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Abstract ASCT2 is an obligate exchanger of neutral amino acids, contributing to cellular amino acid homeostasis. ASCT2 belongs to the same family (SLC1) as Excitatory Amino Acid Transporters (EAATs) that concentrate glutamate in the cytosol. The mechanism that makes ASCT2 an exchanger rather than a concentrator remains enigmatic. Here, we employ cryo-electron microscopy and molecular dynamics simulations to elucidate the structural basis of the exchange mechanism of ASCT2. We establish that ASCT2 binds three Na+ ions per transported substrate and visits a state that likely acts as checkpoint in preventing Na+ ion leakage, both features shared with EAATs. However, in contrast to EAATs, ASCT2 retains one Na+ ion even under Na+-depleted conditions. We demonstrate that ASCT2 cannot undergo the structural transition in TM7 that is essential for the concentrative transport cycle of EAATs. This structural rigidity and the high-affinity Na+ binding site effectively confine ASCT2 to an exchange mode.

Abstract Biomolecular condensates are phase separated systems that play an important role in the spatio-temporal organisation of cells. Their distinct physico-chemical nature offers a unique environment for chemical reactions to occur. The compartmentalisation of chemical reactions is also believed to be central to the development of early life. To demonstrate how molecular dynamics may be used to capture chemical reactions in condensates, here we perform reactive molecular dynamics simulations using the coarse-grained Martini forcefield. We focus on the formation of rings of benzene-1,3-dithiol inside a synthetic peptide-based condensate, and find that the ring size distribution shifts to larger macrocycles compared to when the reaction takes place in an aqueous environment. Moreover, reaction rates are noticeably increased when the peptides simultaneously undergo phase separation, hinting that condensates may act as chaperones in recruiting molecules to reaction hubs.

Abstract Short-cationic alpha-helical antimicrobial peptides (SCHAMPs) are promising candidates to combat the growing global threat of antimicrobial resistance. They are short-sequenced, selective against bacteria, and have rapid action by destroying membranes. A full understanding of their mechanism of action will provide key information to design more potent and selective SCHAMPs. Molecular Dynamics (MD) simulations are invaluable tools that provide detailed insights into the peptide-membrane interaction at the atomic- and meso-scale level. We use atomistic and coarse-grained MD to look into the exact steps that four promising SCHAMPs—BP100, Decoralin, Neurokinin-1, and Temporin L—take when they interact with membranes. Following experimental set-ups, we explored the effects of SCHAMPs on anionic membranes and vesicles at multiple peptide concentrations. Our results showed all four peptides shared similar binding steps, initially binding to the membrane through electrostatic interactions and then flipping on their axes, dehydrating, and inserting their hydrophobic moieties into the membrane core. At higher concentrations, fully alpha-helical peptides induced membrane budding and protrusions. Our results suggest the carpet mode of action is fit for the description of SCHAMPs lysis activity and discuss the importance of large hydrophobic residues in SCHAMPs design and activity.

Summary: Transmembrane transporter proteins are essential for maintaining cellular homeostasis and, as such, are key drug targets. Many transmembrane transporter proteins are known to undergo large structural rearrangements during their functional cycles. Despite the wealth of detailed structural and functional data available for these systems, our understanding of their dynamics and, consequently, how they function is generally limited. We introduce an innovative approach that enables us to directly measure the dynamics and stability of interdomain interactions of transmembrane proteins using optical tweezers. Focusing on the osmoregulatory ATP-binding cassette transporter OpuA from Lactococcus lactis, we examine the mechanical properties and potential interactions of its substrate-binding domains. Our measurements are performed in lipid nanodiscs, providing a native-mimicking environment for the transmembrane protein. The technique provides high spatial and temporal resolution and allows us to study the functionally relevant motions and interdomain interactions of individual transmembrane transporter proteins in real time in a lipid bilayer.

Abstract The energy-coupling factor (ECF) transporters are a family of transmembrane proteins involved in the uptake of vitamins in a wide range of bacteria. Inhibition of the activity of these proteins could reduce the viability of pathogens that depend on vitamin uptake. The central role of vitamin transport in the metabolism of bacteria and absence from humans make the ECF transporters an attractive target for inhibition with selective chemical probes. Here, we report on the identification of a promising class of inhibitors of the ECF transporters. We used coarse-grained molecular dynamics simulations on Lactobacillus delbrueckii ECF-FolT2 and ECF-PanT to profile the binding mode and mechanism of inhibition of this novel chemotype. The results corroborate the postulated mechanism of transport and pave the way for further drug-discovery efforts.

