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.

Hydration of biologically relevant tetramethylammonium cation by neutron scattering and molecular dynamics.

Neutron scattering and molecular dynamics studies were performed on a concentrated aqueous tetramethylammonium (TMA) chloride solution to gain insight into the hydration shell structure of TMA, which is relevant for understanding its behavior in biological contexts of, e.g., properties of phospholipid membrane headgroups or interactions between DNA and histones. Specifically, neutron diffraction with isotopic substitution experiments were performed on TMA and water hydrogens to extract the specific correlation between hydrogens in TMA (HTMA) and hydrogens in water (HW). Classical molecular dynamics simulations were performed to help interpret the experimental neutron scattering data. Comparison of the hydration structure and simulated neutron signals obtained with various force field flavors (e.g. overall charge, charge distribution, polarity of the CH bonds and geometry) allowed us to gain insight into how sensitive the TMA hydration structure is to such changes and how much the neutron signal can capture them. We show that certain aspects of the hydration, such as the correlation of the hydrogen on TMA to hydrogen on water, showed little dependence on the force field. In contrast, other correlations, such as the ion-ion interactions, showed more marked changes. Strikingly, the neutron scattering signal cannot discriminate between different hydration patterns. Finally, ab initio molecular dynamics was used to examine the three-dimensional hydration structure and thus to benchmark force field simulations. Overall, while neutron scattering has been previously successfully used to improve force fields, in the particular case of TMA we show that it has only limited value to fully determine the hydration structure, with other techniques such as ab initio MD being of a significant help.

Influence of electronic polarization on the binding of anions to a chloride-pumping rhodopsin.

The functional properties of some biological ion channels and membrane transport proteins are proposed to exploit anion-hydrophobic interactions. Here, we investigate a chloride-pumping rhodopsin as an example of a membrane protein known to contain a defined anion binding site composed predominantly of hydrophobic residues. Using molecular dynamics simulations, we explore Cl- binding to this hydrophobic site and compare the dynamics arising when electronic polarization is neglected (CHARMM36 [c36] fixed-charge force field), included implicitly (via the prosECCo force field), or included explicitly (through the polarizable force field, AMOEBA). Free energy landscapes of Cl- moving out of the binding site and into bulk solution demonstrate that the inclusion of polarization results in stronger ion binding and a second metastable binding site in chloride-pumping rhodopsin. Simulations focused on this hydrophobic binding site also indicate longer binding durations and closer ion proximity when polarization is included. Furthermore, simulations reveal that Cl- within this binding site interacts with an adjacent loop to facilitate rebinding events that are not observed when polarization is neglected. These results demonstrate how the inclusion of polarization can influence the behavior of anions within protein binding sites and can yield results comparable with more accurate and computationally demanding methods.

Use of Raman and Raman optical activity to extract atomistic details of saccharides in aqueous solution.

Sugars are crucial components in biosystems and industrial applications. In aqueous environments, the natural state of short saccharides or charged glycosaminoglycans is floating and wiggling in solution. Therefore, tools to characterize their structure in a native aqueous environment are crucial but not always available. Here, we show that a combination of Raman/ROA and, on occasions, NMR experiments with Molecular Dynamics (MD) and Quantum Mechanics (QM) is a viable method to gain insights into structural features of sugars in solutions. Combining these methods provides information about accessible ring puckering conformers and their proportions. It also provides information about the conformation of the linkage between the sugar monomers, i.e., glycosidic bonds, allowing for identifying significantly accessible conformers and their relative abundance. For mixtures of sugar moieties, this method enables the deconvolution of the Raman/ROA spectra to find the actual amounts of its molecular constituents, serving as an effective analytical technique. For example, it allows calculating anomeric ratios for reducing sugars and analyzing more complex sugar mixtures to elucidate their real content. Altogether, we show that combining Raman/ROA spectroscopies with simulations is a versatile method applicable to saccharides. It allows for accessing many features with precision comparable to other methods routinely used for this task, making it a viable alternative. Furthermore, we prove that the proposed technique can scale up by studying the complicated raffinose trisaccharide, and therefore, we expect its wide adoption to characterize sugar structural features in solution.

Dynamics of transition dipole moment orientation in representative fluorescent proteins.

