Molecular Models of Nanodiscs

Nanodiscs are discoloidal protein–lipid particles that self-assemble from a mixture of lipids and membrane scaffold proteins. They form a highly soluble membrane mimetic that closely resembles a native-like lipid environment, unlike micelles. Nanodiscs are widely used for experimental studies of membrane proteins. In this work, we present a new method for building arbitrary nanodiscs using a combination of the Martini coarse-grained and all-atom force fields. We model the basic membrane scaffold protein MSP1 and its extended versions, such as MSP1E1 and MSP1E2, using a crystal structure of human apolipoprotein Apo-I. We test our method by generating nanodiscs of different sizes and compositions, including nanodiscs with embedded membrane proteins, such as bacteriorhodopsin, outer membrane protein X, and the glucose transporter. We show that properties of our nanodiscs are in general agreement with experimental data and previous computational studies

Parameterization of Palmitoylated Cysteine, Farnesylated Cysteine, Geranylgeranylated Cysteine, and Myristoylated Glycine for the Martini Force Field

Peripheral membrane proteins go through various post-translational modifications that covalently bind fatty acid tails to specific amino acids. These post-translational modifications significantly alter the lipophilicity of the modified proteins and allow them to anchor to biological membranes. Over 1000 different proteins have been identified to date that require such membrane–protein interactions to carry out their biological functions, including members of the Src and Ras superfamilies that play key roles in cell signaling and carcinogenesis. We have used all-atom simulations with the CHARMM36 force field to parameterize four of the most common post-translational modifications for the Martini 2.2 force field: palmitoylated cysteine, farnesylated cysteine, geranylgeranylated cysteine, and myristoylated glycine. The parameters reproduce the key features of clusters of configurations of the different anchors in lipid membranes as well as the water–octanol partitioning free energies of the anchors, which are crucial for the correct reproduction of the expected biophysical behavior of peripheral membrane proteins at the membrane–water interface. Implementation in existing Martini setup tools facilitates the use of the new parameters

Microsecond Molecular Dynamics Simulations of Lipid Mixing

Molecular dynamics (MD) simulations of membranes are often hindered by the slow lateral diffusion of lipids and the limited time scale of MD. In order to study the dynamics of mixing and characterize the lateral distribution of lipids in converged mixtures, we report microsecond-long all-atom MD simulations performed on the special-purpose machine Anton. Two types of mixed bilayers, POPE:POPG (3:1) and POPC:cholesterol (2:1), as well as a pure POPC bilayer, were each simulated for up to 2 μs. These simulations show that POPE:POPG and POPC:cholesterol are each fully miscible at the simulated conditions, with the final states of the mixed bilayers similar to a random mixture. By simulating three POPE:POPG bilayers at different NaCl concentrations (0, 0.15, and 1 M), we also examined the effect of salt concentration on lipid mixing. While an increase in NaCl concentration is shown to affect the area per lipid, tail order, and lipid lateral diffusion, the final states of mixing remain unaltered, which is explained by the largely uniform increase in Na⁺ ions around POPE and POPG. Direct measurement of water permeation reveals that the POPE:POPG bilayer with 1 M NaCl has reduced water permeability compared with those at zero or low salt concentration. Our calculations provide a benchmark to estimate the convergence time scale of all-atom MD simulations of lipid mixing. Additionally, equilibrated structures of POPE:POPG and POPC:cholesterol, which are frequently used to mimic bacterial and mammalian membranes, respectively, can be used as starting points of simulations involving these membranes

Improving Internal Peptide Dynamics in the Coarse-Grained MARTINI Model: Toward Large-Scale Simulations of Amyloid- and Elastin-like Peptides

We present an extension of the coarse-grained MARTINI model for proteins and apply this extension to amyloid- and elastin-like peptides. Atomistic simulations of tetrapeptides, octapeptides, and longer peptides in solution are used as a reference to parametrize a set of pseudodihedral potentials that describe the internal flexibility of MARTINI peptides. We assess the performance of the resulting model in reproducing various structural properties computed from atomistic trajectories of peptides in water. The addition of new dihedral angle potentials improves agreement with the contact maps computed from atomistic simulations significantly. We also address the question of which parameters derived from atomistic trajectories are transferable between different lengths of peptides. The modified coarse-grained model shows reasonable transferability of parameters for the amyloid- and elastin-like peptides. In addition, the improved coarse-grained model is also applied to investigate the self-assembly of β-sheet forming peptides on the microsecond time scale. The octapeptides SNNFGAIL and (GV)₄ are used to examine peptide aggregation in different environments, in water, and at the water–octane interface. At the interface, peptide adsorption occurs rapidly, and peptides spontaneously aggregate in favor of stretched conformers resembling β-strands