Abstract Microbial synthesis of nutraceutically and pharmaceutically interesting plant polyphenols represents a more environmentally friendly alternative to chemical synthesis or plant extraction. However, most polyphenols are cytotoxic for microorganisms as they are believed to negatively affect cell integrity and transport processes. To increase the production performance of engineered cell factories, strategies have to be developed to mitigate these detrimental effects. Here, we examine the accumulation of the stilbenoid resveratrol in the cell membrane and cell wall during its production using Corynebacterium glutamicum and uncover the membrane rigidifying effect of this stilbenoid experimentally and with molecular dynamics simulations. A screen of free fatty acid supplements identifies palmitelaidic acid and linoleic acid as suitable additives to attenuate resveratrol’s cytotoxic effects resulting in a three-fold higher product titer. This cost-effective approach to counteract membrane-damaging effects of product accumulation is transferable to the microbial production of other polyphenols and may represent an engineering target for other membrane-active bioproducts.

Abstract Energy-coupling factor (ECF)-type transporters mediate the uptake of micronutrients in many bacteria. They consist of a substrate-translocating subunit (S-component) and an ATP-hydrolysing motor (ECF module) Previous data indicate that the S-component topples within the membrane to alternately expose the binding site to either side of the membrane. In many ECF transporters, the substrate-free S-component can be expelled from the ECF module. Here we study this enigmatic expulsion step by cryogenic electron microscopy and reveal that ATP induces a concave-to-convex shape change of two long helices in the motor, thereby destroying the S-component’s docking site and allowing for its dissociation. We show that adaptation of the membrane morphology to the conformational state of the motor may favour expulsion of the substrate-free S-component when ATP is bound and docking of the substrate-loaded S-component after hydrolysis. Our work provides a picture of bilayer-assisted chemo-mechanical coupling in the transport cycle of ECF transporters.

Abstract Antimicrobial resistance is one of the leading concerns in medical care. Here we study the mechanism of action of an antimicrobial cationic tripeptide, AMC-109, by combining high speed-atomic force microscopy, molecular dynamics, fluorescence assays, and lipidomic analysis. We show that AMC-109 activity on negatively charged membranes derived from Staphylococcus aureus consists of two crucial steps. First, AMC-109 self-assembles into stable aggregates consisting of a hydrophobic core and a cationic surface, with specificity for negatively charged membranes. Second, upon incorporation into the membrane, individual peptides insert into the outer monolayer, affecting lateral membrane organization and dissolving membrane nanodomains, without forming pores. We propose that membrane domain dissolution triggered by AMC-109 may affect crucial functions such as protein sorting and cell wall synthesis. Our results indicate that the AMC-109 mode of action resembles that of the disinfectant benzalkonium chloride (BAK), but with enhanced selectivity for bacterial membranes.

ABSTRACTIntestinal microbiota and microbiota-derived metabolites play a key role in regulating the host physiology. Recently, we have identified a gut-bacterial metabolite, namely 5-hydroxyindole, as a potent stimulant of intestinal motility via its modulation of L-type voltage-gated calcium channels located on the intestinal smooth muscle cells. Dysregulation of L-type voltage-gated calcium channels is associated with various gastrointestinal motility disorders, including constipation, making L-type voltage-gated calcium channels an important target for drug development. Nonetheless, the majority of currently available drugs are associated with alteration of the gut microbiota. Using 16S rRNA sequencing this study shows that, when administered orally, 5-hydroxyindole has only marginal effects on the rat cecal microbiota. Molecular dynamics simulations propose potential-binding pockets of 5-hydroxyindole in the α1 subunit of the L-type voltage-gated calcium channels and when its stimulatory effect on the rat colonic contractility was compared to 16 different analogues, ex-vivo, 5-hydroxyindole stood as the most potent enhancer of the intestinal contractility. Overall, the present findings imply a potential role of microbiota-derived metabolites as candidate therapeutics for targeted treatment of slow intestinal motility-related disorders including constipation.