Molecules of fluorescent proteins (FPs) exhibit distinct optical directionality. This optical directionality is characterized by transition dipole moments (TDMs), and their orientation with respect to the molecular structures. Although our recent observations of FP crystals allowed us to determine the mean TDM directions with respect to the framework of representative FP molecules, the dynamics of TDM orientations within FP molecules remain to be ascertained. Here we describe the results of our investigations of the dynamics of TDM directions in the fluorescent proteins eGFP, mTurquoise2 and mCherry, through time-resolved fluorescence polarization measurements and microsecond time scale all-atom molecular dynamics (MD) simulations. The investigated FPs exhibit initial fluorescence anisotropies (r0) consistent with significant differences in the orientation of the excitation and emission TDMs. However, based on MD data, we largely attribute this observation to rapid (sub-nanosecond) fluorophore motions within the FP molecular framework. Our results allow improved determinations of orientational distributions of FP molecules by polarization microscopy, as well as more accurate interpretations of fluorescence resonance energy transfer (FRET) observations.

Ionic Strength and Solution Composition Dictate the Adsorption of Cell-Penetrating Peptides onto Phosphatidylcholine Membranes.

Adsorption of arginine-rich positively charged peptides onto neutral zwitterionic phosphocholine (PC) bilayers is a key step in the translocation of those potent cell-penetrating peptides into the cell interior. In the past, we have shown both theoretically and experimentally that polyarginines adsorb to the neutral PC-supported lipid bilayers in contrast to polylysines. However, comparing our results with previous studies showed that the results often do not match even at the qualitative level. The adsorption of arginine-rich peptides onto 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) may qualitatively depend on the actual experimental conditions where binding experiments have been performed. In this work, we systematically studied the adsorption of R9 and K9 peptides onto the POPC bilayer, aided by molecular dynamics (MD) simulations and fluorescence cross-correlation spectroscopy (FCCS) experiments. Using MD simulations, we tested a series of increasing peptide concentrations, in parallel with increasing Na+ and Ca2+ salt concentrations, showing that the apparent strength of adsorption of R9 decreases upon the increase of peptide or salt concentration in the system. The key result from the simulations is that the salt concentrations used experimentally can alter the picture of peptide adsorption qualitatively. Using FCCS experiments with fluorescently labeled R9 and K9, we first demonstrated that the binding of R9 to POPC is tighter by almost 2 orders of magnitude compared to that of K9. Finally, upon the addition of an excess of either Na+ or Ca2+ ions with R9, the total fluorescence correlation signal is lost, which implies the unbinding of R9 from the PC bilayer, in agreement with our predictions from MD simulations.

Anisotropic diffusion of membrane proteins at experimental timescales.

Single-particle tracking (SPT) experiments of lipids and membrane proteins provide a wealth of information about the properties of biomembranes. Careful analysis of SPT trajectories can reveal deviations from ideal Brownian behavior. Among others, this includes confinement effects and anomalous diffusion, which are manifestations of both the nanoscale structure of the underlying membrane and the structure of the diffuser. With the rapid increase in temporal and spatial resolution of experimental methods, a new aspect of the motion of the particle, namely, anisotropic diffusion, might become relevant. This aspect that so far received only little attention is the anisotropy of the diffusive motion and may soon provide an additional proxy to the structure and topology of biomembranes. Unfortunately, the theoretical framework for detecting and interpreting anisotropy effects is currently scattered and incomplete. Here, we provide a computational method to evaluate the degree of anisotropy directly from molecular dynamics simulations and also point out a way to compare the obtained results with those available from SPT experiments. In order to probe the effects of anisotropic diffusion, we performed coarse-grained molecular dynamics simulations of peripheral and integral membrane proteins in flat and curved bilayers. In agreement with the theoretical basis, our computational results indicate that anisotropy can persist up to the rotational relaxation time [τ=(2Dr)-1], after which isotropic diffusion is observed. Moreover, the underlying topology of the membrane bilayer can couple with the geometry of the particle, thus extending the spatiotemporal domain over which this type of motion can be detected.

Raman Optical Activity of Glucose and Sorbose in Extended Wavenumber Range.

Raman optical activity (ROA) is pursued as a promising method for structural analyses of sugars in aqueous solutions. In the present study, experimental Raman and ROA spectra of glucose and sorbose obtained in an extended range (50-4000 cm-1 ) are interpreted using molecular dynamics and density functional theory, with the emphasis on CH stretching modes. A reasonable theoretical basis for spectral interpretation was obtained already at the harmonic level. Anharmonic corrections led to minor shifts of band positions (up to 25 cm-1 ) below 2000 cm-1 , while the CH stretching bands shifted more, by ∼180 cm-1 , and better reproduced the experiment. However, the anharmonicities could be included on a relatively low approximation level only, and they did not always improve the harmonic band shapes. The dependence on the structure and conformation shows that the CH stretching ROA spectral pattern is a sensitive marker useful in saccharide structure studies.

