Publications by authors named "Leonardo Darré"

16 Publications

  • Page 1 of 1

The ligand-bound state of a G protein-coupled receptor stabilizes the interaction of functional cholesterol molecules.

J Lipid Res 2021 Feb 26;62:100059. Epub 2021 Feb 26.

Univ. Grenoble Alpes, CNRS, CEA, IBS, Grenoble, France. Electronic address:

Cholesterol is a major component of mammalian plasma membranes that not only affects the physical properties of the lipid bilayer but also is the function of many membrane proteins including G protein-coupled receptors. The oxytocin receptor (OXTR) is involved in parturition and lactation of mammals and in their emotional and social behaviors. Cholesterol acts on OXTR as an allosteric modulator inducing a high-affinity state for orthosteric ligands through a molecular mechanism that has yet to be determined. Using the ion channel-coupled receptor technology, we developed a functional assay of cholesterol modulation of G protein-coupled receptors that is independent of intracellular signaling pathways and operational in living cells. Using this assay, we discovered a stable binding of cholesterol molecules to the receptor when it adopts an orthosteric ligand-bound state. This stable interaction preserves the cholesterol-dependent activity of the receptor in cholesterol-depleted membranes. This mechanism was confirmed using time-resolved FRET experiments on WT OXTR expressed in CHO cells. Consequently, a positive cross-regulation sequentially occurs in OXTR between cholesterol and orthosteric ligands.
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http://dx.doi.org/10.1016/j.jlr.2021.100059DOI Listing
February 2021

The Bilayer Collective Properties Govern the Interaction of an HIV-1 Antibody with the Viral Membrane.

Biophys J 2020 01 14;118(1):44-56. Epub 2019 Nov 14.

Instituto Biofisika (CSIC, UPV/EHU), Barrio Sarriena s/n, Leioa, Spain; Centro Nacional de Biotecnología, CSIC, Madrid, Spain; Unidad de Nanobiotecnología, CNB-CSIC-IMDEA Nanociencia Associated Unit, Madrid, Spain. Electronic address:

Efficient engagement with the envelope glycoprotein membrane-proximal external region (MPER) results in robust blocking of viral infection by a class of broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV). Developing an accommodation surface that engages with the viral lipid envelope appears to correlate with the neutralizing potency displayed by these bnAbs. The nature of the interactions established between the antibody and the lipid is nonetheless a matter of debate, with some authors arguing that anti-MPER specificity arises only under pathological conditions in autoantibodies endowed with stereospecific binding sites for phospholipids. However, bnAb-lipid interactions are often studied in systems that do not fully preserve the biophysical properties of lipid bilayers, and therefore, questions on binding specificity and the effect of collective membrane properties on the interaction are still open. Here, to evaluate the specificity of lipid interactions of an anti-MPER bnAb (4E10) in an intact membrane context, we determine quantitatively its association with lipid bilayers by means of scanning fluorescence correlation spectroscopy and all-atom molecular dynamic simulations. Our data support that 4E10 establishes electrostatic and hydrophobic interactions with the viral membrane surface and that the collective physical properties of the lipid bilayer influence 4E10 dynamics therein. We conclude that establishment of peripheral, nonspecific electrostatic interactions with the viral membrane through accommodation surfaces may assist high-affinity binding of HIV-1 MPER epitope at membrane interfaces. These findings highlight the importance of considering antibody-lipid interactions in the design of antibody-based anti-HIV strategies.
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http://dx.doi.org/10.1016/j.bpj.2019.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6950647PMC
January 2020

VeriNA3d: an R package for nucleic acids data mining.

Bioinformatics 2019 12;35(24):5334-5336

Computational Biology Node, Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology.

Summary: veriNA3d is an R package for the analysis of nucleic acids structural data, with an emphasis in complex RNA structures. In addition to single-structure analyses, veriNA3d also implements functions to handle whole datasets of mmCIF/PDB structures that could be retrieved from public/local repositories. Our package aims to fill a gap in the data mining of nucleic acids structures to produce flexible and high throughput analysis of structural databases.

