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Nanosized dispersions of the bicontinuous cubic phase (cubosomes) are emerging carriers for drug delivery. These particles possess well-defined internal structures composed of highly-curved lipid bilayers that can accommodate significant drug payloads. Although cubosomes present promising potential for drug delivery, their physicochemical properties and interactions with cell membranes have not yet been fully understood. To clarify the interactions of the cubosomes with cell membranes, we investigated the changes in the structural and cubic membranes of monoolein (MO) cubosomes when mixed with model cell membranes at different phase states using time-resolved small-angle X-ray scattering (TR-SAXS), cryogenic transmission electron microscopy (cryo-TEM), and fluorescence spectroscopy. TR-SAXS results showed that the cubosomes gradually transitioned from the Im 3 m phase to the lamellar phase after interacting with the liposomes. The time of the structural change of the cubic phase to the lamellar phase was influenced by the fluidity of the liposome bilayers. Mixing the cubosomes with fluid membrane liposomes required less time to transition to the lamellar phase and vice versa. Cryo-TEM images showed that the well-defined internal structure of the cubosomes disappeared, leaving behind lamellar vesicles after the interaction, further confirming the TR-SAXS results. Laurdan fluorescence probe was used to assess the membrane polarity changes occurring to both the cubosomes and liposomes during the interaction. Examination of the normalized fluorescence intensity of the probed cubosomes showed decreasing intensity, followed by a recovery of intensity, which could indicate the disintegration of the cubic membrane and the formation of a mixed membrane. Also, the kinetics of the disintegration of the cubic phase did not seem to be influenced by the composition of the liposomes, which was in line with the normalized SAXS intensity results. Assessing the generalized polarization ( GP 340 ) values of the cubosomes and liposomes after mixing revealed that the fluidity and membrane hydration states of the cubosomes and liposomes transitioned to resemble their counterpart, confirming the exchange of material between the particles. Over time, the hydration states of the cubosomes and liposomes equilibrated toward an intermediate state between the two. The time needed to reach the final intermediate state was influenced by the membrane fluidity and hydration of the liposomes, more particularly the difference in GP 340 values and their membrane phase state. These results highlight the importance of examination of the cubic membrane conditions, such as membrane polarity, and their implications on the changes in the cubic structure during the interaction with liposomal membranes.

Hyaluronan, also known as hyaluronic acid, is a large glycosaminoglycan composed of repeating disaccharide units. It plays a crucial role in providing structural support, hydration, and facilitating cellular processes in connective tissue, skin, and the extracellular matrix in biological systems. We present a coarse-grained (CG) model of hyaluronic acid (HA) and its constituent residues, N -acetyl-d-glucosamine (GlcNAc) and glucuronic acid (GlcA), designed to be compatible with the Martini 3 force field. The model was validated against atomistic molecular dynamics simulations following standard procedures to ensure the accuracy of bonded interactions and, in the case of GlcNAc, the free energies of transfer between octanol and water. For the final HA model, we investigated its properties by simulating the self-assembly of HA chains at varying ion concentrations in solution and comparing the persistence length of single-chain HA with experimental data. We also studied the interactions of HA with lipid bilayers and various HA-binding proteins, demonstrating the ability of the model to accurately reproduce interactions with other biomolecules characteristic of natural biological systems. This extension of the carbohydrate-dedicated branch of the CG Martini 3 force field enables large-scale molecular dynamics simulations of HA-containing systems and contributes to a better understanding of the roles and functions of hyaluronan in natural biomolecular systems.

Selectivity is a key requirement for membrane-active antimicrobials to be viable in therapeutic contexts. Therefore, the rational design or suitable selection of new compounds requires adequate mechanistic understanding of peptide selectivity. In this study, we compare two similar cyclic peptides that differ only in the arrangement of their three hydrophobic tryptophan (W) and three positively charged arginine (R) residues, yet exhibit different selectivities. This family of peptides has previously been shown to target the cytoplasmic membrane of bacteria, but not to act directly by membrane permeabilization. We have systematically studied and compared the interactions of the two peptides with zwitterionic phosphatidylcholine (PC) and negatively charged phosphatidylglycerol/phosphatidylethanolamine (PG/PE) model membranes using various biophysical methods to elucidate the mechanism of the selectivity. Like many antimicrobial peptides, the cyclic, cationic hexapeptides investigated here bind more efficiently to negatively charged membranes than to zwitterionic ones. Consequently, the two peptides induce vesicle leakage, changes in lipid packing, vesicle aggregation, and vesicle fusion predominantly in binary, negatively charged PG/PE membranes. The peptide with the larger hydrophobic molecular surface (three adjacent W residues) causes all these investigated effects more efficiently. In particular, it induces leakage by asymmetry stress and/or leaky fusion in zwitterionic and charged membranes, which may contribute to high activity but reduces selectivity. The unselective type of leakage appears to be driven by the more pronounced insertion into the lipid layer, facilitated by the larger hydrophobic surface of the peptide. Therefore, avoiding local accumulation of hydrophobic residues might improve the selectivity of future membrane-active compounds.

Cells contain thousands of different lipids. Their rapid and redundant metabolism, dynamic movement, and many interactions with other biomolecules have justly earned lipids a reputation as a vexing class of molecules to understand. Further, as the cell's hydrophobic metabolites, lipids assemble into supramolecular structures─most commonly bilayers, or membranes─from which they carry out myriad biological functions. Motivated by this daunting complexity, researchers across disciplines are bringing order to the seeming chaos of biological lipids and membranes. Here, we formalize these efforts as "synthetic lipid biology". Inspired by the idea, central to synthetic biology, that our abilities to understand and build biological systems are intimately connected, we organize studies and approaches across numerous fields to create, manipulate, and analyze lipids and biomembranes. These include construction of lipids and membranes from scratch using chemical and chemoenzymatic synthesis, editing of pre-existing membranes using optogenetics and protein engineering, detection of lipid metabolism and transport using bioorthogonal chemistry, and probing of lipid-protein interactions and membrane biophysical properties. What emerges is a portrait of an incipient field where chemists, biologists, physicists, and engineers work together in proximity─like lipids themselves─to build a clearer description of the properties, behaviors, and functions of lipids and membranes.

In this study, we investigated the stability of the fully activated conformation of the orexin receptor 2 (OX 2 R) embedded in a pure POPC bilayer using MD simulations. Various thermodynamic ensembles (i.e., NPT , NVT , NVE , NPAT , μVT , and NPγT ) were employed to explore the dynamical heterogeneity of the system in a comprehensive way. In addition, informational similarity metrics (e.g., Jensen-Shannon divergence) as well as Markov state modeling approaches were utilized to elucidate the receptor kinetics. Special attention was paid to assessing surface tension within the simulation box, particularly under NPγT conditions, where 21 nominal surface tension constants were evaluated. Our findings suggest that traditional thermodynamic ensembles such as NPT may not adequately control physical properties of the POPC membrane, impacting the plausibility of the OX 2 R model. In general, the performed study underscores the importance of employing the NPγT ensemble for computational investigations of membrane-embedded receptors, as it effectively maintains zero surface tension in the simulated system. These results offer valuable insights for future research aimed at understanding receptor dynamics and designing targeted therapeutics.

Analyzing the cell interface is of paramount importance in understanding how cells interact and communicate with other cells, but an advanced analytical platform that can process complex and networked interactions between cell surface ligands and receptors is lacking. Herein, we developed the cell-interface-deciphering lipid nanotablet (CID-LNT) for multiplexed real-time cell analysis. LNT is a nanoparticle-tethered lipid bilayer chip where freely diffusing plasmonic nanoparticles induce scattering signal changes. The CID-LNT transduces cell surface protein information into DNA data, which operate as nanoparticle logic gates. As a proof of concept, we detected and analyzed programmed death-ligand 1 (PD-L1) and associated immune signals (TNF-α, EGF, and IFN-γ). PD-L1 is an immune checkpoint that suppresses T cell activity with inflammatory biomolecules facilitating its expression. The CID-LNT can serve as a dynamic nanoparticle logic board, enabling the logic gate-based analysis of membrane proteins, and can be expanded to immunological synapse analysis, cell interface engineering, and molecular diagnostics.