Reduced level of docosahexaenoic acid shifts GPCR neuroreceptors to less ordered membrane regions.

G protein-coupled receptors (GPCRs) control cellular signaling and responses. Many of these GPCRs are modulated by cholesterol and polyunsaturated fatty acids (PUFAs) which have been shown to co-exist with saturated lipids in ordered membrane domains. However, the lipid compositions of such domains extracted from the brain cortex tissue of individuals suffering from GPCR-associated neurological disorders show drastically lowered levels of PUFAs. Here, using free energy techniques and multiscale simulations of numerous membrane proteins, we show that the presence of the PUFA DHA helps helical multi-pass proteins such as GPCRs partition into ordered membrane domains. The mechanism is based on hybrid lipids, whose PUFA chains coat the rough protein surface, while the saturated chains face the raft environment, thus minimizing perturbations therein. Our findings suggest that the reduction of GPCR partitioning to their native ordered environments due to PUFA depletion might affect the function of these receptors in numerous neurodegenerative diseases, where the membrane PUFA levels in the brain are decreased. We hope that this work inspires experimental studies on the connection between membrane PUFA levels and GPCR signaling.

Simulation of Raman and Raman optical activity of saccharides in solution.

Structural studies of sugars in solution are challenging for most of the traditional analytical techniques. Raman and Raman optical activity (ROA) spectroscopies were found to be extremely convenient for this purpose. However, Raman and ROA spectra of saccharides are challenging to interpret and model due to saccharides' flexibility and polarity. In this study, we present an optimized computational protocol that enables the simulation of the spectra efficiently. Our protocol, which results in good agreement with experiments, combines molecular dynamics and density functional theory calculations. It further uses a smart optimization procedure and a novel adaptable scaling function. The numerical stability and accuracy of individual computational steps are evaluated by comparing simulated and experimental spectra of d-glucose, d-glucuronic acid, N-acetyl-d-glucosamine, methyl ß-d-glucopyranoside, methyl ß-d-glucuronide, and methyl ß-N-acetyl-d-glucosaminide. Overall, our Raman and ROA simulation protocol allows one to routinely and reliably calculate the spectra of small saccharides and opens the door to advanced applications, such as complete 3-dimensional structural determination by direct interpretation of the experimental spectra.

Binding of divalent cations to acetate: molecular simulations guided by Raman spectroscopy.

In spite of the biological importance of the binding of Zn2+, Ca2+, and Mg2+ to the carboxylate group, cation-acetate binding affinities and binding modes remain actively debated. Here, we report the first use of Raman multivariate curve resolution (Raman-MCR) vibrational spectroscopy to obtain self-consistent free and bound metal acetate spectra and one-to-one binding constants, without the need to invoke any a priori assumptions regarding the shapes of the corresponding vibrational bands. The experimental results, combined with classical molecular dynamics simulations with a force field effectively accounting for electronic polarization via charge scaling and ab initio simulations, indicate that the measured binding constants pertain to direct (as opposed to water separated) ion pairing. The resulting binding constants do not scale with cation size, as the binding constant to Zn2+ is significantly larger than that to either Mg2+ or Ca2+, although Zn2+ and Mg2+ have similar radii that are about 25% smaller than Ca2+. Remaining uncertainties in the metal acetate binding free energies are linked to fundamental ambiguities associated with identifying the range of structures pertaining to non-covalently bound species.

The Role of Temperature and Lipid Charge on Intake/Uptake of Cationic Gold Nanoparticles into Lipid Bilayers.

Understanding the molecular mechanisms governing nanoparticle-membrane interactions is of prime importance for drug delivery and biomedical applications. Neutron reflectometry (NR) experiments are combined with atomistic and coarse-grained molecular dynamics (MD) simulations to study the interaction between cationic gold nanoparticles (AuNPs) and model lipid membranes composed of a mixture of zwitterionic di-stearoyl-phosphatidylcholine (DSPC) and anionic di-stearoyl-phosphatidylglycerol (DSPG). MD simulations show that the interaction between AuNPs and a pure DSPC lipid bilayer is modulated by a free energy barrier. This can be overcome by increasing temperature, which promotes an irreversible AuNP incorporation into the lipid bilayer. NR experiments confirm the encapsulation of the AuNPs within the lipid bilayer at temperatures around 55 °C. In contrast, the AuNP adsorption is weak and impaired by heating for a DSPC-DSPG (3:1) lipid bilayer. These results demonstrate that both the lipid charge and the temperature play pivotal roles in AuNP-membrane interactions. Furthermore, NR experiments indicate that the (negative) DSPG lipids are associated with lipid extraction upon AuNP adsorption, which is confirmed by coarse-grained MD simulations as a lipid-crawling effect driving further AuNP aggregation. Overall, the obtained detailed molecular view of the interaction mechanisms sheds light on AuNP incorporation and membrane destabilization.

