Martini 3: a general purpose force field for coarse-grained molecular dynamics.

The coarse-grained Martini force field is widely used in biomolecular simulations. Here we present the refined model, Martini 3 ( http://cgmartini.nl ), with an improved interaction balance, new bead types and expanded ability to include specific interactions representing, for example, hydrogen bonding and electronic polarizability. The updated model allows more accurate predictions of molecular packing and interactions in general, which is exemplified with a vast and diverse set of applications, ranging from oil/water partitioning and miscibility data to complex molecular systems, involving protein-protein and protein-lipid interactions and material science applications as ionic liquids and aedamers.

Inverse Conformational Selection in Lipid-Protein Binding.

Interest in lipid interactions with proteins and other biomolecules is emerging not only in fundamental biochemistry but also in the field of nanobiotechnology where lipids are commonly used, for example, in carriers of mRNA vaccines. The outward-facing components of cellular membranes and lipid nanoparticles, the lipid headgroups, regulate membrane interactions with approaching substances, such as proteins, drugs, RNA, or viruses. Because lipid headgroup conformational ensembles have not been experimentally determined in physiologically relevant conditions, an essential question about their interactions with other biomolecules remains unanswered: Do headgroups exchange between a few rigid structures, or fluctuate freely across a practically continuous spectrum of conformations? Here, we combine solid-state NMR experiments and molecular dynamics simulations from the NMRlipids Project to resolve the conformational ensembles of headgroups of four key lipid types in various biologically relevant conditions. We find that lipid headgroups sample a wide range of overlapping conformations in both neutral and charged cellular membranes, and that differences in the headgroup chemistry manifest only in probability distributions of conformations. Furthermore, the analysis of 894 protein-bound lipid structures from the Protein Data Bank suggests that lipids can bind to proteins in a wide range of conformations, which are not limited by the headgroup chemistry. We propose that lipids can select a suitable headgroup conformation from the wide range available to them to fit the various binding sites in proteins. The proposed inverse conformational selection model will extend also to lipid binding to targets other than proteins, such as drugs, RNA, and viruses.

Asymmetric CorA Gating Mechanism as Observed by Molecular Dynamics Simulations.

The CorA family of proteins plays a housekeeping role in the homeostasis of divalent metal ions in many bacteria and archaea as well as in mitochondria of eukaryotes, rendering it an important target to study the mechanisms of divalent transport and regulation across different life domains. Despite numerous studies, the mechanistic details of the channel gating and the transport of the metal ions are still not entirely understood. Here, we use all-atom and coarse-grained molecular dynamics simulations combined with in vitro experiments to investigate the influence of divalent cations on the function of CorA. Simulations reveal pronounced asymmetric movements of monomers that enable the rotation of the α7 helix and the cytoplasmic subdomain with the subsequent formation of new interactions and the opening of the channel. These computational results are functionally validated using site-directed mutagenesis of the intracellular cytoplasmic domain residues and biochemical assays. The obtained results infer a complex network of interactions altering the structure of CorA to allow gating. Furthermore, we attempt to reconcile the existing gating hypotheses for CorA to conclude the mechanism of transport of divalent cations via these proteins.

Molecular electrometer and binding of cations to phospholipid bilayers.

Despite the vast amount of experimental and theoretical studies on the binding affinity of cations - especially the biologically relevant Na+ and Ca2+ - for phospholipid bilayers, there is no consensus in the literature. Here we show that by interpreting changes in the choline headgroup order parameters according to the 'molecular electrometer' concept [Seelig et al., Biochemistry, 1987, 26, 7535], one can directly compare the ion binding affinities between simulations and experiments. Our findings strongly support the view that in contrast to Ca2+ and other multivalent ions, Na+ and other monovalent ions (except Li+) do not specifically bind to phosphatidylcholine lipid bilayers at sub-molar concentrations. However, the Na+ binding affinity was overestimated by several molecular dynamics simulation models, resulting in artificially positively charged bilayers and exaggerated structural effects in the lipid headgroups. While qualitatively correct headgroup order parameter response was observed with Ca2+ binding in all the tested models, no model had sufficient quantitative accuracy to interpret the Ca2+:lipid stoichiometry or the induced atomistic resolution structural changes. All scientific contributions to this open collaboration work were made publicly, using nmrlipids.blogspot.fi as the main communication platform.