Transmembrane glucose transport, facilitated by glucose transporters (GLUTs), is commonly understood through the simple mobile carrier model (SMCM), which suggests that the central binding site alternates exposure between the inside and outside of the cell, facilitating glucose exchange. An alternative "multisite model" posits that glucose transport is a stochastic diffusion process between ligand-operated gates within the transporter's central channel. This study aims to test these models by conducting atomistic molecular dynamics simulations of multiple glucose molecules docked along the central cleft of GLUT1 at temperatures both above and below the lipid bilayer melting point. Our results show that glucose exchanges occur on a nanosecond time-scale as glucopyranose rings slide past each other within the channel cavities, with minimal protein conformational movement. While bilayer gelation slows net glucose transit, the frequency of positional exchanges remains consistent across both temperatures. This supports the observation that glucose exchange at 0 °C is much faster than net flux, aligning with experimental data that show approximately 100 times the rate of exchange flux relative to net flux at 0 °C compared to 37 °C.

Antimicrobial resistance has motivated the search for antimicrobial agents with multimodal mechanisms of action. Host defense peptides and bacteriocins hold particular promise in this regard. Among many molecules discovered to date, thanatin appears to represent the properties of the two classes in that it, like bacteriocins, adopts a highly stable fold in solution and, like host defense peptides, exhibits broad-spectrum antibiotic activity. The peptide is believed to depolarize bacterial outer membranes and inhibit lipopolysaccharide transport while restoring bacterial susceptibility to β-lactam antibiotics. However, a direct observation of whether and how thanatin affects membranes is lacking. Here we reason that the peptide should promote bacteriocin-like multimodal poration in phospholipid bilayers. We demonstrate that thanatin induces poration with elements of membrane thinning, fractal ruptures, and transmembrane channels, a phenomenon common for bacteriocin folds but atypical of antimicrobial peptides. The results offer mechanistic insight into the action of antimicrobial agents emerging from different molecular classes but with similar properties.

Squalamine is an aminosterol from dogfish shark which has drawn attention, besides its antimicrobial activity, as a drug candidate in the treatment of Parkinson's disease due to its ability to prevent binding of α-synuclein to lipid membranes. To get insight into the mode of action of this steroid, we studied the influence of squalamine on lipid bilayers and whether it could inhibit the binding of a model peptide. Solid-state 19 F NMR of labeled [KIGAKI] 3 indicated that, indeed, this peptide no longer binds as a flexible chain to the bilayer in the presence of squalamine. When the cationic squalamine was added to lipid vesicles containing phosphatidylglycerol lipids, the aminosterol was found in differential scanning calorimetry and solid-state 31 P NMR experiments to lower the gel-to-fluid phase transition and cause the phase separation of domains enriched in anionic lipids. Squalamine had only a little influence on 2 H NMR relaxation and on the order parameters of the chains. These findings indicate that the aminosterol does not affect the molecular mobility of the hydrophobic core of the bilayer; hence, it does not insert into the membrane, nor causes thinning as found for molecules inserting in the headgroup region. On the other hand, squalamine was found to interact with lipid headgroups through electrostatic interactions, as seen by solid-state 2 H NMR on headgroup-labeled lipids. Furthermore, 31 P NMR showed that squalamine shifted the lamellar-to-hexagonal phase transition of phosphatidylethanolamine lipids to higher temperatures, indicating a preference for positively curved membranes. Altogether, our experiments indicate a strong interaction of the cationic squalamine with lipid headgroups, in particular with anionic lipids. This affinity for membranes is strong enough to efficiently displace cationic polypeptides, confirming the proposed action mechanism in Parkinson treatment. Notably, supported by 1 H- 1 H NOESY experiments, it was found that squalamine does not insert into the bilayer, but rather acts as facial amphiphile binding to the membrane surface. The binding to membranes may be envisaged in the form of oligomeric or micellar assemblies, which can disrupt the membrane at high concentrations, thereby explaining the antimicrobial and antifungal activities of squalamine.

Second harmonic generation (SHG) measurements using SHG-active dye molecules have recently attracted attention as a method to detect the formation of pores in phospholipid bilayers. The bilayers, in which the dye molecules are embedded in the outer leaflet, exhibit a noncentrosymmetric structure, generating SHG signals. However, when pores form, these dye molecules translocate through the pores into the inner leaflet, leading to a more centrosymmetric structure and the subsequent loss of the SHG signals. A decrease in the SHG signals has been experimentally observed in membranes subjected to electrical stimuli. However, the characteristics of the interleaflet translocation of SHG-active dye molecules through pores remain unclear, hindering quantitative estimation of the membrane conditions, such as the pore size and density, based on the SHG signal reduction. In this study, we investigated the interleaflet translocation characteristics of Ap3, an SHG-active dye molecule, using molecular dynamics (MD) simulations and two-dimensional random-walk (RW) simulations. The MD simulations revealed that Ap3 molecules only translocate between the leaflets along the pore sidewalls. We determined the lateral diffusion coefficient of Ap3 within the membrane plane and its propensity for interleaflet movement at the pore wall. Based on these movement characteristics, the RW model successfully reproduced the characteristic time scale of the interleaflet translocation observed in the MD simulations. By varying the pore size and density in the RW simulations, we estimated that the characteristic time scale of interleaflet translocation depends on the -0.31 power of the pore radius and the -1.13 power of the pore density. Using these findings, we estimated the number of pores that probably formed in membranes during previous electroporation experiments. These results indicate the potential of optical measurement of the dye molecule movement for the indirect quantitative estimation of the pore size and number, which are challenging to measure optically.

The enduring pathogenicity of Mycobacterium tuberculosis can be attributed to its lipid-rich cell wall, with mycolic acids (MAs) being a significant constituent. Different MAs' fluidity and structural adaptability within the bacterial cell envelope significantly influence their physicochemical properties, operational capabilities, and pathogenic potential. Therefore, an accurate conformational representation of various MAs in aqueous media can provide insights into their potential role within the intricate structure of the bacterial cell wall. We have carried out MD simulations of MAs in an aqueous solution and shed light on various structural properties such as thickness, order parameters, area-per-MAs, conformational changes, and principle component (PC) in the single-component and mixture MAs monolayer. The different conformational populations in the monolayer were estimated using the distance-based analysis between the function groups represented as W, U, and Z conformations that lead to the fold of the MAs chain in the monolayer. Additionally, we have also simulated the mixture of alpha-MA (α-MA or AMA), methoxy-MA (MMA), and keto-MA (KMA) with 50.90% AMA, 36.36% MMA, and 12.72% KMA composition. The thickness of the MAs monolayer was observed to range from 5 to 7 nm with an average 820 kg/m 3 density for α-MA, MMA, and KMA quantitative agreement with experimental results. The mero chain (long chain), consisting of a functional group at the proximal and distal positions, tends to fold and exhibit a more disordered phase than the short chain. The keto-MA showed the greatest WUZ total conformations (35.32%) with decreasing trend of eZ > eU > aU > aZ folds in both single component and mixture. Our results are in quantitative agreement with the experimental observations. The sZ folds show the lowest conformational probability in monolayer assembly (0.75% in a single component and 1.1% in a mixture). However, eU and aU folds are most probable for AMA and MMA. One striking observation is the abundance of MA conformers beyond the known WUZ convention because of the wide range distribution of intramolecular distances and change in dihedral angles. From a thermodynamic perspective, all mycolic acid monolayers in this study within the microsecond-long simulation, MA molecules self-assembled, and the self-assembled monolayer was found to be stable. The conformation of MAs corresponding to lower free energy minima in the monolayer gives rise to tighter packing and a highly dense self-assembly. Such a highly packed assembly shows higher resistance for drug permeability. Therefore, we concluded that the monolayer formed by AMA will be more densely packed and may cause more resistance for the drug molecules.