Availability And Implementation: http://mmb.irbbarcelona.org/gitlab/dgallego/veriNA3d.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btz553DOI Listing
December 2019

From quantum to subcellular scales: multi-scale simulation approaches and the SIRAH force field.

Interface Focus 2019 Jun 19;9(3):20180085. Epub 2019 Apr 19.

Institut Pasteur de Montevideo, Group of Biomolecular Simulations, Mataojo 2020, CP 11400 Montevideo, Uruguay.

Modern molecular and cellular biology profits from astonishing resolution structural methods, currently even reaching the whole cell level. This is encompassed by the development of computational methods providing a deep view into the structure and dynamics of molecular processes happening at very different scales in time and space. Linking such scales is of paramount importance when aiming at far-reaching biological questions. Computational methods at the interface between classical and coarse-grained resolutions are gaining momentum with several research groups dedicating important efforts to their development and tuning. An overview of such methods is addressed herein, with special emphasis on the SIRAH force field for coarse-grained and multi-scale simulations. Moreover, we provide proof of concept calculations on the implementation of a multi-scale simulation scheme including quantum calculations on a classical fine-grained/coarse-grained representation of double-stranded DNA. This opens the possibility to include the effect of large conformational fluctuations in chromatin segments on, for instance, the reactivity of particular base pairs within the same simulation framework.
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http://dx.doi.org/10.1098/rsfs.2018.0085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6501346PMC
June 2019

Dimerization confers increased stability to nucleases in 5' halves from glycine and glutamic acid tRNAs.

Nucleic Acids Res 2018 09;46(17):9081-9093

Functional Genomics Unit, Institut Pasteur de Montevideo, Montevideo 11400, Uruguay.

We have previously shown that 5' halves from tRNAGlyGCC and tRNAGluCUC are the most enriched small RNAs in the extracellular space of human cell lines, and especially in the non-vesicular fraction. Extracellular RNAs are believed to require protection by either encapsulation in vesicles or ribonucleoprotein complex formation. However, deproteinization of non-vesicular tRNA halves does not affect their retention in size-exclusion chromatography. Thus, we considered alternative explanations for their extracellular stability. In-silico analysis of the sequence of these tRNA-derived fragments showed that tRNAGly 5' halves can form homodimers or heterodimers with tRNAGlu 5' halves. This capacity is virtually unique to glycine tRNAs. By analyzing synthetic oligonucleotides by size exclusion chromatography, we provide evidence that dimerization is possible in vitro. tRNA halves with single point substitutions preventing dimerization are degraded faster both in controlled nuclease digestion assays and after transfection in cells, showing that dimerization can stabilize tRNA halves against the action of cellular nucleases. Finally, we give evidence supporting dimerization of endogenous tRNAGlyGCC 5' halves inside cells. Considering recent reports have shown that 5' tRNA halves from Ala and Cys can form tetramers, our results highlight RNA intermolecular structures as a new layer of complexity in the biology of tRNA-derived fragments.
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http://dx.doi.org/10.1093/nar/gky495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158491PMC
September 2018

Functional mapping of the N-terminal arginine cluster and C-terminal acidic residues of Kir6.2 channel fused to a G protein-coupled receptor.

Biochim Biophys Acta Biomembr 2017 Oct 28;1859(10):2144-2153. Epub 2017 Jul 28.