Lateral membrane organization as target of an antimicrobial peptidomimetic compound.

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.

Membrane manipulation by free fatty acids improves microbial plant polyphenol synthesis.

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.

Membrane thickness, lipid phase and sterol type are determining factors in the permeability of membranes to small solutes.

Cell membranes provide a selective semi-permeable barrier to the passive transport of molecules. This property differs greatly between organisms. While the cytoplasmic membrane of bacterial cells is highly permeable for weak acids and glycerol, yeasts can maintain large concentration gradients. Here we show that such differences can arise from the physical state of the plasma membrane. By combining stopped-flow kinetic measurements with molecular dynamics simulations, we performed a systematic analysis of the permeability of a variety of small molecules through synthetic membranes of different lipid composition to obtain detailed molecular insight into the permeation mechanisms. While membrane thickness is an important parameter for the permeability through fluid membranes, the largest differences occur when the membranes transit from the liquid-disordered to liquid-ordered and/or to gel state, which is in agreement with previous work on passive diffusion of water. By comparing our results with in vivo measurements from yeast, we conclude that the yeast membrane exists in a highly ordered and rigid state, which is comparable to synthetic saturated DPPC-sterol membranes.

Quantitative linear dichroism imaging of molecular processes in living cells made simple by open software tools.

Fluorescence-detected linear dichroism microscopy allows observing various molecular processes in living cells, as well as obtaining quantitative information on orientation of fluorescent molecules associated with cellular features. Such information can provide insights into protein structure, aid in development of genetically encoded probes, and allow determinations of lipid membrane properties. However, quantitating and interpreting linear dichroism in biological systems has been laborious and unreliable. Here we present a set of open source ImageJ-based software tools that allow fast and easy linear dichroism visualization and quantitation, as well as extraction of quantitative information on molecular orientations, even in living systems. The tools were tested on model synthetic lipid vesicles and applied to a variety of biological systems, including observations of conformational changes during G-protein signaling in living cells, using fluorescent proteins. Our results show that our tools and model systems are applicable to a wide range of molecules and polarization-resolved microscopy techniques, and represent a significant step towards making polarization microscopy a mainstream tool of biological imaging.

Adaptive response to wine selective pressures shapes the genome of a Saccharomyces interspecies hybrid.

During industrial processes, yeasts are exposed to harsh conditions, which eventually lead to adaptation of the strains. In the laboratory, it is possible to use experimental evolution to link the evolutionary biology response to these adaptation pressures for the industrial improvement of a specific yeast strain. In this work, we aimed to study the adaptation of a wine industrial yeast in stress conditions of the high ethanol concentrations present in stopped fermentations and secondary fermentations in the processes of champagne production. We used a commercial Saccharomyces cerevisiae × S. uvarum hybrid and assessed its adaptation in a modified synthetic must (M-SM) containing high ethanol, which also contained metabisulfite, a preservative that is used during wine fermentation as it converts to sulfite. After the adaptation process under these selected stressful environmental conditions, the tolerance of the adapted strain (H14A7-etoh) to sulfite and ethanol was investigated, revealing that the adapted hybrid is more resistant to sulfite compared to the original H14A7 strain, whereas ethanol tolerance improvement was slight. However, a trade-off in the adapted hybrid was found, as it had a lower capacity to ferment glucose and fructose in comparison with H14A7. Hybrid genomes are almost always unstable, and different signals of adaptation on H14A7-etoh genome were detected. Each subgenome present in the adapted strain had adapted differently. Chromosome aneuploidies were present in S. cerevisiae chromosome III and in S. uvarum chromosome VII-XVI, which had been duplicated. Moreover, S. uvarum chromosome I was not present in H14A7-etoh and a loss of heterozygosity (LOH) event arose on S. cerevisiae chromosome I. RNA-sequencing analysis showed differential gene expression between H14A7-etoh and H14A7, which can be easily correlated with the signals of adaptation that were found on the H14A7-etoh genome. Finally, we report alterations in the lipid composition of the membrane, consistent with conserved tolerance mechanisms.