Biological memory is the ability to develop, retain, and retrieve information over time. Currently, it is widely accepted that memories are stored in synapses (i.e., connections between brain cells throughout the brain) through a process known as synaptic plasticity, which leads to either long-term potentiation (LTP) or long-term depression (LTD). However, the strengthening (LTP) and weakening (LTD) of synapses involve post-translational modifications to neural networks requiring de novo gene expression, a lengthy and energetically expensive process. Recently, we observed that lipid bilayers in the absence of peptides/proteins are capable of LTP, not unlike what has been observed in mammals and birds. As such, this finding has prompted us to postulate that the lipid bilayer provides a good model for understanding the molecular basis of biological memory. In this article, we discuss the status, challenges, and opportunities of neuronal plasma membranes as structures for biological memory and learning, therapeutic targets for various brain disorders, and platforms for neural network developments.

The aggregation and accumulation of amyloid β 42 (Aβ42) peptides on the surface of brain cells is associated with Alzheimer's disease (AD); however, the underlying molecular mechanisms remain unclear. Herein, we used a unique brain-mimetic open system that continuously flows Aβ42 solution to analyze the initial aggregation and adsorptive nature of Aβ42 at physiological concentrations on the lipid membrane. The open system accelerated the adsorption and dimerization kinetics. Upon the addition of Aβ42, monomeric Aβ42 was dominant on the lipid bilayer surface in the closed system with no flow, whereas dimers and high-order oligomers were dominant in the open system. Closed and open systems exhibited different oligomerization kinetics, lipid-Aβ42 interactions, and diffusive properties of monomers and oligomers. These results indicate the specific adsorptive and diffusive nature of Aβ42 on the cell membrane. The open system may help in elucidating the molecular mechanisms underlying AD progression.

We demonstrate, using non-equilibrium molecular dynamics simulations, that lipid membrane capacitance varies with surface charge accumulation linked to membrane shape and curvature changes. Specifically, we show that lipid membranes exhibit a hysteretic response when exposed to oscillatory electric fields. The electromechanical coupling in these membranes leads to hysteretic buckling, in which the membrane can spontaneously buckle in one of two distinct directions along the electric field, even for the same ionic charge accumulation at the water-membrane interface. In this regard, these binary buckled membrane states suggest potential applications in neuromorphic computing. Their bistable nature, characterized by two distinct and stable configurations, could serve as a foundation for implementing memory storage systems and logic operations. Furthermore, we introduce a circuit model that captures these dynamic effects, offering insights into emergent memory effects in electrically stimulated lipid membranes. Finally, this work presents lipid bilayers as dynamic, adaptable elements and suggests a new platform for exploring energy storage, information processing, and memory encoding at the lipid membrane level.

Nanodiscs have become a popular membrane mimetic system offering a well-defined bilayer environment to stabilize membrane proteins for in vitro analyses using a range of analytical methods; however, lipid compositions common to their deployment are simplistic and often fail to model native membrane complexity. Furthermore, there has been a general lack of rigorous analytical and biophysical characterization of nanodiscs comprising more than one lipid. To address these challenges, we coupled a nanodisc formation and purification workflow with targeted LC-MS/MS analysis to quantify lipids in nanodiscs made with different compositions. We screened lipids with a variety of headgroups and acyl chains and found that lipids did not always incorporate into nanodiscs at expected levels. Disparities in lipid incorporation were found to increase upon the addition of lipids known to induce curvature or rigidity to the membrane. Additionally, we found that adding just one additional type of lipid to nanodiscs changes the particle diameter and dispersity compared to nanodiscs containing a single lipid. We also formed and characterized nanodiscs using a complex starting composition inspired by the endoplasmic reticulum membrane and observed native-like cholesterol dynamics that modulated the lipid fluidity in the model bilayer system. Taken together, this work serves as a foundation for understanding nonstoichiometric lipid incorporation into nanodiscs and provides a basis for more thorough nanodisc characterization and quality control, which is critical to ensure multilipid nanodiscs synthesized accurately model the biological system of interest, enabling robust characterization of how the lipid landscape affects membrane protein structure and activity.

Cell-penetrating peptides (CPPs) can translocate into cells without inducing cytotoxicity. The internalization process implies several steps at different time scales ranging from microseconds to minutes. We combine adaptive Steered Molecular Dynamics (aSMD) with conventional Molecular Dynamics (cMD) to observe nonequilibrium and equilibrium states to study the early mechanisms of peptide-bilayer interaction leading to CPPs internalization. We define three membrane compositions representing bilayer sections, neutral lipids (i.e., upper leaflet), neutral lipids with cholesterol (i.e., hydrophobic core), and neutral/negatively charged lipids with cholesterol (i.e., lower leaflet) to study the energy barriers and disruption mechanisms of Arg9, MAP, and TP2, representing cationic, amphiphilic, and hydrophobic CPPs, respectively. Cholesterol and negatively charged lipids increase the energetic barriers for the peptide-bilayer crossing. TP2 interacts with the bilayer by hydrophobic insertion, while Arg9 disrupts the bilayer by forming transient or stable pores. MAP has shown both behaviors. Collectively, these findings underscore the significance of innovative computational approaches in studying membrane-disruptive peptides and, more specifically, in harnessing their potential for cell penetration.

The assembly of proteins in membranes plays a key role in many crucial cellular pathways. Despite their importance, characterizing transmembrane assembly remains challenging for experiments and simulations. Equilibrium molecular dynamics simulations do not cover the time scales required to sample the typical transmembrane assembly. Hence, most studies rely on enhanced sampling schemes that steer the dynamics of transmembrane proteins along a collective variable that should encode all slow degrees of freedom. However, given the complexity of the condensed-phase lipid environment, this is far from trivial, with the consequence that free energy profiles of dimerization can be poorly converged. Here, we introduce an alternative approach, which relies only on simulating short, dynamically unbiased paths, avoiding using collective variables or biasing forces. By merging all paths, we obtain free energy profiles, rates, and mechanisms of transmembrane dimerization with the same set of simulations. We showcase our algorithm by sampling the spontaneous association and dissociation of a transmembrane protein in a lipid bilayer, the popular coarse-grained Martini force field. Our algorithm represents a promising way to investigate assembly processes in biologically relevant membranes, overcoming some of the challenges of conventional methods.

PGLa, an antimicrobial peptide (AMP), primarily exerts its antibacterial effects by disrupting bacterial cell membrane integrity. Previous theoretical studies mainly focused on the binding mechanism of PGLa with membranes, while the mechanism of water pore formation induced by PGLa peptides, especially the role of structural flexibility in the process, remains unclear. In this study, using all-atom simulations, we investigated the entire process of membrane deformation caused by the interaction of PGLa with an anionic cell membrane composed of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG). Using a deep learning-based key intermediate identification algorithm, we found that the C-terminal tail plays a crucial role for PGLa insertion into the membrane, and that with its assistance, a variety of water pores formed inside the membrane. Mutation of the tail residues revealed that, in addition to electrostatic and hydrophobic interactions, the flexibility of the tail residues is crucial for peptide insertion and pore formation. The full extension of these flexible residues enhances peptide-peptide and peptide-membrane interactions, guiding the transmembrane movement of PGLa and the aggregation of PGLa monomers within the membrane, ultimately leading to the formation of water-filled pores in the membrane. Overall, this study provides a deep understanding of the transmembrane mechanism of PGLa and similar AMPs, particularly elucidating for the first time the importance of C-terminal flexibility in both insertion and oligomerization processes.

During the blood coagulation cascade, coagulation factor VIII (FVIII) is activated by thrombin to form activated factor VIII (FVIIIa). FVIIIa associates with platelet surfaces at the site of vascular damage to form an intrinsic tenase complex with activated factor IX. A working model for FVIII membrane binding involves the association of positively charged FVIII residues with negatively charged lipid headgroups and the burial of hydrophobic residues into the membrane interior. Currently, the atomic details of the FVIII lipid binding interactions and membrane orientation are lacking. This study reports residue-specific FVIII C domain interactions with 1,2-dioleoyl- sn -glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl- sn -glycero-3-phospho-l-serine (DOPS) in atomistic detail. Contact maps between residues in the C domains with different lipid moieties support prior structural data describing how the C domains associate with membranes through electrostatic and hydrophobic interactions. Solvent-accessible surface area analysis quantified the extent to which residues in the C1 and C2 domains bury into the membrane. Calculations of the potential energy between the C domains and DOPC and DOPS revealed an FVIII membrane-binding orientation that agrees with previous experimental data. This study expands our knowledge of the structural basis of FVIII membrane association, which may be critical for the development of next-generation FVIII replacement constructs with improved activity.