Computational models of cells cannot be considered complete unless they include the most fundamental process of life, the replication and inheritance of genetic material. By creating a computational framework to model systems of replicating bacterial chromosomes as polymers at 10 bp resolution with Brownian dynamics, we investigate changes in chromosome organization during replication and extend the applicability of an existing whole-cell model (WCM) for a genetically minimal bacterium, JCVI-syn3A, to the entire cell-cycle. To achieve cell-scale chromosome structures that are realistic, we model the chromosome as a self-avoiding homopolymer with bending and torsional stiffnesses that capture the essential mechanical properties of dsDNA in Syn3A. In addition, the conformations of the circular DNA must avoid overlapping with ribosomes identitied in cryo-electron tomograms. While Syn3A lacks the complex regulatory systems known to orchestrate chromosome segregation in other bacteria, its minimized genome retains essential loop-extruding structural maintenance of chromosomes (SMC) protein complexes (SMC-scpAB) and topoisomerases. Through implementing the effects of these proteins in our simulations of replicating chromosomes, we find that they alone are sufficient for simultaneous chromosome segregation across all generations within nested theta structures. This supports previous studies suggesting loop-extrusion serves as a near-universal mechanism for chromosome organization within bacterial and eukaryotic cells. Furthermore, we analyze ribosome diffusion under the influence of the chromosome and calculate in silico chromosome contact maps that capture inter-daughter interactions. Finally, we present a methodology to map the polymer model of the chromosome to a Martini coarse-grained representation to prepare molecular dynamics models of entire Syn3A cells, which serves as an ultimate means of validation for cell states predicted by the WCM.

Protein kinases function as pivotal regulators in biological events, governing essential cellular processes through the transfer of phosphate groups from ATP molecules to substrates. Dysregulation of kinase activity is frequently associated with cancer, ocasionally arising from chromosomal translocation events that relocate genes encoding kinases. Fusion proteins resulting from such events, particularly those involving the proto-oncogene tyrosine-protein kinase ROS (ROS1), manifest as constitutively active kinases, emphasizing their role in oncogenesis. Notably, the chromosomal reallocation of the ros1 gene leads to fusion of proteins with the ROS1 kinase domain, implicated in various cancer types. Despite their prevalence, targeted inhibition of these fusion proteins relies on repurposed kinase inhibitors. This review comprehensively surveys experimentally determined ROS1 structures, emphasizing the pivotal role of X-ray crystallography in providing high-quality insights. We delve into the intricate interactions between ROS1 and kinase inhibitors, shedding light on the structural basis for inhibition. Additionally, we explore point mutations identified in patients, employing molecular modeling to elucidate their structural impact on the ROS1 kinase domain. By integrating structural insights with in vitro and in silico data, this review advances our understanding of ROS1 kinase in cancer, offering potential avenues for targeted therapeutic strategies.

Abstract Based on recent findings indicating that metabolism might be governed by a limit on the rate at which cells can dissipate Gibbs energy, in this Perspective, we propose a new mechanism of how metabolic activity could globally regulate biomolecular processes in a cell. Specifically, we postulate that Gibbs energy released in metabolic reactions is used to perform work, allowing enzymes to self‐propel or to break free from supramolecular structures. This catalysis‐induced enzyme movement will result in increased intracellular motion, which in turn can compromise biomolecular functions. Once the increased intracellular motion has a detrimental effect on regulatory mechanisms, this will establish a feedback mechanism on metabolic activity, and result in the observed thermodynamic limit. While this proposed explanation for the identified upper rate limit on cellular Gibbs energy dissipation rate awaits experimental validation, it offers an intriguing perspective of how metabolic activity can globally affect biomolecular functions and will hopefully spark new research.

Membrane permeability of small molecules depends on the composition of the lipid bilayer. Here, authors compare permeability measured on membranes in different physical states and conclude that the yeast membrane exists in a highly ordered phase.

To facilitate the rational design of (nano)-materials and biomacromolecules by MD simulations, the authors present the polyply suite, featuring a graph matching algorithm and a random walk protocol for generating multi-scale polymeric topologies and initial coordinates.

