Contact area size and number of water molecules near vesicle interface.

<p>The size of the vesicle-vesicle contact area is plotted in green and the number of water molecules near the interface is plotted in blue. After the vesicles first come together, a metastable contact patch is formed where the lipid headgroups make patchy contact through a thinned water layer. The transition state for stalk formation occurs within this patch structure; later, as the stalk expands the polar contact is replaced by the nascent hemifusion structure. Contact area was measured as the difference between the solvent-accessible surface areas of the individual vesicles and the joint structure, computed with NACCESS <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1000829#pcbi.1000829-Hubbard1" target="_blank">[64]</a>. Water molecules were counted in a cylinder of radius 2 nm and height 1 nm along the axis between the vesicle centers of mass. Lipid headgroup tilt was not measurably correlated with proximity to the vesicle-vesicle interface in our simulations (r<0.03).</p

Influenza hemagglutinin peptides increase lipid tail protrusion probability.

<p>Lipid tail protrusion probability is plotted as a function of distance from the nearest fusion peptide, with values averaged in 1-Å bins. Tails within 5 Å of the fusion peptides have a significantly greater probability of protrusion than those >20 Å away (p<0.02, Kolmogorov-Smirnov test).</p

Orientation of lipid tails in a nascent fusion stalk.

<p>Most of the lipids near in this early stalk structure are either radially or tangentially aligned, not antiparallel. The stalk forms a hydrophobic “narrow bridge” between contacting vesicle outer leaflets, similar to that proposed by Kozlov and Markin<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1000829#pcbi.1000829-Kozlov1" target="_blank">[5]</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1000829#pcbi.1000829-Markin1" target="_blank">[12]</a>. Outer leaflet lipids from both vesicles are shown in stick form with the lipid tails colored by the tail orientation relative to the vesicle radius. Tails aligned with the vesicle radius are colored dark blue, tails aligned antiparallel to the radius are colored dark red, and tails tangent to the vesicle are colored white. A few antiparallel tails can be seen in red, but these are generally far from the stalk. Head groups are shown in transparent brown.</p

The transition state for stalk formation occurs when a pair of lipid tails make contact through the polar interface layer.

<p>In our simulations, contact between a single pair of hydrophobic tails is sufficient to nucleate stalk formation. This pair of lipids is rendered at the time of first encounter, when hydrophobic contact forms a transition state, and in the nascent stalk shortly after commitment.</p

Contact patch formation is accompanied by thinning of the water layer between vesicles.

<p>Panels (a–d) show slices through the vesicle-vesicle interface at intervals around the transition state. As a contact patch forms, the water layer thins to allow contact between lipid polar headgroups. The transition state, however, does not occur until hydrophobic tails make contact. Lipids from opposing vesicles are rendered in green and teal, with head-groups rendered as lines, tails as sticks, and water in surface form. After stalk formation, the growing hydrophobic stalk excludes water from the interface region, resulting in non-leaky fusion.</p

Committor analysis for transition state identification.

<p>Plots show percent commitment to the stalk state at varying time lags and thresholds for defining a stalk. To calculate these commitment probabilities, 20 snapshots were taken from a single fusion trajectory, and 20 independent simulations were started from each of these snapshots. Committor theory states that any snapshot with a 50% probability of proceeding to stalk formation is a member of the transition state ensemble <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1000829#pcbi.1000829-Du1" target="_blank">[23]</a>. To ensure robust measurement, commitment probabilities are plotted here as a function of both the length in each of these independent simulations after which stalk formation was assessed and the cutoff used to define stalk formation. Stalk formation was measured by taking 1-Å slices perpendicular to the vesicle:vesicle center-of-mass axis and measuring the number of lipids in the most sparsely populated slice, which corresponded to the polar interface region between vesicles. A stalk was judged to have formed if at least 1 lipid (<b>a</b>), 2 lipids (<b>b</b>), 3 lipids (<b>c</b>), or 4 lipids (<b>d</b>) were present in this minimal slice. The equi-commitment point or transition state occurred at approximately 75 ns (range: 65–80 ns) for all cutoffs and time lags tested.</p

Formation of an extended contact patch between vesicles precedes fusion.

<p>Shown in (a) is a free-energy schematic for vesicle fusion, illustrated with snapshots from a fusion simulation at atomic resolution. In addition to the canonical intermediates, we find a metastable contact patch between vesicles to precede fusion. This patch is shown in (b) before (left) and after (right) stalk formation. Lipid tails are rendered in green and teal, head groups in line form. Water molecules are omitted for clarity.</p

Understanding Drug Skin Permeation Enhancers Using Molecular Dynamics Simulations

Our skin constitutes an effective permeability barrier that protects the body from exogenous substances but concomitantly severely limits the number of pharmaceutical drugs that can be delivered transdermally. In topical formulation design, chemical permeation enhancers (PEs) are used to increase drug skin permeability. In vitro skin permeability experiments can measure net effects of PEs on transdermal drug transport, but they cannot explain the molecular mechanisms of interactions between drugs, permeation enhancers, and skin structure, which limits the possibility to rationally design better new drug formulations. Here we investigate the effect of the PEs water, lauric acid, geraniol, stearic acid, thymol, ethanol, oleic acid, and eucalyptol on the transdermal transport of metronidazole, caffeine, and naproxen. We use atomistic molecular dynamics (MD) simulations in combination with developed molecular models to calculate the free energy difference between 11 PE-containing formulations and the skin’s barrier structure. We then utilize the results to calculate the final concentration of PEs in skin. We obtain an RMSE of 0.58 log units for calculated partition coefficients from water into the barrier structure. We then use the modified PE-containing barrier structure to calculate the PEs’ permeability enhancement ratios (ERs) on transdermal metronidazole, caffeine, and naproxen transport and compare with the results obtained from in vitro experiments. We show that MD simulations are able to reproduce rankings based on ERs. However, strict quantitative correlation with experimental data needs further refinement, which is complicated by significant deviations between different measurements. Finally, we propose a model for how to use calculations of the potential of mean force of drugs across the skin’s barrier structure in a topical formulation design

Molecular PUFA-VSD interactions in the <i>Shaker</i> channel.

<p><b>(A)</b> Close-up of a PUFA (DHA) in its initial conformation. The numbers marked in grey depict the carbons forming the <i>cis</i> double-bonds. <b>(B)</b> Side-view of one VSD equilibrated in a POPC lipid bilayer represented by a yellow iso-density surface corresponding to the positions of lipid nitrogens in the simulation at 5% occupancy. The residues shown in experimental studies to be close to the interaction site of DHA, namely residues I325, T329 located on S3, and A359, and I360 located on S4, are colored in cyan [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004704#pcbi.1004704.ref029" target="_blank">29</a>]. <b>(C)</b> Top-view of the <i>Shaker</i> tetramer with PUFAs in their starting positions. The PUFA carboxyl head group and carbon tail are colored in blue and green, respectively. The simulated dynamics of the PUFAs surrounding the <i>Shaker</i> tetramer is represented by a brown mesh iso-density surface at 27% occupancy. The cut-off was chosen to visualize the differences between the PD and VSD interactions with the PUFAs.</p

SFA contacts to the <i>Shaker</i> tetramer in the open and closed states.

<p>The contact frequencies of amino acid residues within 3.5 Å of SFA carboxyl head groups and carbon tails are displayed for the open <b>(AB)</b> and closed <b>(CD)</b> states of the channel. The red dotted line differentiates between helices S1 and S2 or helices S3 and S4 of the VSD. Side-view of a VSD and interacting residues are displayed separately for the PUFA carboxyl head groups and tails for each state of the channel (insets).</p