The structural response of 1,2-dimyristoyl- sn- glycero-3-phosphatidylcholine (DMPC)/water bilayers to addition and subsequent solvation of a small amphiphilic molecule - an anesthetic benzyl alcohol - was studied by means of solid-state NMR ( 2 H NMR, 31 P NMR) spectroscopy and low-angle X-ray diffraction. The sites of binding of this solute molecule within the bilayer were determined - the solute was shown to partition between several sites in the bilayer and the equilibrium was shown to be dynamic and dependent on the level of hydration and temperature. At the same time, it was shown that solubilization of benzyl alcohol reached a solubility limit and was terminated when the ordering profile of DMPC hydrocarbon chains adopted finite limiting values throughout the whole chain. Such findings were made probably for the first time for any lipid bilayer system and possibly have more general implications for dissolution of other small-molecule amphiphilic solutes in lipid bilayer systems other than DMPC. The limit to the hydrocarbon chain profile is probably a more general property and corresponds to the balance of intrabilayer and interbilayer forces established in combination with the elastic properties of the bilayer system that still consists of one single phase just before the solute forms an excess phase. It is not necessary to quantify the contribution of each individual intrabilayer and interbilayer force acting within such a bilayer system. A model of the dependence of surface density of lipid chains on the chain segment order parameter was also developed - an empirical mathematical model based on experimental data was derived and it was proposed to represent a relationship between intrinsic bilayer forces and bilayer deformation characteristics and might be proven to be of more general significance in the future.

Understanding the evolution of protocells, primitive compartments that distinguish self from nonself, is crucial for exploring the origin of life. Fatty acids and monoglycerides have been proposed as key components of protocell membranes due to their ability to self-assemble into bilayers and vesicles capable of nutrient exchange. In this study, we investigate the electrophysiological properties of planar bilayers composed of monoglyceride and fatty acid mixtures, using a droplet interface bilayer system. Three fatty acids with varying hydrocarbon chain lengths─oleic acid (C18), palmitoleic acid (C16), and myristoleic acid (C14)─in combination with monoolein (C18) are examined to evaluate the influence of chain length and composition on bilayer stability, thickness, and ion permeability. The results show that pure monoolein bilayers exhibit enhanced ion permeability compared to phospholipid bilayers, which are characteristic of modern cellular membranes. Furthermore, the incorporation of fatty acids into monoolein bilayers destabilizes the membrane structure and further increases ion permeability. We consider that this increased permeability is likely driven by three molecular characteristics. First, the wedge-like shape of monoolein may disrupt bilayer packing and induce transient pore formation. Second, the rapid flip-flop of fatty acids between bilayer leaflets likely facilitates ion transport. Third, the chain-length mismatch between monoolein and myristoleic acid further destabilizes the bilayer, promoting the formation of structural defects. These findings suggest that compositional motifs in monoglyceride-fatty acid bilayers may provide an alternative ion transport mechanism, such as the flip-flop of amphiphilic molecules, in early protocell membranes before the evolution of protein-based transporters.

We performed all-atom and coarse-grained simulations of lipid bilayer mixtures of the unsaturated lipid DOPC, with saturated lipids having the same 18-carbon acyl tails and different headgroups, to understand their mechanical properties. The secondary lipids were DSPG, DSPA, DSPS, DSPC, and DSPE. The DOPC:DSPG system with 65:35 molar ratio was the softest, with area compressibility modulus K A ∼ 22% smaller than the pure DOPC value. Raising the mole % of DOPC leads to increases in K A , yet at any given composition the K A trend is DSPG < DSPA < DSPS < DSPC < DSPE. Lipid-lipid interactions are weaker in DOPC:DSPG mixtures and stronger in DSPE systems. The head and phosphate groups of the secondary lipids DSPG, DSPA, and DSPS interact strongly with salt ions. Adding secondary lipids leads to DOPC having more ordered acyl tails relative to pure DOPC systems. No evidence of phase separation or inhomogeneities was observed in our simulations. We synthesized three liposomal formulations, L-DOPC (pure DOPC) and L-DOPC/DSPG and L-DOPC/DSPA, both with 15 mol % of secondary lipid. L-DOPC/DSPA had approximately 3- and 2-times higher in vitro internalization by normal epithelial (EpH4-Ev) and metastatic breast cancer (4T1) cells, compared with L-DOPC. The uptake of L-DOPC/DSPG by EpH4-Ev cells was almost 2-fold compared to L-DOPC, but both liposomes had similar uptakes by cancer cells. As L-DOPC/DSPG and L-DOPC/DSPA have similar K A values, we presumed that the mechanical properties, possibly in combination with the higher negative surface charges in L-DOPC/DSPA and differences in effective liposome diameters and diffusivities, contributed to these observations.

Solid supported lipid bilayers (SLBs) serve as an excellent platform for biophysical studies. However, the formation of highly negatively charged SLBs on negatively charged surfaces remains a challenge due to electrostatic repulsion. Here, we study the effects of ionic strength and osmotic stress on the formation of highly negatively charged SLBs on the silica surface. We used quartz crystal microbalance-dissipation to study the adsorption and rupture of highly negatively charged small unilamellar vesicles on the silica surface in different concentrations of NaCl and under different osmotic stresses. It was demonstrated that an increase in the ionic strength of the solution enhances SLB formation. Both hypertonic and moderate hypotonic osmotic stress can promote the formation of SLBs. However, the SLB cannot be formed under high hypotonic osmotic stress. Importantly, osmotic stress alone without a change in ionic strength is insufficient to promote SLB formation. Moreover, the topographical images obtained by atomic force microscopy showed that complete bilayers were formed under hypertonic osmotic stress and high ionic strength, whereas defects were noticed in the bilayers formed under hypotonic osmotic stress. Furthermore, the fluidity of the lipid bilayers was studied by fluorescence recovery after photobleaching. A higher membrane fluidity was observed for the complete lipid bilayers compared to that of the lipid bilayers with defects. Our findings further the understanding of how ionic strength and osmotic stress affect the formation of highly negatively charged SLBs on negatively charged surfaces, providing insights for preparing model biological membranes.

Lipid membranes can control the permeability of a pharmaceutical drug, whereas the drug can induce changes in the structural and biophysical properties of the membranes. Understanding this interplay of drug-lipid membrane interactions can be of great importance in drug design. Here, we present a molecular dynamics study to provide insights into the interactions between the antidepressant fluoxetine and 1,2-dipalmitoyl- sn -glycero-3-phosphocholine (DPPC) or 1,2-dipalmitoyl- sn -glycero-3-phosphoglycerol (DPPG) bilayers. It was found that, due to the electrostatic interaction, the headgroup of the zwitterionic DPPC lipid is more stable than that of the negatively charged DPPG lipid, allowing the gel phase to persist even at the elevated temperature. At 25 °C, fluoxetine cannot penetrate into the gel-phase DPPC bilayer, while the electrostatic interaction between positively charged fluoxetine and negatively charged DPPG bilayer retains the drug within the lipid headgroup domain. When the temperature is increased to 45 °C, both neutral and charged forms of fluoxetine can partition into the DPPC and DPPG bilayers spontaneously. Analysis of the biophysical and structural changes in both DPPC and DPPG bilayers in the presence of fluoxetine revealed a phase-dependent effect. The binding of fluoxetine to the lipid bilayers limits the movement and orientation of the drug. These findings shed light on the interactions between a commonly prescribed antidepressant and lipid membranes, and such information can be beneficial to the development of potential therapeutic agents.