We previously presented a computational protocol to predict the enzymatic (enantio)selectivity of an ω-transaminase towards a set of ligands (Ramírez-Palacios et al. (2021) Journal of Chemical Information and Modeling 61(11), 5569–5580) by counting the number of binding poses present in molecular dynamics (MD) simulations that met a defined set of geometric criteria. The geometric criteria consisted of a hand-crafted set of distances, angles and dihedrals deemed to be important for the enzymatic reaction to take place. In this work, the MD trajectories are reanalysed using a deep-learning approach to predict the enantiopreference of the enzyme without the need for hand-crafted criteria. We show that a convolutional neural network is capable of classifying the trajectories as belonging to the ‘reactive’ or ‘non-reactive’ enantiomer (binary classification) with a good accuracy (>0.90). The new method reduces the computational cost of the methodology, because it does not necessitate the sampling approach from the previous work. We also show that analysing how neural networks reach specific decisions can aid hand-crafted approaches (e.g. definition of near-attack conformations, or binding poses).

The ultimate microscope, directed at a cell, would reveal the dynamics of all the cell’s components with atomic resolution. In contrast to their real-world counterparts, computational microscopes are currently on the brink of meeting this challenge. In this perspective, we show how an integrative approach can be employed to model an entire cell, the minimal cell, JCVI-syn3A, at full complexity. This step opens the way to interrogate the cell’s spatio-temporal evolution with molecular dynamics simulations, an approach that can be extended to other cell types in the near future.

The ASCT1 transporter of the SLC1 family is largely involved in equilibration of neutral amino acids’ pools across the plasma membrane and plays a prominent role in the transport of both L- and D-isomers of serine, essential for the normal functioning of the central nervous system in mammals. A number of mutations in ASCT1 (E256K, G381R, R457W) have been linked to severe neurodevelopmental disorders, however in the absence of ASCT1 structure it is hard to understand their impact on substrate transport. To ameliorate that we have determined a cryo-EM structure of human ASCT1 at 4.2 Å resolution and performed functional transport assays and molecular dynamics simulations, which revealed that given mutations lead to the diminished transport capability of ASCT1 caused by instability of transporter and impeded transport cycle.

Achieved high thermoelectric figure of merit (ZT) in organic thermoelectric materials remains a challenge due to their low packing order and poor host/dopant miscibility. Here, the authors report side chain-engineered n-doped fullerene derivatives with record ZT >0.3 for organic thermoelectrics.

Computer-aided design of protein-ligand binding is important for the development of novel drugs. Here authors present an approach to use the recently re-parametrized coarse-grained Martini model to perform unbiased millisecond sampling of protein-ligand binding interactions of small drug-like molecules.

Computer simulations of large-scale changes in membrane shape are challenging since they occur across a wide range of spatiotemporal scales. Here, authors present a multiscale algorithm that backmaps a continuum membrane model represented as a dynamically triangulated surface to its corresponding molecular model based on the coarse-grained Martini force field.

Energy coupling factor (ECF) transporters are responsible for the uptake of micronutrients and consist of an integral membrane unit, the S-component, which confers substrate specificity. Here, authors present multi-scale molecular dynamics simulations and in vitro experiments to study the molecular toppling mechanism of the S-component of a folate-specific ECF transporter.

Ceramides are lipids that act directly on mitochondria to trigger apoptosis, but the underlying mechanism remains largely unclear. Here authors use a photoactivatable ceramide probe combined with a computation approach and functional studies to identify the voltage-dependent anion channel VDAC2 as a direct effector of ceramide-mediated cell death.

Molecular docking is central to rational drug design. Current docking techniques suffer, however, from limitations in protein flexibility and solvation models and by the use of simplified scoring functions. All-atom molecular dynamics simulations, on the other hand, feature a realistic representation of protein flexibility and solvent, but require knowledge of the binding site. Recently we showed that coarse-grained molecular dynamics simulations, based on the most recent version of the Martini force field, can be used to predict protein/ligand binding sites and pathways, without requiring any a priori information, and offer a level of accuracy approaching all-atom simulations. Given the excellent computational efficiency of Martini, this opens the way to high-throughput drug screening based on dynamic docking pipelines. In this opinion article, we sketch the roadmap to achieve this goal.