Rapid diffusion of cholesterol along polyunsaturated membranes via deep dives.

Cholesterol regulates the function of membrane proteins either via direct or membrane-mediated effects. Therefore, its ready availability is crucial for many protein-governed cellular processes. Recent studies suggest that cholesterol can partition to the core of polyunsaturated membranes, where cholesterol binding sites of many membrane proteins are also located. This core region is characterized by a lower viscosity. Therefore, we hypothesized that cholesterol partitioning into the membrane interior increases the rate of its diffusion in polyunsaturated membrane environments. We studied the behavior of cholesterol in membranes with increasing level of lipid chain unsaturation using a combination of atomistic and coarse-grained molecular dynamics simulations. Our simulations suggest a strong correlation between entropy-driven enhanced cholesterol partitioning to the membrane core and its faster lateral diffusion, which indicates that the less viscous membrane core indeed provides an efficient means for cholesterol movement in polyunsaturated membrane environments.

Atomistic fingerprint of hyaluronan-CD44 binding.

Hyaluronan is a polyanionic, megadalton-scale polysaccharide, which initiates cell signaling by interacting with several receptor proteins including CD44 involved in cell-cell interactions and cell adhesion. Previous studies of the CD44 hyaluronan binding domain have identified multiple widespread residues to be responsible for its recognition capacity. In contrast, the X-ray structural characterization of CD44 has revealed a single binding mode associated with interactions that involve just a fraction of these residues. In this study, we show through atomistic molecular dynamics simulations that hyaluronan can bind CD44 with three topographically different binding modes that in unison define an interaction fingerprint, thus providing a plausible explanation for the disagreement between the earlier studies. Our results confirm that the known crystallographic mode is the strongest of the three binding modes. The other two modes represent metastable configurations that are readily available in the initial stages of the binding, and they are also the most frequently observed modes in our unbiased simulations. We further discuss how CD44, fostered by the weaker binding modes, diffuses along HA when attached. This 1D diffusion combined with the constrained relative orientation of the diffusing proteins is likely to influence the aggregation kinetics of CD44. Importantly, CD44 aggregation has been suggested to be a possible mechanism in CD44-mediated signaling.

Efficient preparation and analysis of membrane and membrane protein systems.

Molecular dynamics (MD) simulations have become a highly important technique to consider lipid membrane systems, and quite often they provide considerable added value to laboratory experiments. Rapid development of both software and hardware has enabled the increase of time and size scales reachable by MD simulations to match those attainable by several accurate experimental techniques. However, until recently, the quality and maturity of software tools available for building membrane models for simulations as well as analyzing the results of these simulations have seriously lagged behind. Here, we discuss the recent developments of such tools from the end-users' point of view. In particular, we review the software that can be employed to build lipid bilayers and other related structures with or without embedded membrane proteins to be employed in MD simulations. Additionally, we provide a brief critical insight into force fields and MD packages commonly used for membrane and membrane protein simulations. Finally, we list analysis tools that can be used to study the properties of membrane and membrane protein systems. In all these points we comment on the respective compatibility of the covered tools. We also share our opinion on the current state of the available software. We briefly discuss the most commonly employed tools and platforms on which new software can be built. We conclude the review by providing a few ideas and guidelines on how the development of tools can be further boosted to catch up with the rapid pace at which the field of membrane simulation progresses. This includes improving the compatibility between software tools and promoting the openness of the codes on which these applications rely. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

doGlycans-Tools for Preparing Carbohydrate Structures for Atomistic Simulations of Glycoproteins, Glycolipids, and Carbohydrate Polymers for GROMACS.

Carbohydrates constitute a structurally and functionally diverse group of biological molecules and macromolecules. In cells they are involved in, e.g., energy storage, signaling, and cell-cell recognition. All of these phenomena take place in atomistic scales, thus atomistic simulation would be the method of choice to explore how carbohydrates function. However, the progress in the field is limited by the lack of appropriate tools for preparing carbohydrate structures and related topology files for the simulation models. Here we present tools that fill this gap. Applications where the tools discussed in this paper are particularly useful include, among others, the preparation of structures for glycolipids, nanocellulose, and glycans linked to glycoproteins. The molecular structures and simulation files generated by the tools are compatible with GROMACS.