The selective insertion of membrane-impermeant amphiphiles such as detergents, (lipo)peptides, drugs, etc. into the cis leaflet of a membrane causes an imbalance between the intrinsic areas of the cis and trans leaflet, referred to as asymmetry stress or differential stress. The literature provides individual mechanisms of how membranes respond to such stress, which are relevant to membrane remodeling processes and leakage phenomena. By studying vesicle budding, membrane leakage, and isothermal titration calorimetry of liposomes interacting with digitonin, alkyl maltosides, miltefosine, and octyl glucoside, we developed a roadmap linking the stress-response mechanisms to each other. Initially, lateral compression or stretching of the leaflets accommodates a minor asymmetry stress. Then, either molecules flip to the trans leaflet or the membrane bends to form buds. Fast flip leads to the classic three-stage model. Budding proceeds up to its limit at 20-40% of the lipid. Beyond, insertion of further detergent is opposed by the pressure in the overpopulated leaflet. This "staying out" state can persist over hours or days and up to high detergent concentrations before detergent micelles induce "micellar solubilization". Alternatively, the stress can be reduced by a transient failure of the membrane, allowing for "cracking in" of molecules, transferring them to the trans side.

The influenza A M2 homotetrameric channel consists of four transmembrane (TM) and four amphipathic helices (AHs). This viral proton channel is suggested to form clusters in the catenoid budding neck areas in raft-like domains of the plasma membrane, resulting in cell membrane scission and viral release. The channel clustering environment is rich in cholesterol. Previous experiments have shown that cholesterol significantly contributes to lipid bilayer undulations in viral buds. However, a clear explanation of membrane curvature from the distribution of cholesterol around the M2TM-AH clusters is lacking. Using coarse-grained molecular dynamics simulations of M2TM-AH in bilayers, we observed that M2 channels form specific, C2-symmetric, clusters with conical shapes driven by the attraction of their AHs. We showed that cholesterol stabilized the formation of M2 channel clusters by filling and bridging the conical gap between M2 channels at specific sites in the N-termini of adjacent channels or via the C-terminal region of TM and AHs, with the latter sites displaying a longer interaction time and higher stability. The potential of mean force calculations showed that when cholesterols occupy the identified interfacial binding sites between two M2 channels, the dimer is stabilized by 11 kJ/mol. This translates to the cholesterol-bound dimer being populated by almost 2 orders of magnitude compared to a dimer lacking cholesterol. We demonstrated that the cholesterol-bridged M2 channels can exert a lateral force on the surrounding membrane to induce the necessary negative Gaussian curvature profile, which permits spontaneous scission of the catenoid membrane neck and leads to viral buds and scission.

Plasma membrane (PM) simulations at longer length and time scales at nearly atomistic resolution can provide invaluable insights into cell signaling, apoptosis, lipid trafficking, and lipid raft formation. We propose a coarse-grained (CG) model of a mammalian PM considering major lipid head groups distributed asymmetrically across the membrane bilayer and validate the model against bilayer structural properties from atomistic simulation. Using the proposed CG model, we identify a recurring pattern in the passive collective cholesterol transbilayer motion and study the individual cholesterol flip-flop events and associated pathways along with lateral ordering in the bilayer during a flip-flop event. We identify two discrete cholesterol flip-flop pathways: (i) a systematic rototranslational pathway and (ii) intraleaflet inversion followed by interleaflet translation (or reverse). We observe a periodic cholesterol enrichment in the exoplasmic leaflet of the PM bilayer and examine the underlying cholesterol-lipid affinities. We observe closer association between cholesterol and palmitoylsphingomyelin (PSM) lipid, relative to other lipids, and conclude that the cholesterol enrichment in the exoplasmic leaflet can be attributed to higher PSM content in that leaflet, together leading to formation of short-lived PSM-cholesterol-rich domains.

Alkylphospholipids are single-chain lipid amphiphiles that possess clinically relevant biological activities driven by membrane-destabilizing interactions. Subtle variations in alkylphospholipid structure can lead to significant differences in their biological effects, yet corresponding membrane interactions remain underexplored. Herein, we employed the quartz crystal microbalance-dissipation (QCM-D) technique to characterize the real-time membrane interactions of three alkylphospholipids-edelfosine, miltefosine, and perifosine-on supported lipid bilayers with varying cholesterol fractions. Our findings reveal that the tested alkylphospholipids had distinct membrane-interaction profiles: (1) edelfosine exhibited irreversible binding and caused weak membrane disruption; (2) miltefosine caused major disruption by affecting membrane packing; and (3) perifosine exhibited reversible binding while triggering structural rearrangements and modest disruption. Overall, alkylphospholipid micelles showed greater activity than monomers, and higher membrane cholesterol fractions resulted in more extensive disruption, highlighting the interplay between membrane stiffness and responsiveness. These results provide biophysical evidence that different alkylphospholipids have distinct membrane-interaction behaviors that align well with reported biological activities. Our supported lipid bilayer approach offers a valuable platform for designing and assessing alkylphospholipids with tailored membrane-interaction profiles.

Understanding the molecular mechanisms of pore formation is crucial for elucidating fundamental biological processes and developing therapeutic strategies, such as the design of drug delivery systems and antimicrobial agents. Although experimental methods can provide valuable information, they often lack the temporal and spatial resolution necessary to fully capture the dynamic stages of pore formation. In this study, we present two novel collective variables (CVs) designed to characterize membrane pore behavior, particularly its energetics, through molecular dynamics (MD) simulations. The first CV─termed Full-Path─effectively tracks both the nucleation and expansion phases of pore formation. The second CV─called Rapid─is tailored to accurately assess pore expansion in the limit of large pores, providing quick and reliable method for evaluating membrane line tension under various conditions. Our results clearly demonstrate that the line tension predictions from both our CVs are in excellent agreement. Moreover, these predictions align qualitatively with available experimental data. Specifically, they reflect higher line tension of 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC) membranes containing 1-palmitoyl-2-oleoyl- sn -glycero-3-phospho-l-serine (POPS) lipids compared to pure POPC, the decrease in line tension of POPC vesicles as the 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoglycerol (POPG) content increases, and higher line tension when ionic concentration is increased. Notably, these experimental trends are accurately captured only by the all-atom CHARMM36 and prosECCo75 force fields. In contrast, the all-atom Slipids force field, along with the coarse-grained Martini 2.2, Martini 2.2 polarizable, and Martini 3 models, show varying degrees of agreement with experiments. Our developed CVs can be adapted to various MD simulation engines for studying pore formation, with potential implications in membrane biophysics. They are also applicable to simulations involving external agents, offering an efficient alternative to existing methodologies.

All-atom molecular dynamics simulations are powerful tools for studying cell membranes and their interactions with proteins and other molecules. However, these processes occur on time scales determined by the diffusion rate of phospholipids, which are challenging to achieve in all-atom models. Here, we present a new all-atom model that accelerates lipid diffusion by splitting phospholipid molecules into head and tail groups. The bilayer structure is maintained by using external lateral potentials, which compensate for the lipid split. This split model enhances lateral lipid diffusion more than ten times, allowing faster and cheaper equilibration of large systems with different phospholipid types. The current model has been tested on membranes containing PSM, POPC, POPS, POPE, POPA, and cholesterol. We have also evaluated the interaction of the split model membranes with the Disheveled DEP domain and amphiphilic helix motif of the transcriptional repressor Opi1 as representative of peripheral proteins as well as the dimeric fragment of the epidermal growth factor receptor transmembrane domain and the Human A2A Adenosine of G protein-coupled receptors as representative of transmembrane proteins. The split model can predict the interaction sites of proteins and their preferred phospholipid type. Thus, the model could be used to identify lipid binding sites and equilibrate large membranes at an affordable computational cost.

The light-harvesting pigment-protein complex II (LHCII) from plants can be used as a component for biohybrid photovoltaic devices, acting as a photosensitizer to increase the photocurrent generated when devices are illuminated with sunlight. LHCII is effective at photon absorption in the red and blue regions of the visible spectrum, however, it has low absorption in the green region (550-650 nm). Previous studies have shown that synthetic chromophores can be used to fill this spectral gap and transfer additional energy to LHCII, but it was uncertain whether this would translate into an improved performance for photovoltaics. In this study, we demonstrate amplified photocurrent generation from LHCII under green light illumination by coupling this protein to Texas Red (TR) chromophores that are coassembled into a lipid bilayer deposited onto electrodes. Absorption spectroscopy shows that LHCII and lipid-linked TR are successfully incorporated into lipid membranes and maintained on electrode surfaces. Photocurrent action spectra show that the increased absorption due to TR directly translates into a significant increase of photocurrent output from LHCII. However, the absolute magnitude of the photocurrent appears to be limited by the lipid bilayer acting as an insulator and the TR enhancement effect reaches a maximum due to protein, lipid or substrate-related quenching effects. Future work should be performed to optimize the use of extrinsic chromophores within novel biophotovoltaic devices.