We performed coarse-grained simulations of the antimicrobial peptides Magainin-2, BP100, MSI-103, and MSI-78 on a phase-separated membrane to study their preference for the different domains. All the peptides displayed a clear preference for the liquid-disordered (Ld) phase over the liquid-ordered (Lo) one. For BP100, MSI-103, and MSI-78 there was a further preference of the peptides for the domain interface. The peptides’ preference toward the disordered phase was shown to reflect a penalization of lipid–lipid interaction enthalpy in the Lo phase, when in the vicinity of peptides. Similar results were observed at the two studied concentrations, although Ld phase saturation at the higher concentration drove some of the peptide excess to the Lo phase. Magainin-2 and MSI-103 were found to dimerize, in agreement with available experimental data. Interestingly, at high concentrations of Magainin-2 toroidal pores spontaneously formed in the Ld phase. We performed additional simulations to characterize this phenomenon, which is likely related to Magainin-2’s membranolytic action.

The co-localization of Cluster-of-Differentiation-44 protein (CD44) and cytoplasmic adaptors in specific membrane environments is crucial for cell adhesion and migration. The process is controlled by two different pathways: On the one hand palmitoylation keeps CD44 in lipid raft domains and disables the linking to the cytoplasmic adaptor, whereas on the other hand, the presence of phosphatidylinositol-4,5-biphosphate (PIP2) lipids accelerates the formation of the CD44-adaptor complex. The molecular mechanism explaining how CD44 is migrating into and out of the lipid raft domains and its dependence on both palmitoylations and the presence of PIP2 remains, however, elusive. In this study, we performed extensive molecular dynamics simulations to study the raft affinity and translocation of CD44 in phase separated model membranes as well as more realistic plasma membrane environments. We observe a delicate balance between the influence of the palmitoylations and the presence of PIP2 lipids: whereas the palmitoylations of CD44 increases the affinity for raft domains, PIP2 lipids have the opposite effect. Additionally, we studied the association between CD44 and the membrane adaptor FERM in dependence of these factors. We find that the presence of PIP2 lipids allows CD44 and FERM to associate in an experimentally observed binding mode whereas the highly palmitoylated species shows no binding affinity. Together, our results shed light on the sophisticated mechanism on how membrane translocation and peripheral protein association can be controlled by both protein modifications and membrane composition.

The use of non-viral vectors for in vivo gene therapy could drastically increase safety, whilst reducing the cost of preparing the vectors. A promising approach to non-viral vectors makes use of DNA/cationic liposome complexes (lipoplexes) to deliver the genetic material. Here we use coarse-grained molecular dynamics simulations to investigate the molecular mechanism underlying efficient DNA transfer from lipoplexes. Our computational fusion experiments of lipoplexes with endosomal membrane models show two distinct modes of transfection: parallel and perpendicular. In the parallel fusion pathway, DNA aligns with the membrane surface, showing very quick release of genetic material shortly after the initial fusion pore is formed. The perpendicular pathway also leads to transfection, but release is slower. We further show that the composition and size of the lipoplex, as well as the lipid composition of the endosomal membrane, have a significant impact on fusion efficiency in our models.

Dimerization free energies are fundamental quantities that describe the strength of interaction of different molecules. Obtaining accurate experimental values for small molecules and disentangling the conformations that contribute most to the binding can be extremely difficult, due to the size of the systems and the small energy differences. In many cases, one has to resort to computational methods to calculate such properties. In this work, we used molecular dynamics simulations in conjunction with metadynamics to calculate the free energy of dimerization of small aromatic rings, and compared three models from popular online servers for atomistic force fields, namely G54a7, CHARMM36 and OPLS. We show that, regardless of the force field, the profiles for the dimerization free energy of these compounds are very similar. However, significant care needs to be taken when studying larger molecules, since the deviations from the trends increase with the size of the molecules, resulting in force field dependent preferred stacking modes; for example, in the cases of pyrene and tetracene. Our results provide a useful background study for using topology builders to model systems which rely on stacking of aromatic moieties, and are relevant in areas ranging from drug design to supramolecular assembly.

Hydrophobic hydrocarbons are absorbed by cell membranes. The effects of hydrocarbons on biological membranes have been studied extensively, but less is known how these compounds affect lipid phase separation. Here, we show that pyrene and pyrene-like hydrocarbons can dissipate lipid domains in phase separating giant unilamellar vesicles at room temperature. In contrast, related aromatic compounds left the phase separation intact, even at high concentration. We hypothesize that this behavior is because pyrene and related compounds lack preference for either the liquid-ordered (Lo) or liquid-disordered (Ld) phase, while larger molecules prefer Lo, and smaller, less hydrophobic molecules prefer Ld. In addition, our data suggest that localization in the bilayer (depth) and the shape of the molecules might contribute to the effects of the aromatic compounds. Localization and shape of pyrene and related compounds are similar to cholesterol and therefore these molecules could behave as such.