Improved Cation Binding to Lipid Bilayers with Negatively Charged POPS by Effective Inclusion of Electronic Polarization.

Phosphatidylserine (PS) lipids are important signaling molecules and the most common negatively charged lipids in eukaryotic membranes. The signaling can be often regulated by calcium, but its interactions with PS headgroups are not fully understood. Classical molecular dynamics (MD) simulations can potentially give detailed description of lipid-ion interactions, but the results strongly depend on the used force field. Here, we apply the electronic continuum correction (ECC) to the Amber Lipid17 parameters of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS) lipid to improve its interactions with K+, Na+, and Ca2+ ions. The partial charges of the headgroup, glycerol backbone, and carbonyls of POPS, bearing a unit negative charge, were scaled with a factor of 0.75, derived for monovalent ions, and the Lennard-Jones σ parameters of the same segments were scaled with a factor of 0.89. The resulting ECC-POPS model gives more realistic interactions with Na+ and Ca2+ cations than the original Amber Lipid17 parameters when validated using headgroup order parameters and the "electrometer concept". In ECC-lipids simulations, populations of complexes of Ca2+ cations with more than two PS lipids are negligible, and interactions of Ca2+ cations with only carboxylate groups are twice more likely than with only phosphate groups, while interactions with carbonyls almost entirely involve other groups as well. Our results pave the way for more realistic MD simulations of biomolecular systems with anionic membranes, allowing signaling processes involving PS and Ca2+ to be elucidated.

Dual Resolution Membrane Simulations Using Virtual Sites.

All-atomistic (AA) and coarse-grain (CG) simulations have been successfully applied to investigate a broad range of biomolecular processes. However, the accessible time and length scales of AA simulation are limited and the specific molecular details of CG simulation are simplified. Here, we propose a virtual site (VS) based hybrid scheme that can concurrently couple AA and CG resolutions in a single membrane simulation, mitigating the shortcomings of either representation. With some adjustments to make the AA and CG force fields compatible, we demonstrate that lipid bilayer properties are well kept in our hybrid approach. Our VS hybrid method was also applied to simulate a small lipid vesicle, with the inner leaflet and interior solvent represented in AA, and the outer leaflet together with exterior solvent at the CG level. Our multiscale method opens the way to investigate biomembrane properties at increased computational efficiency, in particular applications involving large solvent filled regions.

Accurate Biomolecular Simulations Account for Electronic Polarization.

In this perspective, we discuss where and how accounting for electronic many-body polarization affects the accuracy of classical molecular dynamics simulations of biomolecules. While the effects of electronic polarization are highly pronounced for molecules with an opposite total charge, they are also non-negligible for interactions with overall neutral molecules. For instance, neglecting these effects in important biomolecules like amino acids and phospholipids affects the structure of proteins and membranes having a large impact on interpreting experimental data as well as building coarse grained models. With the combined advances in theory, algorithms and computational power it is currently realistic to perform simulations with explicit polarizable dipoles on systems with relevant sizes and complexity. Alternatively, the effects of electronic polarization can also be included at zero additional computational cost compared to standard fixed-charge force fields using the electronic continuum correction, as was recently demonstrated for several classes of biomolecules.

Headgroup Structure and Cation Binding in Phosphatidylserine Lipid Bilayers.