Eukaryotic plasma membranes exhibit nanoscale lateral lipid heterogeneity, a feature that is thought to be central to their function. Studying these heterogeneities is challenging since few biophysical methods are capable of detecting domains at submicron length scales. We recently showed that cryogenic electron microscopy (cryo-EM) can directly image nanoscale liquid-liquid phase separation in extruded liposomes due to its ability to resolve the intrinsic thickness and electron density differences of ordered and disordered phases. However, the intensity contrast between these phases is poor compared with conventional fluorescence microscopy and is thus both a limiting factor and a focal point for optimization. Because the fundamental source of intensity contrast is the spatial variation in electron density within the bilayer, lipid modifications aimed at selectively increasing the electron density of one phase might enhance the ability to resolve coexisting phases. To this end, we investigated model membrane mixtures of DPPC/DOPC/cholesterol in which one hydrogen of cholesterol's C19 methyl group was replaced by an electron-rich halogen atom (either bromine or iodine). We characterized the phase behavior as a function of composition and temperature using fluorescence microscopy, Förster resonance energy transfer, and cryo-EM. Our data suggest that halogenated cholesterol variants distribute approximately evenly between liquid-ordered and liquid-disordered phases and are thus ineffective at enhancing the intensity difference between them. Furthermore, replacing more than half of the native cholesterol with halogenated cholesterol variants dramatically reduces the size of the membrane domains. Our results reinforce how small changes in the sterol structure can have a large impact on the lateral organization of membrane lipids.

Plasma membranes are known to segregate into liquid disordered and ordered nanoscale phases, the latter being called lipid rafts. The structure, lipid composition, and function of lipid rafts have been the subject of numerous studies using a variety of experimental and computational methods. Double electron-electron resonance (DEER, also known as PELDOR) is a member of the pulsed dipole EPR spectroscopy (PDS) family of techniques, allowing the study of nanoscale distances between spin-labeled molecules. To extend the possibilities of DEER in the study of molecule clusters, its joint application with the simple two-pulse electron spin echo (2p ESE) method is carried out here. We studied spin-labeled ibuprofen (ibuprofen-SL) diluted in bilayers composed of equimolar mixtures of dioleoyl-glycero-phosphocholine (DOPC) and dipalmitoyl-glycero-phosphocholine (DPPC) phospholipids, with added cholesterol, a system known as a raft-mimicking. The data obtained show that ibuprofen-SL molecules in this system form isolated clusters of about 4 nm in size, containing 6-8 molecules spaced at least 1.3 nm apart. These results indicate the interaction of ibuprofen-SL molecules with lipid rafts, for which the existence of nanoscale substructures at the boundaries of which adsorption of these molecules occurs is suggested.

Independent methods show that sub-microMolar concentrations of perfluorooctanoic acid (PFOA), a member of the PFAS family of "forever chemicals", change the properties of DPPC vesicle bilayers. Specifically, calorimetry measurements show that PFOA at concentrations as low as 0.1 nM lowers DPPC's gel-liquid crystalline transition enthalpy by several J/g without changing the transition temperature ( T gel-LC ), and dynamic light scattering (DLS) data illustrate that PFOA markedly broadens the size distribution of DPPC vesicles. Furthermore, DLS results from PFOA-containing, DPPC vesicle solutions also contain smaller objects having diameters of 30-50 nm. Close inspection of cryo-EM images reveals that DPPC vesicles formed in the presence of PFOA are multilamellar and the smaller objects have a clear bilayer structure similar to niosomes. A consequence of these PFOA-induced changes to DPPC bilayer structure is that the bilayers themselves are more susceptible to secondary solute accumulation. Time resolved emission measurements of Coumarin 152 (C152) report that C152 is 3-fold more likely to partition into the bilayer's acyl chain, hydrophobic interior when PFOA is present, and fluorescence lifetimes from C152 partitioned into the polar region of the lipid bilayer show evidence of PFOA-induced membrane hydration below T gel-LC .

Molecular oxygen (O 2 ) is essential for life, and continuous effort has been made to understand its pathways in cellular respiration with all-atom (AA) molecular dynamics (MD) simulations of, e.g., membrane permeation or binding to proteins. To reach larger length scales with models, such as curved membranes in mitochondria or caveolae, coarse-grained (CG) simulations could be used at much lower computational cost than AA simulations. Yet a CG model for O 2 is lacking. In this work, a CG model for O 2 is therefore carefully selected from the Martini 3 force field based on criteria including size, zero charge, nonpolarity, solubility in nonpolar organic solvents, and partitioning in a phospholipid membrane. This chosen CG model for O 2 (TC3 bead) is then further evaluated through the calculation of its diffusion constant in water and hexadecane, its permeability rate across pure phospholipid- and cholesterol-containing membranes, and its binding to the T4 lysozyme L99A protein. Our CG model shows semiquantitative agreement between CG diffusivity and permeation rates with the corresponding AA values and available experimental data. Additionally, it captures the binding to hydrophobic cavities of the protein, aligning well with the AA simulation of the same system. Thus, the results show that our O 2 model approximates the behavior observed in the AA simulations. The CG O 2 model is compatible with the widely used multifunctional Martini 3 force field for biological simulations, which will allow for the simulation of large biomolecular systems involved in O 2 's transport in the body.

The present work focuses on one of the possible target mechanisms of action of the anionic antimicrobial peptide β-lg 125-135 derived from trypsin hydrolysis of β-lactoglobulin. After confirmation of bactericidal activity against a pathogenic Gram(+) strain and demonstration of the innocuousness on a eukaryotic cell line, we investigated the interaction of β-lg 125-135 with monolayers and bilayers of dpPC and dpPC:dpPG as model membranes of eukaryotic and bacterial membranes, respectively. In monolayers, compared to zwitterionic dpPC, in the negatively charged dpPC-dpPG, β-lg 125-135 injected into the subphase penetrated up to higher surface pressures and showed greater extents of penetration with increasing concentration in the subphase. Additionally, the rate constants for β-lg 125-135 adsorption and desorption were 1 order of magnitude higher, and the resultant thermodynamic association constant was 1 order of magnitude lower. In turn, the compression isotherms of monolayers prepared with the β- lg 125-135 present in the mixture spread over the air-water interface, remained in the monolayer and showed positive deviations from ideality, a greater decrease in the surface compressibility modulus, and an increase in the surface potential of both interfaces, more pronounced on dpPC:dpPG. In SUVs, fluorescence anisotropy (FA) assays using DPH and TMA-DPM indicated that β- lg 125-135 tended to disrupt the gel phase of dpPC bilayers. Conversely, in dpPC:dpPG, the peptide increased the FA of both probes. These results reflect a relatively high tendency of the β- lg 125-135 to approach the negative interface, with a favorable electrostatic orientation but low stability and short residence time. Once inside the membrane, it stiffens dpPG-containing bilayers.

pH-sensitive lipids are important components of lipid nanoparticles, which enable the targeted delivery and controlled release of drugs. Understanding the mechanism of pH-triggered drug release at the molecular level is important for the rational design of ionizable lipids. Based on a recently reported pH-switchable lipid, named SL2, molecular dynamics (MD) simulations were employed to explore the microscopic mechanism behind the membrane destabilization induced by the conformational change of pH-switchable lipids. The simulated results showed that, at neutral pH, the neutral SL2 lipids assembled with other components (helper lipids and cholesterol) to form a structurally ordered bilayer structure. At this moment, the two hydrocarbon chains of SL2 were closely aligned and inserted in an orderly manner inside of the membrane. With a decrease in pH, the protonation of the pyridinium ring caused a large degree of molecular structural change. The pyridinium ring preferred to form intramolecular H-bonds with the methoxy groups and intermolecular H-bonds with water, resulting in the flip of the pyridinium ring. Meanwhile, due to the structural flip, the two alkane chains showed a more open state, which perturbed the arrangement of molecules within the membrane. The perturbations caused local collapse of the membrane and the formation of water molecule channels, which contributed to the pH-induced drug release. Our results verified the experimentally proposed mechanism at the molecular level and provided more complementary information, which are expected to have deeper insights into the pH-triggered drug release.