Antimicrobial Peptide Simulations and the Influence of Force Field on the Free Energy for Pore Formation in Lipid Bilayers

Due to antimicrobial resistance, the development of new drugs to combat bacterial and fungal infections is an important area of research. Nature uses short, charged, and amphipathic peptides for antimicrobial defense, many of which disrupt the lipid membrane in addition to other possible targets inside the cell. Computer simulations have revealed atomistic details for the interactions of antimicrobial peptides and cell-penetrating peptides with lipid bilayers. Strong interactions between the polar interface and the charged peptides can induce bilayer deformations – including membrane rupture and peptide stabilization of a hydrophilic pore. Here, we performed microsecond-long simulations of the antimicrobial peptide CM15 in a POPC bilayer expecting to observe pore formation (based on previous molecular dynamics simulations). We show that caution is needed when interpreting results of equilibrium peptide-membrane simulations, given the length of time single trajectories can dwell in local energy minima for 100’s of ns to microseconds. While we did record significant membrane perturbations from the CM15 peptide, pores were not observed. We explain this discrepancy by computing the free energy for pore formation with different force fields. Our results show a large difference in the free energy barrier (ca. 40 kJ/mol) against pore formation predicted by the different force fields that would result in orders of magnitude differences in the simulation time required to observe spontaneous pore formation. This explains why previous simulations using the Berger lipid parameters reported pores induced by charged peptides, while with CHARMM based models pores were not observed in our long time-scale simulations. We reconcile some of the differences in the distance dependent free energies by shifting the free energy profiles to account for thickness differences between force fields. The shifted curves show that all the models describe small defects in lipid bilayers in a consistent manner, suggesting a common physical basis

The Role of Atomic Polarization in the Thermodynamics of Chloroform Partitioning to Lipid Bilayers

In spite of extensive research and use in medical practice, the precise molecular mechanism of volatile anesthetic action remains unknown. The distribution of anesthetics within lipid bilayers and potential targeting to membrane proteins is thought to be central to therapeutic function. Therefore, obtaining a molecular level understanding of volatile anesthetic partitioning into lipid bilayers is of vital importance to modern pharmacology. In this study we investigate the partitioning of the prototypical anesthetic, chloroform, into lipid bilayers and different organic solvents using molecular dynamics simulations with potential models ranging from simplified coarse-grained MARTINI to additive and polarizable CHARMM all-atom force fields. Many volatile anesthetics display significant inducible dipole moments, which correlate with their potency, yet the exact role of molecular polarizability in their stabilization within lipid bilayers remains unknown. We observe that explicit treatment of atomic polarizability makes it possible to accurately reproduce solvation free energies in solvents with different polarities, allowing for quantitative studies in heterogeneous molecular distributions, such as lipid bilayers. We calculate the free energy profiles for chloroform crossing lipid bilayers to reveal a role of polarizability in modulating chloroform partitioning thermodynamics via the chloroform-induced dipole moment and highlight competitive binding to the membrane core and toward the glycerol backbone that may have significant implications for understanding anesthetic action

Two-Dimensional Potentials of Mean Force of Nile Red in Intact and Damaged Model Bilayers. Application to Calculations of Fluorescence Spectra

Fluorescent dyes revolutionized and expanded our understanding of biological membranes. The interpretation of experimental fluorescence data in terms of membrane structure, however, requires detailed information about the molecular environment of the dyes. Nile red is a fluorescent molecule whose excitation and emission maxima depend on the polarity of the solvent. It is mainly used as a probe to study lipid microenvironments, for example in imaging the progression of damage to the myelin sheath in multiple sclerosis. In this study, we determine the position and orientation of Nile red in lipid bilayers by calculating two-dimensional Potential of Mean Force (2D-PMF) profiles in a defect-free 1-palmitoyl-2-oleoyl-<i>sn</i>-glycero-3-phosphocholine (POPC) bilayer and in damaged bilayers containing two mixtures of the oxidized lipid 1-palmitoyl-2-(9′-oxo-nonanoyl)-<i>sn</i>-glycero-3-phosphocholine and POPC. From 2D-PMF simulations we obtain positions and orientations of Nile Red corresponding to the minimum on the binding free energy surface in three different membrane environments with increasing amounts of water, mimicking damage in biological tissue. Using representative snapshots from the simulations, we use combined quantum mechanical/molecular mechanical (QM/MM) models to calculate the emission spectrum of Nile red as a function of its local solvation environment. The results of QM and QM/MM computations are in qualitative agreement with the experimentally observed shift in fluorescence for the dye moving from aqueous solution to the more hydrophobic environment of the lipid interiors. The range of the conformation dependent values of the computed absorption-emission spectra and the lack of solvent relaxation effects in the QM/MM calculations made it challenging to delineate specific differences between the intact and damaged bilayers