Institut de Biologie Structurale (IBS), Univ. Grenoble Alpes, CEA, CNRS, LabEx ICST, 71, avenue des Martyrs, CS10090, F-38044 Grenoble, France. Electronic address:

Ion channel-coupled receptors (ICCRs) are original man-made ligand-gated ion channels created by fusion of G protein-coupled receptors (GPCRs) to the inward-rectifier potassium channel Kir6.2. GPCR conformational changes induced by ligand binding are transduced into electrical current by the ion channel. This functional coupling is closely related to the length of the linker region formed by the GPCR C-terminus (C-ter) and Kir6.2N-terminus (N-ter). Manipulating the GPCR C-ter length allows to finely tune the channel regulation, both in amplitude and sign (opening or closing Kir6.2). In this work, we demonstrate that the primary sequence of the channel N-terminal domain is an additional parameter for the functional coupling with GPCRs. As for all Kir channels, a cluster of basic residues is present in the N-terminal domain of Kir6.2 and is composed of 5 arginines which are proximal to the GPCR C-ter in the fusion proteins. Using a functional mapping approach, we demonstrate the role of specific arginines (R27 and R32) for the function of ICCRs, indicating that the position and not the cluster of positively-charged arginines is critical for the channel regulation by the GPCR. Following observations provided by molecular dynamics simulation, we explore the hypothesis of interaction of these arginines with acidic residues, and using site-directed mutagenesis, we identified aspartate D307 and glutamate E308 residues as critical for the function of ICCRs. These results demonstrate the critical role of the N-terminal and C-terminal charged residues of Kir6.2 for its allosteric regulation by the fused GPCR.
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http://dx.doi.org/10.1016/j.bbamem.2017.07.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5599172PMC
October 2017

Compaction of Duplex Nucleic Acids upon Native Electrospray Mass Spectrometry.

ACS Cent Sci 2017 May 26;3(5):454-461. Epub 2017 Apr 26.

INSERM, CNRS, Université de Bordeaux, Acides Nucléiques Régulations Naturelle et Artificielle (ARNA, U1212, UMR5320), IECB, 2 rue Robert Escarpit, 33607 Pessac, France.

We report on the fate of nucleic acids conformation in the gas phase as sampled using native mass spectrometry coupled to ion mobility spectrometry. On the basis of several successful reports for proteins and their complexes, the technique has become popular in structural biology, and the conformation survival becomes more and more taken for granted. Surprisingly, we found that DNA and RNA duplexes, at the electrospray charge states naturally obtained from native solution conditions (≥100 mM aqueous NHOAc), are significantly more compact in the gas phase compared to the canonical solution structures. The compaction is observed for all duplex sizes (gas-phase structures are more compact than canonical B-helices by ∼20% for 12-bp, and by up to ∼30% for 36-bp duplexes), and for DNA and RNA alike. Molecular modeling (density functional calculations on small helices, semiempirical calculations on up to 12-bp, and molecular dynamics on up to 36-bp duplexes) demonstrates that the compaction is due to phosphate group self-solvation prevailing over Coulomb repulsion. Molecular dynamics simulations starting from solution structures do not reproduce the experimental compaction. To be experimentally relevant, molecular dynamics sampling should reflect the progressive structural rearrangements occurring during desolvation. For nucleic acid duplexes, the compaction observed for low charge states results from novel phosphate-phosphate hydrogen bonds formed across both grooves at the very late stages of electrospray.
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http://dx.doi.org/10.1021/acscentsci.7b00084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5445532PMC
May 2017

Small Details Matter: The 2'-Hydroxyl as a Conformational Switch in RNA.

J Am Chem Soc 2016 12 13;138(50):16355-16363. Epub 2016 Dec 13.

Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology , 08028 Barcelona, Spain.

While DNA is mostly a primary carrier of genetic information and displays a regular duplex structure, RNA can form very complicated and conserved 3D structures displaying a large variety of functions, such as being an intermediary carrier of the genetic information, translating such information into the protein machinery of the cell, or even acting as a chemical catalyst. At the base of such functional diversity is the subtle balance between different backbone, nucleobase, and ribose conformations, finely regulated by the combination of hydrogen bonds and stacking interactions. Although an apparently simple chemical modification, the presence of the 2'OH in RNA has a profound effect in the ribonucleotide conformational balance, adding an extra layer of complexity to the interactions network in RNA. In the present work, we have combined database analysis with extensive molecular dynamics, quantum mechanics, and hybrid QM/MM simulations to provide direct evidence on the dramatic impact of the 2'OH conformation on sugar puckering. Calculations provide evidence that proteins can modulate the 2'OH conformation to drive sugar repuckering, leading then to the formation of bioactive conformations. In summary, the 2'OH group seems to be a primary molecular switch contributing to specific protein-RNA recognition.
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http://dx.doi.org/10.1021/jacs.6b09471DOI Listing
December 2016

Lateral Fenestrations in K(+)-Channels Explored Using Molecular Dynamics Simulations.