Lysophospholipids (LPLs) and host defense peptides (HDPs) are naturally occurring membrane-active agents that disrupt key membrane properties, including the hydrocarbon thickness, intrinsic curvature, and molecular packing. Although the membrane activity of these agents has been widely examined separately, their combined effects are largely unexplored. Here, we use experimental and computational tools to investigate how lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE), an LPL of lower positive spontaneous curvature, influence the membrane activity of piscidin 1 (P1), an α-helical HDP from fish. Four membrane systems are probed: 75:25 C16:0-C18:1 PC (POPC)/C16:0-C18:1 phosphoglycerol (POPG), 50:25:25 POPC/POPG/16:0 LPC, 75:25 C16:0-C18:1 PE (POPE)/POPG, and 50:25:25 POPE/POPG/14:0 LPE. Dye leakage, circular dichroism, and NMR experiments demonstrate that while the presence of LPLs alone does not induce leakage-proficient defects, it boosts the permeabilization capability of P1, resulting in an efficacy order of POPC/POPG/16:0 LPC > POPE/POPG/14:0 LPE > POPC/POPG > POPE/POPG. This enhancement occurs without altering the membrane affinity and conformation of P1. Molecular dynamics simulations feature two types of asymmetric membranes to represent the imbalanced ("area stressed") and balanced ("area relaxed") distribution of lipids and peptides in the two leaflets. The simulations capture the membrane thinning effects of P1, LPC, and LPE, and the positive curvature strain imposed by both LPLs is reflected in the lateral pressure profiles. They also reveal a higher number of membrane defects for the P1/LPC than P1/LPE combination, congruent with the permeabilization experiments. Altogether, these results show that P1 and LPLs disrupt membranes in a concerted fashion, with LPC, the more disruptive LPL, boosting the permeabilization of P1 more than LPE. This mechanistic knowledge is relevant to understanding biological processes where multiple membrane-active agents such as HDPs and LPLs are involved.

Neuronal cell death induced by cell membrane damage is one of the major hallmarks of neurodegenerative diseases. Neuroinflammation precedes the loss of neurons; however, whether and how inflammation-related proteins contribute to the loss of membrane integrity remains unknown. We employed a range of biophysical tools, including high-speed atomic force microscopy, fluorescence spectroscopy, and electrochemical impedance spectroscopy, to ascertain whether the pro-inflammatory protein S100A8 induces alterations in biomimetic lipid membranes upon interaction. Our findings underscore the crucial roles played by divalent cations and membrane charge. We found that apo-S100A8 selectively interacts with anionic lipid membranes composed of phosphatidylserine (PS), causing membrane disruption through a detergent-like mechanism, primarily affecting regions where phospholipids are less tightly packed. Interestingly, the introduction of Ca 2+ ions inhibited S100A8-induced membrane disruption, suggesting that the disruptive effects of S100A8 are most pronounced under conditions mimicking intracellular compartments, where calcium levels are low, and PS concentrations in the inner leaflet of the membrane are high. Overall, our results present a mechanistic basis for understanding the molecular interactions between S100A8 and the plasma membrane, emphasizing S100A8 as a potential contributor to the onset of neurodegenerative diseases.

RNA-lipid interactions directly influence RNA activity, which plays a crucial role in the development of new applications in medicine and biotechnology. However, while specific preferential behaviors between RNA and lipid bilayers have been identified experimentally, their molecular origin remains unexplored. Here we use molecular dynamics simulations to investigate the interaction between RNA and membranes composed of zwitterionic lipids at the atomistic level. Our data reproduce and rationalize previous experimental observations, including that short-chain RNAs rich in guanine have a higher affinity for gel-phase membranes compared to RNA sequences rich in other nucleotides and that RNA prefers gel-phase membranes to fluid bilayers. Our simulations reveal that RNA order is a key molecular determinant of RNA-zwitterionic phospholipid interactions. Our data provide a wealth of information at the atomic level that will help accelerate research on RNA-lipid assemblies for task-specific applications such as designing lipid-based nanocarriers for RNA delivery.

Phosphatidylinositol phosphates (PIPs) are a family of seven different eukaryotic membrane lipids that have a large role in cell viability, despite their minor concentration in eukaryotic cellular membranes. PIPs tightly regulate cellular processes, such as cellular growth, metabolism, immunity, and development through direct interactions with partner proteins. Understanding the biophysical properties of PIPs in the complex membrane environment is important to understand how PIPs selectively regulate a partner protein. Here, we investigate the structure and dynamics of PIP 3 in lipid bilayers that are simplified models of the natural membrane environment. We probe the effects of the anionic lipid phosphatidylserine (PS) and the divalent cation Ca 2+ by using full-length lipids in well-formed bilayers. We used solution and solid-state NMR on naturally abundant 1 H, 31 P, and 13 C atoms combined with molecular dynamics (MD) simulations to characterize the structure and dynamics of PIPs. 1 H and 31 P 1D spectra show good resolution at temperatures above the phase transition with isolated peaks in the headgroup, interfacial, and bilayer regions. Site-specific assignment of the chemical shifts of these reporters enables the measurement of the effects of Ca 2+ and PS at the single atom level. In particular, the resolved 31 P signals of the PIP 3 headgroup allow for extremely well-localized information about PIP 3 phosphate dynamics, which the MD simulations can further explain. A quantitative assessment of cross-polarization kinetics provides additional dynamics measurements for the PIP 3 headgroups.

The dual capability of conductive polymers to conduct ions and electrons, in combination with their flexible mechanical properties, makes them ideal for bioelectronic applications. This study explores the in situ enzymatic polymerization of water-soluble π-conjugated monomers on native lipid bilayers derived from the F11 cell line, mimicking mammalian neural membranes. Enzymatic polymerization was catalyzed using horseradish peroxidase (HRP) in the presence of oxidant hydrogen peroxide (H 2 O 2 ) and monitored via electrochemical quartz crystal microbalance with dissipation (EQCM-D) and electrochemical impedance spectroscopy (EIS). Results showed polymerization with HRP. The structural properties of the formed polymer films were characterized ex situ using atomic force microscopy (AFM), while the quality of the F11 native lipid vesicles and bilayer was respectively assessed through dynamic light scattering (DLS) and fluorescence recovery after photobleaching (FRAP) techniques. This work demonstrates, for the first time, the feasibility of the in situ formation of conductive polymers on native lipid membranes, offering a promising approach for the development of minimally invasive neural electrodes to diagnose and treat neurological disorders.

A fundamental understanding of how the HIV-1 envelope (Env) protein facilitates fusion is still lacking. The HIV-1 fusion peptide, consisting of 15 to 22 residues, is the N-terminus of the gp41 subunit of the Env protein. Further, this peptide, a promising vaccine candidate, initiates viral entry into target cells by inserting and anchoring into human immune cells. The influence of membrane lipid reorganization and the conformational changes of the fusion peptide during the membrane insertion and anchoring processes, which can significantly affect HIV-1 cell entry, remains largely unexplored due to the limitations of experimental measurements. In this work, we investigate the insertion of the fusion peptide into an immune cell membrane mimic through multiscale molecular dynamics simulations. We mimic the native T-cell by constructing a nine-lipid asymmetric membrane, along with geometrical restraints accounting for insertion in the context of gp41. To account for the slow time scale of lipid mixing while enabling conformational changes, we implement a protocol to go back and forth between atomistic and coarse-grained simulations. Our study provides a molecular understanding of the interactions between the HIV-1 fusion peptide and the T-cell membrane, highlighting the importance of the conformational flexibility of fusion peptides and local lipid reorganization in stabilizing the anchoring of gp41 into the targeted host membrane during the early events of HIV-1 cell entry. Importantly, we identify a motif within the fusion peptide critical for fusion that can be further manipulated in future immunological studies.

