Supplementary data are available at Bioinformatics online.

CHARMM27 is a widespread and popular force field for biomolecular simulation, and several recent algorithms such as implicit solvent models have been developed specifically for it. We have here implemented the CHARMM force field and all necessary extended functional forms in the GROMACS molecular simulation package, to make CHARMM-specific features available and to test them in combination with techniques for extended time steps, to make all major force fields available for comparison studies in GROMACS, and to test various solvent model optimizations, in particular the effect of Lennard-Jones interactions on hydrogens. The implementation has full support both for CHARMM-specific features such as multiple potentials over the same dihedral angle and the grid-based energy correction map on the ϕ, ψ protein backbone dihedrals, as well as all GROMACS features such as virtual hydrogen interaction sites that enable 5 fs time steps. The medium-to-long time effects of both the correction maps and virtual sites have been tested by performing a series of 100 ns simulations using different models for water representation, including comparisons between CHARMM and traditional TIP3P. Including the correction maps improves sampling of near native-state conformations in our systems, and to some extent it is even able to refine distorted protein conformations. Finally, we show that this accuracy is largely maintained with a new implicit solvent implementation that works with virtual interaction sites, which enables performance in excess of 250 ns/day for a 900-atom protein on a quad-core desktop computer.

The thermodynamics of hydrogen bond breaking and formation was studied in solutions of alcohol (methanol, ethanol, 1-propanol) molecules. An extensive series of over 400 molecular dynamics simulations with an aggregate length of over 900 ns was analyzed using an analysis technique in which hydrogen bond (HB) breaking is interpreted as an Eyring process, for which the Gibbs energy of activation DeltaG can be determined from the HB lifetime. By performing simulations at different temperatures, we were able to determine the enthalpy of activation DeltaH and the entropy of activation TDeltaS for this process from the Van't Hoff relation. The equilibrium thermodynamics was determined separately, based on the number of donor hydrogens that are involved in hydrogen bonds. Results (DeltaH) are compared to experimental data from Raman spectroscopy and found to be in good agreement for pure water and methanol. The DeltaG as well as the DeltaG are smooth functions of the composition of the mixtures. The main result of the calculations is that DeltaG is essentially independent of the environment (around 5 kJ/mol), suggesting that buried hydrogen bonds (e.g., in proteins) do not contribute significantly to protein stability. Enthalpically HB formation is a downhill process in all substances; however, for the alcohols there is an entropic barrier of 6-7 kJ/mol, at 298.15 K, which cannot be detected in pure water.

Fusion peptides from influenza hemagglutinin act on membranes to promote membrane fusion, but the mechanism by which they do so remains unknown. Recent theoretical work has suggested that contact of protruding lipid tails may be an important feature of the transition state for membrane fusion. If this is so, then influenza fusion peptides would be expected to promote tail protrusion in proportion to the ability of the corresponding full-length hemagglutinin to drive lipid mixing in fusion assays. We have performed molecular dynamics simulations of influenza fusion peptides in lipid bilayers, comparing the X-31 influenza strain against a series of N-terminal mutants. As hypothesized, the probability of lipid tail protrusion correlates well with the lipid mixing rate induced by each mutant. This supports the conclusion that tail protrusion is important to the transition state for fusion. Furthermore, it suggests that tail protrusion can be used to examine how fusion peptides might interact with membranes to promote fusion. Previous models for native influenza fusion peptide structure in membranes include a kinked helix, a straight helix, and a helical hairpin. Our simulations visit each of these conformations. Thus, the free energy differences between each are likely low enough that specifics of the membrane environment and peptide construct may be sufficient to modulate the equilibrium between them. However, the kinked helix promotes lipid tail protrusion in our simulations much more strongly than the other two structures. We therefore predict that the kinked helix is the most fusogenic of these three conformations.