Mol Pharm 2016 07 26;13(7):2263-73. Epub 2016 May 26.

Department of Chemistry, King's College London , Britannia House, 7 Trinity Street, London SE1 1DB, U.K.

Potassium channels are of paramount physiological and pathological importance and therefore constitute significant drug targets. One of the keys to rationalize the way drugs modulate ion channels is to understand the ability of such small molecules to access their respective binding sites, from which they can exert an activating or inhibitory effect. Many computational studies have probed the energetics of ion permeation, and the mechanisms of voltage gating, but little is known about the role of fenestrations as possible mediators of drug entry in potassium channels. To explore the existence, structure, and conformational dynamics of transmembrane fenestrations accessible by drugs in potassium channels, molecular dynamics simulation trajectories were analyzed from three potassium channels: the open state voltage-gated channel Kv1.2, the G protein-gated inward rectifying channel GIRK2 (Kir3.2), and the human two-pore domain TWIK-1 (K2P1.1). The main results of this work were the identification of the sequence identity of four main lateral fenestrations of similar length and with bottleneck radius in the range of 0.9-2.4 Å for this set of potassium channels. It was found that the fenestrations in Kv1.2 and Kir3.2 remain closed to the passage of molecules larger than water. In contrast, in the TWIK-1 channel, both open and closed fenestrations are sampled throughout the simulation, with bottleneck radius shown to correlate with the random entry of lipid membrane molecules into the aperture of the fenestrations. Druggability scoring function analysis of the fenestration regions suggests that Kv and Kir channels studied are not druggable in practice due to steric constraining of the fenestration bottleneck. A high (>50%) fenestration sequence identity was found in each potassium channel subfamily studied, Kv1, Kir3, and K2P1. Finally, the reported fenestration sequence of TWIK-1 compared favorably with another channel, K2P channel TREK-2, reported to possess open fenestrations, suggesting that K2P channels could be druggable via fenestrations, for which we reported atomistic detail of the fenestration region, including the flexible residues M260 and L264 that interact with POPC membrane in a concerted fashion with the aperture and closure of the fenestrations.
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http://dx.doi.org/10.1021/acs.molpharmaceut.5b00942DOI Listing
July 2016

SIRAH: a structurally unbiased coarse-grained force field for proteins with aqueous solvation and long-range electrostatics.

J Chem Theory Comput 2015 Feb;11(2):723-39

Institut Pasteur de Montevideo , Montevideo, Uruguay.

Modeling of macromolecular structures and interactions represents an important challenge for computational biology, involving different time and length scales. However, this task can be facilitated through the use of coarse-grained (CG) models, which reduce the number of degrees of freedom and allow efficient exploration of complex conformational spaces. This article presents a new CG protein model named SIRAH, developed to work with explicit solvent and to capture sequence, temperature, and ionic strength effects in a topologically unbiased manner. SIRAH is implemented in GROMACS, and interactions are calculated using a standard pairwise Hamiltonian for classical molecular dynamics simulations. We present a set of simulations that test the capability of SIRAH to produce a qualitatively correct solvation on different amino acids, hydrophilic/hydrophobic interactions, and long-range electrostatic recognition leading to spontaneous association of unstructured peptides and stable structures of single polypeptides and protein-protein complexes.
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http://dx.doi.org/10.1021/ct5007746DOI Listing
February 2015

Binding of Capsaicin to the TRPV1 Ion Channel.