Membrane budding is vital for various cellular processes such as synaptic activity regulation, vesicle transport and release, and endocytosis/exocytosis. Although protein-mediated membrane budding has been extensively investigated, the effects of the lipid asymmetry of the two leaflets and the asymmetrically electrical environments of the cellular membrane on membrane budding remain elusive. In this work, using coarse-grained molecular dynamics simulations, we systematically investigate the impacts of lipid bilayer asymmetry and external electric fields mimicking the asymmetric membrane potential on the membrane budding. The results show that the differential stress induced by the asymmetric distribution of lipids in the two leaflets is a crucial factor for the membrane budding. The unidirectional flip of cholesterol induced by the membrane curvature and the asymmetric ion adsorption induced by the anionic lipids promote the budding process. Furthermore, the external electric field applied perpendicularly to the bilayer plane increases the transmembrane potential and produces an additional differential stress across the leaflets by imposing an asymmetric torque on the lipid headgroups in the two leaflets, facilitating the membrane budding. These findings offer insights into how the structural and the environmental asymmetry in natural cellular membranes influence membrane budding in cellular processes.

Despite their wide use and far-reaching implications, molecular dynamics (MD) simulations suffer from a lack of both traceability and reproducibility. We introduce Martignac: computational workflows for the coarse-grained (CG) Martini force field. Martignac describes Martini CG MD simulations as an acyclic directed graph, providing the entire history of a simulation─from system preparation to property calculations. Martignac connects to NOMAD, such that all simulation data generated are automatically normalized and stored according to the FAIR principles. We present several prototypical Martini workflows, including system generation of simple liquids and bilayers, as well as free-energy calculations for solute solvation in homogeneous liquids and drug permeation in lipid bilayers. By connecting to the NOMAD database to automatically pull existing simulations and push any new simulation generated, Martignac contributes to improving the sustainability and reproducibility of molecular simulations.

Methane emissions from livestock contribute to global warming. Seaweeds used as food additive offer a promising emission mitigation strategy because seaweeds are enriched in bromoform─a methanogenesis inhibitor. Therefore, understanding bromoform storage and production in seaweeds and particularly in a cell-like environment is crucial. As a first step toward this aim, we present an atomistic description of bromoform dynamics, diffusion, and aggregation in the presence of lipid membranes. Using all-atom molecular dynamics simulations with customized CHARMM-formatted bromoform force field files, we investigate the interactions of bromoform and lipid bilayer across various concentrations. Bromoform penetrates membranes and at high concentrations forms aggregates outside the membrane without affecting membrane thickness or lipid tail order. Aggregates outside the membrane influence the membrane curvature. Within the membrane, bromoform preferentially localizes in the membrane hydrophobic core and diffuses the slowest along the membrane normal. Employing general local-atomic descriptors and unsupervised machine learning, we demonstrate the similarity of bromoform local structures between the liquid and aggregated forms.

Atomistic molecular dynamics (MD) simulations are a much-used tool for investigating the structure and dynamics of biomembranes with atomic resolution. The validity of the representations obtained is determined by the accuracy and realism of the MD model (force field). Here, we evaluated the proprietary OPLS4 force field of Schrödinger, Inc. against atomic-resolution experimental data, and compared its performance to CHARMM36, one of the best-performing openly available force fields. As a benchmark, we used high-resolution nuclear magnetic resonance (NMR) order parameters for C-H bonds─directly and reliably calculable from MD simulations─measured in phosphatidylcholine (PC) lipid bilayers under varying hydration conditions. Comparisons were made with two dehydration data sets: for saturated (1,2-dimyristoylphosphatidylcholine, DMPC) lipid bilayers from the literature and for unsaturated (1-palmitoyl-2-oleoylphosphatidylcholine, POPC) lipid bilayers measured here. Our findings indicate that OPLS4 reproduces the structure and dehydration response of PC-lipid bilayers fairly well, even slightly outperforming CHARMM36. Both models' main inaccuracies appear in (1) the order parameter magnitudes in the glycerol backbone and unsaturated carbon segments and (2) the qualitatively differing structural response of the PC headgroup to dehydration compared to experiments. In summary, this work underscores the importance of independent validation for (proprietary) force fields and highlights the striking similarities and nuanced differences between OPLS4 and CHARMM36 in describing biomembranes.

Terpenes are widely used in cosmetic formulations as chemical penetration enhancers (CPEs). However, the molecular mechanisms underlying the skin-penetration-enhancing effect have not been completely understood. In this work, molecular dynamics (MD) simulations were used to explore the influence of terpineol (TER), 1,8-cineole (CIN), and limonene (LIM) on the stratum corneum (SC) model. The results indicated that terpenes affected lipid membranes in a concentration-dependent manner and promoted skin permeation by disorganizing the cholesterol (CHOL) portion. The penetration of CPEs across the skin membrane also differed, with diffusion rates of limonene > 1,8-cineole > terpineol. The limonene molecules could penetrate the bilayer's center, forming a "triple layer" membrane structure. Furthermore, constrained simulations showed that the most favorable penetration pathway is via areas rich in CHOL. Terpineol could lower the energy barrier of the hydrophilic molecule (caffeic acid) across the cholesterol region. For the lipophilic molecule (osthole), limonene and 1,8-cineole could reduce the energy barrier across the cholesterol region. Each of the results provides novel insights into the mechanism of penetration of CPEs in the skin.

Fluorescence probes play crucial roles in unraveling the structure and dynamics of cell membranes including membrane fluidity, polarity, and lipid molecule ordering. The fluorescence lifetime of probes describes the average duration of time that a fluorescent molecule remains in an excited state before returning to the ground state, which is sensitive to environmental changes. However, the molecular mechanism and inherent properties to determine the fluorescence lifetimes remain unexplored and inadequately studied. Furthermore, the effects of the probe on the membrane are also unclear. In this study, we investigated the interactions between probes and lipids, as well as the structural properties of probes within the outer and inner membrane of Mycobacterium smegmatis ( Msm ) by combining molecular dynamics (MD) simulations, enhanced sampling methods, fluorescence lifetime imaging microscopy (FLIM), and time-correlated single photon counting (TCSPC). The results show that even though the probes have very little effect on the membrane lipids, different membrane environments significantly affect the fluorescence lifetime of the probes. The analysis based on the all-atom simulations shows a strong correlation between the probe's immersion depth within the membrane and its fluorescence lifetime. Specifically, probes buried in the membrane environment shielded from rapid water molecule collisions exhibit longer fluorescence lifetimes. The molecular basis of the fluorescence lifetime of probes in cell membranes revealed in this work would enhance the comprehension of fluorescence probes and facilitate the rational design of novel efficient probes.

Nucleic acid mimics (NAMs) have demonstrated high potential as antibacterial drugs. However, very few studies have assessed their possible diffusion across the bacterial envelope. In this work, we studied NAMs' diffusion in lipid bilayer systems that mimic the bacterial outer membrane using molecular dynamics (MD) simulations. Additionally, we examined the interactions of a NAM sequence with lipid membranes and ascertained the partition constants ( K p ) through MD and spectroscopic investigations. The NAM sequences were composed of locked nucleic acid (LNA) and 2'-O-methyl (2'-OMe) residues, whereas the membrane models were composed of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) or 1-palmitoyl-2-oleoyl- sn -glycero-3-phospho-(1'-rac-glycerol) (POPG) phospholipids. The parametrization protocol followed was validated against literature data and demonstrated the reliability of our approach for simulating NAM sequences. Investigation into the interaction of the sequences with zwitterionic and anionic membranes revealed a preference for hydrogen bond formation with the anionic model over the zwitterionic one. Additionally, potential of mean force (PMF) calculations unveiled a lower free energy barrier for translocation across the zwitterionic bilayer model. Contrarily, the partition constants derived suggested a slightly higher partitioning within the anionic membrane, emphasizing a nuanced interplay of factors. Finally, spectroscopic partition measurements with liposomes presented challenges in quantifying the partition of NAMs due to minimal signal variations. However, a tendency for quenching in anionic vesicles suggested a potential, albeit small, partitioning effect that warrants further investigation. In summary, our study revealed that NAMs will not, in principle, be able to cross an intact bacterial outer membrane by passive diffusion.