Phosphatidylserine (PS) is a negatively charged lipid type commonly found in eukaryotic membranes, where it interacts with proteins via nonspecific electrostatic interactions as well as via specific binding. Moreover, in the presence of calcium ions, PS lipids can induce membrane fusion and phase separation. Molecular details of these phenomena remain poorly understood, partly because accurate models to interpret the experimental data have not been available. Here we gather a set of previously published experimental NMR data of C-H bond order parameter magnitudes, |SCH|, for pure PS and mixed PS:PC (phosphatidylcholine) lipid bilayers and augment this data set by measuring the signs of SCH in the PS headgroup using S-DROSS solid-state NMR spectroscopy. The augmented data set is then used to assess the accuracy of the PS headgroup structures in, and the cation binding to, PS-containing membranes in the most commonly used classical molecular dynamics (MD) force fields including CHARMM36, Lipid17, MacRog, Slipids, GROMOS-CKP, Berger, and variants. We show large discrepancies between different force fields and that none of them reproduces the NMR data within experimental accuracy. However, the best MD models can detect the most essential differences between PC and PS headgroup structures. The cation binding affinity is not captured correctly by any of the PS force fields-an observation that is in line with our previous results for PC lipids. Moreover, the simulated response of the PS headgroup to bound ions can differ from experiments even qualitatively. The collected experimental data set and simulation results will pave the way for development of lipid force fields that correctly describe the biologically relevant negatively charged membranes and their interactions with ions. This work is part of the NMRlipids open collaboration project ( nmrlipids.blogspot.fi ).

Accurate Binding of Sodium and Calcium to a POPC Bilayer by Effective Inclusion of Electronic Polarization.

Binding affinities and stoichiometries of Na+ and Ca2+ ions to phospholipid bilayers are of paramount significance in the properties and functionality of cellular membranes. Current estimates of binding affinities and stoichiometries of cations are, however, inconsistent due to limitations in the available experimental and computational methods. In this work, we improve the description of the binding details of Na+ and Ca2+ ions to a 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayer by implicitly including electronic polarization as a mean field correction, known as the electronic continuum correction (ECC). This is applied by scaling the partial charges of a selected state-of-the-art POPC lipid model for molecular dynamics simulations. Our improved ECC-POPC model reproduces not only the experimentally measured structural parameters for the ion-free membrane, but also the response of lipid headgroup to a strongly bound cationic amphiphile, as well as the binding affinities of Na+ and Ca2+ ions. With our new model, we observe on the one side negligible binding of Na+ ions to POPC bilayer, while on the other side stronger interactions of Ca2+ primarily with phosphate oxygens, which is in agreement with the previous interpretations of the experimental spectroscopic data. The present model results in Ca2+ ions forming complexes with one to three POPC molecules with almost equal probabilities, suggesting more complex binding stoichiometries than those from simple models used to interpret the NMR data previously. The results of this work pave the way to quantitative molecular simulations with realistic electrostatic interactions of complex biochemical systems at cellular membranes.

Transmembrane Potential Modeling: Comparison between Methods of Constant Electric Field and Ion Imbalance.

Two approaches for modeling of the transmembrane potential, as present in all eukaryotic cells, are examined in detail and compared with each other. One approach uses an externally applied electric field, whereas the other maintains an imbalance of ions on the two sides of a membrane. We demonstrate that both methods provide converged results concerning structural parameters of the membrane which are practically indistinguishable from each other, at least for monovalent ions. Effects of the electric field on the detailed molecular structure of the phospholipid bilayer are also presented and discussed. In addition, we achieve a considerable speed-up of the underlying molecular dynamics simulations by implementing the virtual interaction sites method for the Slipids force field.

Estimation of Transition-Metal Empirical Parameters for Molecular Mechanical Force Fields.

Force-field parameters of the first row transition metals together with a few additional common elements such as those from the second (Rh, Ru) and third (Hg, Pt) rows of elements in ligated forms were determined based on the density functional theory calculations. Bonding characteristics were determined by averaging metal-ligand force constants in optimal geometries from several chosen complexes of each metal in the most common oxidation numbers and structural arrangements. Parameters of Lennard-Jones potential were determined based on a supermolecular model. Our determined molecular mechanical parameters are compared with presently available parameters published by other groups. We performed two different kinds of testing in order to demonstrate the reliability of these parameters in the case of ligated metallo complexes. First, the nonbonding potential was constructed for an additional set of 19 larger systems containing common complexes with organic molecules. The second test compares the Pt-O and Pt-H radial distribution functions for cisplatin in a box of TIP3P water with lately published studies.