Atomistic simulations of anionic Au144(SR)60 nanoparticles interacting with asymmetric model lipid membranes.

Experimental observations indicate that the interaction between nanoparticles and lipid membranes varies according to the nanoparticle charge and the chemical nature of their protecting side groups. We report atomistic simulations of an anionic Au nanoparticle (AuNP(-)) interacting with membranes whose lipid composition and transmembrane distribution are to a large extent consistent with real plasma membranes of eukaryotic cells. To this end, we use a model system which comprises two cellular compartments, extracellular and cytosolic, divided by two asymmetric lipid bilayers. The simulations clearly show that AuNP(-) attaches to the extracellular membrane surface within a few tens of nanoseconds, while it avoids contact with the membrane on the cytosolic side. This behavior stems from several factors. In essence, when the nanoparticle interacts with lipids in the extracellular compartment, it forms relatively weak contacts with the zwitterionic head groups (in particular choline) of the phosphatidylcholine lipids. Consequently, AuNP(-) does not immerse deeply in the leaflet, enabling, e.g., lateral diffusion of the nanoparticle along the surface. On the cytosolic side, AuNP(-) remains in the water phase due to Coulomb repulsion that arises from negatively charged phosphatidylserine lipids interacting with AuNP(-). A number of structural and dynamical features resulting from these basic phenomena are discussed. We close the article with a brief discussion of potential implications.

MEMBPLUGIN: studying membrane complexity in VMD.

SUMMARY: Computer simulations are giving way to more complex and accurate studies of biological membranes by molecular dynamics (MD) simulations. The analysis of MD trajectories comprises the biophysical characterization of membrane properties or the study of protein-lipid interactions and dynamics. However, there is a lack of automated tools to analyse MD simulations of complex membrane or membrane-protein systems. Here we present MEMBPLUGIN, a plugin for the Visual Molecular Dynamics package that provides algorithms to measure a host of essential biophysical properties in simulated membranes. MEMBPLUGIN features are accessible both through a user-friendly graphical interface and as command-line procedures to be invoked in analysis scripts. AVAILABILITY AND IMPLEMENTATION: MEMBPLUGIN is a VMD extension written in Tcl. Multi-platform source code, documentation and tutorials are freely available at http://membplugin.sourceforge.net. CONTACT: toni.giorgino@isib.cnr.it or jana.selent@upf.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Building Water Models Compatible with Charge Scaling Molecular Dynamics.

Charge scaling has proven to be an efficient way to account in a mean-field manner for electronic polarization by aqueous ions in force field molecular dynamics simulations. However, commonly used water models with dielectric constants over 50 are not consistent with this approach leading to "overscaling", i.e., generally too weak ion-ion interactions. Here, we build water models fully compatible with charge scaling, i.e., having the correct low-frequency dielectric constant of about 45. To this end, we employ advanced optimization and machine learning schemes in order to explore the vast parameter space of four-site water models efficiently. As an a priori unwarranted positive result, we find a sizable range of force field parameters that satisfy the above dielectric constant constraint providing at the same time accuracy with respect to experimental data comparable with the best existing four-site water models such as TIP4P/2005, TIP4P-FB, or OPC. The present results thus open the way to the development of a consistent charge scaling force field for modeling ions in aqueous solutions.

Foldamers controlled by functional triamino acids: structural investigation of α/γ-hybrid oligopeptides.

Peptide-like foldamers controlled by normal amide backbone hydrogen bonding have been extensively studied, and their folding patterns largely rely on configurational and conformational constraints induced by the steric properties of backbone substituents at appropriate positions. In contrast, opportunities to influence peptide secondary structure by functional groups forming individual hydrogen bond networks have not received much attention. Here, peptide-like foldamers consisting of alternating α,ß,γ-triamino acids 3-amino-4-(aminomethyl)-2-methylpyrrolidine-3-carboxylate (AAMP) and natural amino acids glycine and alanine are reported, which were obtained by solution phase peptide synthesis. They form ordered secondary structures, which are dominated by a three-dimensional bridged triazaspiranoid-like hydrogen bond network involving the non-backbone amino groups, the backbone amide hydrogen bonds, and the relative configuration of the α,ß,γ-triamino and α-amino acid building blocks. This additional stabilization leads to folding in both nonpolar organic as well as in aqueous environments. The three-dimensional arrangement of the individual foldamers is supported by X-ray crystallography, NMR spectroscopy, chiroptical methods, and molecular dynamics simulations.