Plastoquinone (PLQ) shuttles electrons between photosystem II (PSII) and cytochrome b6f. Here the authors perform molecular dynamics simulations and propose that PLQ enters the exchange cavity of PSII by a promiscuous diffusion mechanism whereby three different channels each act as entry and exit points.

Although DNA nanopores are widely explored as synthetic membrane proteins, it is still unclear how the anionic DNA assemblies stably reside within the hydrophobic core of a lipid bilayer. Here, the authors use molecular dynamics simulations to reveal the key dynamic interactions and energetics stabilizing the nanopore-membrane interaction.

The migration of circulating leukocytes toward damaged tissue is absolutely fundamental to the inflammatory response, and transendothelial migration (TEM) describes the first cellular barrier that is breached in this process. Human CD14+ inflammatory monocytes express L-selectin, bestowing a non-canonical role in invasion during TEM. In vivo evidence supports a role for L-selectin in regulating TEM and chemotaxis, but the intracellular mechanism is poorly understood. The ezrin-radixin-moesin (ERM) proteins anchor transmembrane proteins to the cortical actin-based cytoskeleton and additionally act as signaling adaptors. During TEM, the L-selectin tail within transmigrating pseudopods interacts first with ezrin to transduce signals for protrusion, followed by moesin to drive ectodomain shedding of L-selectin to limit protrusion. Collectively, interaction of L-selectin with ezrin and moesin fine-tunes monocyte protrusive behavior in TEM. Using FLIM/FRET approaches, we show that ERM binding is absolutely required for outside-in L-selectin clustering. The cytoplasmic tail of human L-selectin contains two serine (S) residues at positions 364 and 367, and here we show that they play divergent roles in regulating ERM binding. Phospho-S364 blocks direct interaction with ERM, whereas molecular modeling suggests phospho-S367 likely drives desorption of the L-selectin tail from the inner leaflet of the plasma membrane to potentiate ERM binding. Serine-to-alanine mutagenesis of S367, but not S364, significantly reduced monocyte protrusive behavior in TEM under flow conditions. Our data propose a model whereby L-selectin tail desorption from the inner leaflet of the plasma membrane and ERM binding are two separable steps that collectively regulate protrusive behavior in TEM.

We present a multi-scale simulation procedure to describe membrane-related biological processes that span over a wide range of length scales. At macroscopic length-scale, a membrane is described as a flexible thin film modeled by a dynamic triangulated surface with its spatial conformations governed by an elastic energy containing only a few model parameters. An implicit protein model allows us to include complex effects of membrane-protein interactions in the macroscopic description. The gist of this multi-scale approach is a scheme to calibrate the implicit protein model using finer scale simulation techniques e.g., all atom and coarse grain molecular dynamics. We previously used this approach and properly described the formation of membrane tubular invaginations upon binding of B-subunit of Shiga toxin. Here, we provide a perspective of our multi-scale approach, summarizing its main features and sketching possible routes for future development.

In neurons and glia, glutamate transporters catalyse the reuptake of this neurotransmitter by coupling it with cation transport. Here the authors combine X-ray crystallography and molecular dynamics simulations of the archeal glutamate transporter GltTkto get insight into the coupled transport mechanism.

Amphotericin B is the most potent antimycotic known to date. However due to its large collateral toxicity, its use, although long standing, had been limited. Many attempts have been made to produce derivatives with reduced collateral damage. The molecular mechanism of polyene has also been closely studied for this purpose and understanding it would contribute to the development of safe derivatives. Our study examined polyene action, including chemical synthesis, electrophysiology, pharmacology, toxicology and molecular dynamics. The results were used to support a novel Amphotericin B derivative with increased selectivity: L-histidine methyl ester of Amphotericin B. We found that this derivative has the same form of action as Amphotericin B, i.e. pore formation in the cell membrane. Its reduced dimerization in solution, when compared to Amphotericin B, is at least partially responsible for its increased selectivity. Here we also present the results of preclinical tests, which show that the derivative is just as potent as Amphotericin B and has increased safety.