Graphical abstract

When researchers build high-quality models of protein structure from sequence homology, it is today common to use several alternative target-template alignments. Several methods can, at least in theory, utilize information from multiple templates, and many examples of improved model quality have been reported. However, to our knowledge, thus far no study has shown that automatic inclusion of multiple alignments is guaranteed to improve models without artifacts. Here, we have carried out a systematic investigation of the potential of multiple templates to improving homology model quality. We have used test sets consisting of targets from both recent CASP experiments and a larger reference set. In addition to Modeller and Nest, a new method (Pfrag) for multiple template-based modeling is used, based on the segment-matching algorithm from Levitt's SegMod program. Our results show that all programs can produce multi-template models better than any of the single-template models, but a large part of the improvement is simply due to extension of the models. Most of the remaining improved cases were produced by Modeller. The most important factor is the existence of high-quality single-sequence input alignments. Because of the existence of models that are worse than any of the top single-template models, the average model quality does not improve significantly. However, by ranking models with a model quality assessment program such as ProQ, the average quality is improved by ;5% in the CASP7 test set.Keywords: protein structure/folding; structure; protein structure prediction; homology modeling Supplemental material: see www.proteinscience.orgThe gap between the number of known protein sequences in genome databases and corresponding threedimensional structures is rapidly increasing, and for the vast majority of proteins we will likely never determine experimental structures. One important tool to bridge this gap and deduce structural properties from sequence is theoretical modeling based on homology. Even if the quality of these models cannot yet compete with experimental structures, they are extremely cheap to produce and can be applied on a much larger scale. Homology modeling methods use the fact that evolutionarily related proteins frequently share a similar structure. Therefore, if the sequence identity is high enough a three-dimensional model of a protein with unknown structure (target) can be built using a sequence alignment to a protein of known structure (template). Improving these model-building algorithms is important not only for decreasing the structuresequence gap, but also to achieve higher-quality individual models that, e.g., are accurate enough for drug design.The accuracy of homology models is directly related to how similar the target is to the template sequence, and there is pretty solid consensus that the two most important factors are to (1) choose the best possible template and then (2) optimally align the target sequence onto this Reprint requests to: Arne Elofsson, Center for Biomembran...

Ebolavirus is an enveloped virus causing severe hemorrhagic fever. Its surface glycoproteins undergo proteolytic cleavage and rearrangements to permit membrane fusion and cell entry. Here we focus on the glycoprotein's internal fusion loop (FL), critical for lowpH-triggered fusion in the endosome. Alanine mutations at L529 and I544 and particularly the L529 I544 double mutation compromised viral entry and fusion. The nuclear magnetic resonance (NMR) structures of the I544A and L529A I544A mutants in lipid environments showed significant disruption of a three-residue scaffold that is required for the formation of a consolidated fusogenic hydrophobic surface at the tip of the FL. Biophysical experiments and molecular simulation revealed the position of the wild-type (WT) FL in membranes and showed the inability of the inactive double mutant to reach this position. Consolidation of hydrophobic residues at the tip of FLs may be a common requirement for internal FLs of class I, II, and III fusion proteins. IMPORTANCEMany class I, II, and III viral fusion proteins bear fusion loops for target membrane insertion and fusion. We determined structures of the Ebolavirus fusion loop and found residues critical for forming a consolidated hydrophobic surface, membrane insertion, and viral entry. E bolavirus (Ebov) is a filovirus that causes severe hemorrhagic fever with mortality rates of between 25 and 90% (1, 2). Outbreaks involving human fatalities have occurred in sub-Saharan Africa since 1976, the last three being in Uganda and the Democratic Republic of the Congo in 2012 and in Guinea and Liberia in 2014 (3). Ebolavirus is also a much feared potential agent of bioterrorism. However, there are still no FDA-approved treatments or vaccines for this devastatingly morbid infectious agent. One area of therapeutic interest is to target the viral entry machinery that governs virus-host membrane fusion. This would halt infection before initiation of viral replication and subsequent cell destruction. To guide therapeutic design, a detailed knowledge of Ebov entry and membrane fusion is needed, and currently little is known about Ebov virus-host membrane interactions.Entry of Ebov is mediated by glycoprotein (GP) spikes that protrude from the virus particle (4-8). Like most other class I viral fusion proteins, GP is composed of a receptor binding unit (GP1) and a fusion (GP2) subunit. After binding to cell surface receptors, GP mediates virus uptake through a macropinocytosis-like process (9, 10). Viral fusion ultimately occurs in endosomes, where GP1 is cleaved by cathepsins B and L to an ϳ19-kDa species (11, 12), which engages Niemann-Pick C1, a late endosomal protein essential for Ebov entry (13-15). A final unknown trigger causes conformational changes in GP (16, 17) that expose the fusion loop (FL) in GP2. The Ebov FL is clamped by a disulfide bond and has a hydrophobic region at its tip. It is thought to be functionally equivalent to the linear hydrophobic and glycine-rich fusion peptides found at the N termini of mo...

The structure and dynamics of Opa proteins, which we report herein, are responsible for the receptor-mediated engulfment of Neisseria gonorrheae or Neisseria meningitidis by human cells and can offer deep understanding into the molecular recognition of pathogen–host receptor interactions. Such interactions are vital to understanding bacterial pathogenesis as well as the mechanism of foreign body entry to a human cell, which may provide insights for the development of targeted pharmaceutical delivery systems. The size and dynamics of the extracellular loops of Opa60 required a hybrid refinement approach wherein membrane and distance restraints were used to generate an initial NMR structural ensemble, which was then further refined using molecular dynamics in a DMPC bilayer. The resulting ensemble revealed that the extracellular loops, which bind host receptors, occupy compact conformations, interact with each other weakly, and are dynamic on the nanosecond time scale. We predict that this conformational sampling is critical for enabling diverse Opa loop sequences to engage a common set of receptors.