configurations. Simulations were conducted using three different membrane systems: a pure POPC bilayer, a mammalian membrane model (DOPC:Cholesterol, 9:1), and a bacterial membrane model (DOPE:DOPG, 3:1). For each system, we examined four different starting configurations, in which six melittin peptides were arranged in a star-like pattern. Our results demonstrated that the pore formation process strongly depends on the initial peptide configuration. In one specific initial arrangement (termed as Conf.I), pore formation consistently occurred within 100 nanoseconds, regardless of membrane composition. Furthermore, the simulations revealed that pore formation was more challenging in the mammalian membrane model and even more so in the bacterial membrane model, in comparison with the pure POPC bilayer. These findings are in line with previously reported minimum inhibitory concentration (MIC) and the 50% hemolysis concentration (HC50) of melittin in the literature. Additionally, we identified lysine-7 (K7) as the key residue in determining whether a stable pore can form. In configurations where the K7 side chain formed electrostatic interactions with the phosphate group of a lipid, melittin were anchored to the membrane surface, thereby preventing pore formation. In contrast, simulations of melittin mutants K7A and K7Q showed no such anchoring effect, and thus pore formation was possible in multiple initial configurations. Notably, the K7Q mutation showed a preference for pore formation in bacterial membranes over mammalian membranes, suggesting that reducing toxicity while maintaining antimicrobial efficacy is possible.

The machinery involved in cytotoxic T-cell activation requires three main characters such as: the major histocompatibility complex class I (MHC I) bound to the peptide (p), the T-cell receptor (TCR), and the CD3-complex which is a multidimer interfaced with the intracellular side. The pMHC:TCR interaction has been largely studied both in experimental and computational models, giving a contribution in understanding the complexity of the TCR triggering process. Nevertheless, a detailed study of the structural and dynamical characterization of the full complex (pMHC:TCR:CD3-complex) is still missing, due to insufficient data available on the CD3-chains arrangement around the TCR. The recent determination of the TCR:CD3-complex structure by means of Cryo-EM technique has given a chance to build the entire proteins system essential in the activation of T-cell, and thus in the adaptive immune response. Here, we present the first full model of the pMHC interacting with the TCR:CD3-complex, built in a lipid environment. To describe the conformational behaviour associated with the unbound and the bound states, all atoms Molecular Dynamics simulations were performed for the TCR:CD3-complex and for two pMHC:TCR:CD3-complex systems, bound to two different peptides. Our data point out that a conformational change affecting the TCR Constant {beta} (C{beta}) region occurs after the binding to the pMHC, revealing a key role of such a region in the propagation of the signal. Moreover, we found that the TCR reduces the flexibility of the MHC I binding groove, confirming our previous results.

Peptide nanodiscs are promising anti-atherosclerosis therapeutics, drug delivery particles and structural biology tools. However, the lack of experimental methods for structural and dynamical characterization of these particles hinders their further development. Here we integrated nuclear magnetic resonance (NMR), small-angle x-ray scattering, and small-angle neutron scattering experiments with molecular dynamics (MD) simulations to investigate the structure and dynamics of peptide nanodiscs stabilized by the apolipoprotein A-I mimetic peptide 22A with therapeutic activity against atherosclerosis. This multi-technique approach takes advantage of combining average size and shape information from small-angle scattering, peptide site-specific information from NMR spectroscopy, and interpretative power of MD simulations. Our results reveal the intrinsic polydispersity in size of peptide nanodiscs, highlighting the importance of careful interpretation when using averaged experimental parameters. Our consensus model suggests that 22A peptides are predominantly in -helical configuration with a disordered inter-helical orientation around the lipid matrix. The terminal regions of the peptides display greater flexibility relative to the peptide core and an enhanced C-terminal exposure to solvent, which could facilitate interaction with the enzyme LCAT. Interestingly, our results indicate that peptides and lipids rotate together as a rigid body. The methodological approach described in this paper paves the way for the design of more stable and effective therapeutic nanodiscs and for the characterization of other biomolecular aggregates that are beyond the scope of current structural biology techniques. Significance StatementNanodiscs stabilized by 22A apoA-I mimetic peptides hold significant pharmaceutical potential for treating cardiovascular diseases by mimicking HDL functions, yet their development is hindered by the difficulty of characterizing disordered biomolecular systems. Standard structural biology techniques cannot readily resolve the structure and dynamics of these peptide nanodiscs, which is essential for rational therapeutic design. Here, we integrate complementary biophysical experiments with MD simulations to establish a consensus model of 22A peptide nanodisc structure, dynamics, and interactions with biological partners at molecular resolution. Beyond advancing peptide nanodisc design, our integrative methodology provides a generalizable framework for characterizing other disordered biomolecular assemblies that are similarly challenging to conventional structural approaches.

The Aurein 1.2 is an anuran antimicrobial peptide (AMP) with a proven lytical activity against bacterial membranes. Previously, we found a differential action of aurein by both experimental and computational methods. This differential action was over membranes of two related probiotic strains, where the main differences between membranes were the number of glycolipids on lipid composition. In the present work, we focused on the differential behavior of the aurein 1.2 at its interaction with bacterium model membranes with different amounts of glycolipids in their composition. We carried out extensive molecular dynamics (MD) simulations by using the coarse-grain force field MARTINI and raising up differential mixtures of phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and monogalactosylglycerol (MG). We found a correlation between the presence of MG in PG/PE mixtures and the difficulty of aurein to stabilize pore structures, suggesting an AMPresistance factor encoded in the lipid composition of the membrane. Through this study, we hope to shed light on a possible resistance mechanism to AMPs related with the glycolipid content of diverse types of bacterial membranes.

Staphylococcus aureus readily adapts to various environments and quickly develops antibiotic resistance, which has led to an increase in multidrug-resistant infections. Hence, S. aureus presents a significant global health issue and its adaptations to the host environment are crucial for understanding pathogenesis and antibiotic susceptibility. When S. aureus is grown conventionally, its membrane lipids contain a mix of branched-chain and straight-chain saturated fatty acids. However, when unsaturated fatty acids are present in the growth medium, they become a major part of the total fatty acid composition. This study explores the biophysical effects of incorporating straight-chain unsaturated fatty acids into S. aureus membrane lipids. Membrane preparations from cultures supplemented with oleic acid showed more complex differential scanning calorimetry scans than those grown in tryptic soy broth alone. When grown in the presence of oleic acid, the cultures exhibited a transition significantly above the growth temperature, attributed to the presence of glycolipids with long-chain fatty acids causing acyl chain packing frustration within the bilayer. Functional aspects of the membrane were assessed by studying the kinetics of dye release from unilamellar vesicles induced by the antimicrobial peptide mastoparan X. Dye release was slower from liposomes prepared from cells grown in oleic acid-supplemented cultures, suggesting that changes in membrane lipid composition and biophysics protect the cell membrane against peptide-induced lysis. These findings underscore the intricate relationship between the growth environment, membrane lipid composition, and the physical properties of the bacterial membrane, which should be considered when developing new strategies against S. aureus infections.