Cell membranes have a complex lateral organization featuring domains with distinct composition, also known as rafts, which play an essential role in cellular processes such as signal transduction and protein trafficking. In vivo, perturbations of membrane domains (e.g., by drugs or lipophilic compounds) have major effects on the activity of raft-associated proteins and on signaling pathways, but they are difficult to characterize because of the small size of the domains, typically below optical resolution. Model membranes, instead, can show macroscopic phase separation between liquid-ordered and liquid-disordered domains, and they are often used to investigate the driving forces of membrane lateral organization. Studies in model membranes have shown that some lipophilic compounds perturb membrane domains, but it is not clear which chemical and physical properties determine domain perturbation. The mechanisms of domain stabilization and destabilization are also unknown. Here we describe the effect of six simple hydrophobic compounds on the lateral organization of phase-separated model membranes consisting of saturated and unsaturated phospholipids and cholesterol. Using molecular simulations, we identify two groups of molecules with distinct behavior: aliphatic compounds promote lipid mixing by distributing at the interface between liquid-ordered and liquid-disordered domains; aromatic compounds, instead, stabilize phase separation by partitioning into liquid-disordered domains and excluding cholesterol from the disordered domains. We predict that relatively small concentrations of hydrophobic species can have a broad impact on domain stability in model systems, which suggests possible mechanisms of action for hydrophobic compounds in vivo.

The mechanosensitive channel of large conductance (MscL) has become a model system in which to understand mechanosensation, a process involved in osmoregulation and many other physiological functions. While a high resolution closed state structure is available, details of the open structure and the gating mechanism remain unknown. In this study we combine coarse grained simulations with restraints from EPR and FRET experiments to study the structural changes involved in gating with much greater level of conformational sampling than has previously been possible. We generated a set of plausible open pore structures that agree well with existing open pore structures and gating models. Most interestingly, we found that membrane thinning induces a kink in the upper part of TM1 that causes an outward motion of the periplasmic loop away from the pore centre. This previously unobserved structural change might present a new mechanism of tension sensing and might be related to a functional role in osmoregulation.

The depletion of cholesterol from membranes, mediated by β-cyclodextrin (β-CD) is well known and documented, but the molecular details of this process are largely unknown. Using molecular dynamics simulations, we have been able to study the CD mediated extraction of cholesterol from model membranes, in particular from a pure cholesterol monolayer, at atomic resolution. Our results show that efficient cholesterol extraction depends on the structural distribution of the CDs on the surface of the monolayer. With a suitably oriented dimer, cholesterol is extracted spontaneously on a nanosecond time scale. Additional free energy calculations reveal that the CDs have a strong affinity to bind to the membrane surface, and, by doing so, destabilize the local packing of cholesterol molecules making their extraction favorable. Our results have implications for the interpretation of experimental measurements, and may help in the rational design of efficient CD based nano-carriers.

Coarse-grained (CG) simulations have become an essential tool to study a large variety of biomolecular processes, exploring temporal and spatial scales inaccessible to traditional models of atomistic resolution. One of the major simplifications of CG models is the representation of the solvent, which is either implicit or modeled explicitly as a van der Waals particle. The effect of polarization, and thus a proper screening of interactions depending on the local environment, is absent. Given the important role of water as a ubiquitous solvent in biological systems, its treatment is crucial to the properties derived from simulation studies. Here, we parameterize a polarizable coarse-grained water model to be used in combination with the CG MARTINI force field. Using a three-bead model to represent four water molecules, we show that the orientational polarizability of real water can be effectively accounted for. This has the consequence that the dielectric screening of bulk water is reproduced. At the same time, we parameterized our new water model such that bulk water density and oil/water partitioning data remain at the same level of accuracy as for the standard MARTINI force field. We apply the new model to two cases for which current CG force fields are inadequate. First, we address the transport of ions across a lipid membrane. The computed potential of mean force shows that the ions now naturally feel the change in dielectric medium when moving from the high dielectric aqueous phase toward the low dielectric membrane interior. In the second application we consider the electroporation process of both an oil slab and a lipid bilayer. The electrostatic field drives the formation of water filled pores in both cases, following a similar mechanism as seen with atomistically detailed models.