Mol Pharm 2015 Dec 3;12(12):4454-65. Epub 2015 Nov 3.

Department of Chemistry, King's College London , Britannia House, 7 Trinity Street, London SE1 1DB, U.K.

Transient receptor potential (TRP) ion channels constitute a notable family of cation channels involved in the ability of an organisms to detect noxious mechanical, thermal, and chemical stimuli that give rise to the perception of pain, taste, and changes in temperature. One of the most experimentally studied agonist of TRP channels is capsaicin, which is responsible for the burning sensation produced when chili pepper is in contact with organic tissues. Thus, understanding how this molecule interacts and regulates TRP channels is essential to high impact pharmacological applications, particularly those related to pain treatment. The recent publication of a three-dimensional structure of the vanilloid receptor 1 (TRPV1) in the absence and presence of capsaicin from single particle electron cryomicroscopy experiments provides the opportunity to explore these questions at the atomic level. In the present work, molecular docking and unbiased and biased molecular dynamics simulations were employed to generate a structural model of the capsaicin-channel complex. In addition, the standard free energy of binding was estimated using alchemical transformations coupled with conformational, translational, and orientational restraints on the ligand. Key binding modes consistent with previous experimental data are identified, and subtle but essential dynamical features of the binding site are characterized. These observations shed some light into how TRPV1 interacts with capsaicin, and may help to refine design parameters for new TRPV1 antagonists, and potentially guide further developments of TRP channel modulators.
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http://dx.doi.org/10.1021/acs.molpharmaceut.5b00641DOI Listing
December 2015

Surfactin at the Water/Air Interface and in Solution.

Langmuir 2015 Oct 1;31(40):11097-104. Epub 2015 Oct 1.

Department of Chemistry, King's College London , Britannia House, 7 Trinity Street, London SE1 1DB, U.K.

The lipopeptide surfactin produced by certain strains of Bacillus subtillis is a potent biosurfactant with high amphiphilicity and a strong tendency for self-aggregation. Surfactin possesses a number of valuable biological properties such as antiviral, antibacterial, antifungal, and hemolytic activities. Owing to these properties, in addition to the general advantages of biosurfactants over synthetic surfactants, surfactin has potential biotechnological and biomedical applications. Here, the aggregation properties of surfactin in solution together with its behavior at the water/air interface were studied using classical molecular dynamics simulations (MD) at three different pH values. Validation of the MD structural data was performed by comparing neutron reflectivity and volume fraction profiles computed from the simulations with their experimental counterparts. Analysis of the MD trajectories supported conclusions about the distribution, conformations, and interactions of surfactin in solution and at the water-air interface. Considering altogether, the work presented provides atomistic models for the rationalization of some of the structural and dynamic characteristics as well as the modes of action of surfactin at different pH values.
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http://dx.doi.org/10.1021/acs.langmuir.5b02305DOI Listing
October 2015

In silico identification of PAP-1 binding sites in the Kv1.2 potassium channel.

Mol Pharm 2015 Apr 16;12(4):1299-307. Epub 2015 Mar 16.

†Department of Chemistry, Britannia House, 7 Trinity Street, King's College London, London SE1 1DB, U.K.

Voltage-gated potassium channels of the Kv1 family play a crucial role in the generation and transmission of electrical signals in excitable cells affecting neuronal and cardiac activities. Small-molecule blockage of these channels has been proposed to occur via a cooperative mechanism involving two main blocking sites: the inner-pore site located below the selectivity filter, and a side-pocket cavity located between the pore and the voltage sensor. Using 0.5 μs molecular dynamics simulation trajectories complemented by docking calculations, the potential binding sites of the PAP-1 (5-(4-phenoxybutoxy)psoralen) blocker to the crystal structure of Kv1.2 channel have been studied. The presence of both mentioned blocking sites at Kv1.2 is confirmed, adding evidence in favor of a cooperative channel blockage mechanism. These observations provide insight into drug modulation that will guide further developments of Kv inhibitors.
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http://dx.doi.org/10.1021/acs.molpharmaceut.5b00023DOI Listing
April 2015

Permeation and dynamics of an open-activated TRPV1 channel.