Going Backward: A Flexible Geometric Approach to Reverse Transformation from Coarse Grained to Atomistic Models

The conversion of coarse-grained to atomistic models is an important step in obtaining insight about atomistic scale processes from coarse-grained simulations. For this process, called backmapping or reverse transformation, several tools are available, but these commonly require libraries of molecule fragments or they are linked to a specific software package. In addition, the methods are usually restricted to specific molecules and to a specific force field. Here, we present an alternative method, consisting of geometric projection and subsequent force-field based relaxation. This method is designed to be simple and flexible, and offers a generic solution for resolution transformation. For simple systems, the conversion only requires a list of particle correspondences on the two levels of resolution. For special cases, such as nondefault protonation states of amino acids and virtual sites, a target particle list can be specified. The mapping uses simple building blocks, which list the particles on the different levels of resolution. For conversion to higher resolution, the initial model is relaxed with several short cycles of energy minimization and position-restrained MD. The reconstruction of an atomistic backbone from a coarse-grained model is done using a new dedicated algorithm. The method is generic and can be used to map between any two particle based representations, provided that a mapping can be written. The focus of this work is on the coarse-grained MARTINI force field, for which mapping definitions are written to allow conversion to and from the higher-resolution force fields GROMOS, CHARMM, and AMBER, and to and from a simplified three-bead lipid model. Together, these offer the possibility to simulate mesoscopic membrane structures, to be transformed to MARTINI and subsequently to an atomistic model for investigation of detailed interactions. The method was tested on a set of systems ranging from a simple, single-component bilayer to a large protein–membrane–solvent complex. The results demonstrate the efficiency and the efficacy of the new approach

Computational Lipidomics with <i>insane</i>: A Versatile Tool for Generating Custom Membranes for Molecular Simulations

For simulations of membranes and membrane proteins, the generation of the lipid bilayer is a critical step in the setup of the system. Membranes comprising multiple components pose a particular challenge, because the relative abundances need to be controlled and the equilibration of the system may take several microseconds. Here we present a comprehensive method for building membrane containing systems, characterized by simplicity and versatility. The program uses preset, coarse-grain lipid templates to build the membrane, and also allows on-the-fly generation of simple lipid types by specifying the headgroup, linker, and lipid tails on the command line. The resulting models can be equilibrated, after which a relaxed atomistic model can be obtained by reverse transformation. For multicomponent membranes, this provides an efficient means for generating equilibrated atomistic models. The method is called <i>insane</i>, an acronym for INSert membrANE. The program has been made available, together with the complementary method for reverse transformation, at http://cgmartini.nl/. This work highlights the key features of <i>insane</i> and presents a survey of properties for a large range of lipids as a start of a computational lipidomics project

Conformational Changes of the Antibacterial Peptide ATP Binding Cassette Transporter McjD Revealed by Molecular Dynamics Simulations

The ATP binding cassette (ABC) transporters form one of the largest protein superfamilies. They use the energy of ATP hydrolysis to transport chemically diverse ligands across membranes. An alternating access mechanism in which the transporter switches between inward- and outward-facing conformations has been proposed to describe the translocation process. One of the main open questions in this process is the degree of opening of the transporter at different stages of the transport cycle, as crystal structures and biochemical data have suggested a wide range of distances between nucleotide binding domains. Recently, the crystal structure of McjD, an antibacterial peptide ABC transporter from <i>Escherichia coli</i>, revealed a new occluded intermediate state of the transport cycle. The transmembrane domain is closed on both sides of the membrane, forming a cavity that can accommodate its ligand, MccJ25, a lasso peptide of 21 amino acids. In this work, we investigate the degree of opening of the transmembrane cavity required for ligand translocation. By means of steered molecular dynamics simulations, the ligand was pulled from the internal cavity to the extracellular side. This resulted in an outward-facing state. Comparison with existing outward-facing crystal structures shows a smaller degree of opening in the simulations, suggesting that the large conformational changes in some crystal structures may not be necessary even for a large substrate like MccJ25