J Mol Biol 2015 Jan 3;427(2):537-49. Epub 2014 Dec 3.

Department of Chemistry, King's College London, Britannia House, 7 Trinity Street, London SE1 1DB, UK; Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK. Electronic address:

Transient receptor potential (TRP) ion channels constitute a large and diverse protein family, found in yeast and widespread in the animal kingdom. TRP channels work as sensors for a wide range of cellular and environmental signals. Understanding how these channels respond to physical and chemical stimuli has been hindered by the limited structural information available until now. The three-dimensional structure of the vanilloid receptor 1 (TRPV1) was recently determined by single particle electron cryo-microscopy, offering for the first time the opportunity to explore ionic conduction in TRP channels at atomic detail. In this study, we present molecular dynamics simulations of the open-activated pore domain of TRPV1 in the presence of three cationic species: Na(+), Ca(2+) and K(+). The dynamics of these ions while interacting with the channel pore allowed us to rationalize their permeation mechanism in terms of a pathway involving three binding sites at the intracellular cavity, as well as the extracellular and intracellular entrance of the selectivity filter. Furthermore, conformational analysis of the pore in the presence of these ions reveals specific ion-mediated structural changes in the selectivity filter, which influences the permeability properties of the TRPV1 channel.
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http://dx.doi.org/10.1016/j.jmb.2014.11.016DOI Listing
January 2015

Transferable mixing of atomistic and coarse-grained water models.

J Phys Chem B 2013 Nov 12;117(46):14438-48. Epub 2013 Nov 12.

Institut Pasteur de Montevideo , Mataojo 2020, Montevideo 11400, Uruguay.

Dual-resolution approaches for molecular simulations combine the best of two worlds, providing atomic details in regions of interest and coarser but much faster descriptions of less-relevant parts of molecular systems. Given the abundance of water in biomolecular systems, reducing the computational cost of simulating bulk water without perturbing the solute's properties is a very attractive strategy. Here we show that the coarse-grained model for water called WatFour (WT4) can be combined with any of the three most used water models for atomistic simulations (SPC, TIP3P, and SPC/E) without modifying the characteristics of the atomistic solvent and solutes. The equivalence of fully atomistic and hybrid solvation approaches is assessed by comparative simulations of pure water, electrolyte solutions, and the β1 domain of streptococcal protein G, for which comparisons between experimental and calculated chemical shifts at (13)Cα are equivalent.
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http://dx.doi.org/10.1021/jp4079579DOI Listing
November 2013

Mixing Atomistic and Coarse Grain Solvation Models for MD Simulations: Let WT4 Handle the Bulk.

J Chem Theory Comput 2012 Oct 15;8(10):3880-94. Epub 2012 Jun 15.

Institut Pasteur de Montevideo, Mataojo 2020, CP 11400, Uruguay.

Accurate simulation of biomolecular systems requires the consideration of solvation effects. The arrangement and dynamics of water close to a solute are strongly influenced by the solute itself. However, as the solute-solvent distance increases, the water properties tend to those of the bulk liquid. This suggests that bulk regions can be treated at a coarse grained (CG) level, while keeping the atomistic details around the solute. Since water represents about 80% of any biological system, this approach may offer a significant reduction in the computational cost of simulations without compromising atomistic details. We show here that mixing the popular SPC water model with a CG model for solvation (called WatFour) can effectively mimic the hydration, structure, and dynamics of molecular systems composed of pure water, simple electrolyte solutions, and solvated macromolecules. As a nontrivial example, we present simulations of the SNARE membrane fusion complex, a trimeric protein-protein complex embedded in a double phospholipid bilayer. Comparison with a fully atomistic reference simulation illustrates the equivalence between both approaches.
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http://dx.doi.org/10.1021/ct3001816DOI Listing
October 2012