Multisite Ion Models That Improve Coordination and Free Energy Calculations in Molecular Dynamics Simulations

Current ion models in molecular mechanics are simple spheres, and their interactions are solely determined from the van der Waals radius of the sphere and the total charge. Here, we introduce a model where we distribute the total charge of the ion into <i>n</i>-dummy centers that are placed in the direction of the coordinating atoms. We have parametrized this model for two divalent cations, Ca²⁺ and Mg²⁺, and have tested the model’s accuracy in a variety of simulations. With this model we are not only able to correctly predict the free energies and selectivity for cation binding sites in proteins and nucleic acids, but we achieve better coordination geometries and can capture more subtle effects such as the exchange of inner shell waters. Additionally, this model does not employ higher-order electrostatics and thus can be easily used with standard force fields

Multivariate Analyses of Amyloid-Beta Oligomer Populations Indicate a Connection between Pore Formation and Cytotoxicity

<div><p>Aggregates of amyloid-beta (Aβ) peptides are thought to be involved in the development of Alzheimer’s disease because they can change synaptic plasticity and induce neuronal cell death by inflammation, oxidative damage, and transmembrane pore formation. Exactly which oligomeric species underlie these cytotoxic effects remains unclear. The work presented here established well-controlled aggregation conditions of Aβ_1–40 or Aβ_1–42 peptides over a 20-day period and characterized these preparations with regard to their β-sheet content, degree of fibril formation, relative abundance of various oligomer sizes, and propensity to induce membrane pore formation and cytotoxicity. Using this multivariate data set, a systematic and inherently unbiased partial least squares (PLS) approach showed that for both peptides the abundance of oligomers in the tetramer to 13-mer range contributed positively to both pore formation and cytotoxicity, while monomers, dimers, trimers, and the largest oligomers (>210 kDa) were negatively correlated to both phenomena. Multivariate PLS analysis is ideally suited to handle complex data sets and interdependent variables such as relative oligomer concentrations, making it possible to elucidate structure function relationships in complex mixtures. This approach, therefore, introduces an enabling tool to the field of amyloid research, in which it is often difficult to interpret the activity of individual species within a complex mixture of bioactive species.</p> </div

Pore formation by Aβ_1–40 and Aβ_1–42 in planar lipid bilayers.

<p><b>A)</b> Cartoon of the experimental setup. <b>B)</b> Example of transmembrane ion flux induced by 15 µM Aβ_1–40 prepared by one day incubation in water (method A, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047261#pone.0047261.s010" target="_blank">Table S1</a>). <b>C)</b> Example of transmembrane current induced by 15 µM Aβ_1–42 prepared by three day incubation (method A). Addition of 10 mM Zn²⁺ (arrows) inhibited Aβ-induced ion flux.</p

Pore formation, cytotoxicity, ThT fluorescence and β-sheet content as a function of aggregation time of Aβ samples in water (method A).

<p>Red and blue curves in each panel are the two component predictions from PLS regression for Aβ_1–40 or Aβ_1–42, respectively. <b>A)</b> Percentage of experiments that showed pore formation in planar lipid bilayers. Each point represents 10–15 experiments in the presence of 15–25 µM Aβ_1–40 or Aβ_1–42; error bars represent the error of proportion. <b>B)</b> Cell death of human neuroblastoma SH-SY5Y cells 24 h after exposure to serum-free media containing 20 µM Aβ prepared by method A. Each point represents 5–15 independent experiments. <b>C)</b> Degree of fibril formation as determined by ThT fluorescence. Each point represents an average from 5–20 experiments, error bars represent standard errors of the mean. <b>D)</b> Beta-sheet formation as determined by CD spectroscopy. Each point represents an average from 3–5 experiments, error bars represent standard errors of the mean.</p

Separation of Aβ samples by SDS-PAGE followed by Western blot and densitometry analysis.

<p><b>A)</b> Example of a Western blot of after SDS-PAGE of Aβ_1–40 (left) and Aβ_1–42 (right) preparations that had been incubated in water for 0, 1, 2, 3, 10, and 20 days (method A). All samples were cross-linked with 12 mM glutaraldehyde before electrophoresis through a 16.5% Tris-Tricine gel. Grouping of oligomers is indicated on the right. <b>B)</b> Average relative abundance of aggregated Aβ_1–40 (red) and Aβ_1–42 (blue) species of different size as a function of aggregation time. Each point represents the mean value of the relative abundance of each species from 4 to 6 gels; error bars represent the standard error of the mean. Red and blue curves are best curve fits of equation S1 to the data (see Supporting Information).</p

Synthetic Chondramide A Analogues Stabilize Filamentous Actin and Block Invasion by <i>Toxoplasma gondii</i>

Apicomplexan parasites such as <i>Toxoplasma gondii</i> rely on actin-based motility to cross biological barriers and invade host cells. Key structural and biochemical differences in host and parasite actins make this an attractive target for small-molecule inhibitors. Here we took advantage of recent advances in the synthesis of cyclic depsipeptide compounds that stabilize filamentous actin to test the ability of chondramides to disrupt growth of <i>T. gondii in vitro</i>. Structural modeling of chondramide A (<b>2</b>) binding to an actin filament model revealed variations in the binding site between host and parasite actins. A series of 10 previously synthesized analogues (<b>2b</b>–<b>k</b>) with substitutions in the β-tyrosine moiety blocked parasite growth on host cell monolayers with EC₅₀ values that ranged from 0.3 to 1.3 μM. <i>In vitro</i> polymerization assays using highly purified recombinant actin from <i>T. gondii</i> verified that synthetic and natural product chondramides target the actin cytoskeleton. Consistent with this, chondramide treatment blocked parasite invasion into host cells and was more rapidly effective than pyrimethamine, a standard therapeutic agent. Although the current compounds lack specificity for parasite vs host actin, these studies provide a platform for the future design and synthesis of synthetic cyclic peptide inhibitors that selectively disrupt actin dynamics in parasites

Dependence of pore formation, cytotoxicity, β-sheet content and ThT fluorescence on the oligomer levels of Aβ_1–40 (top) and Aβ_1–42 (bottom).

<p>The sign of the regression coefficients indicates whether a particular species contributes positively or negatively to a particular observation. The error bars are the standard deviation of the coefficient estimated through a jackknife procedure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047261#pone.0047261-Shao1" target="_blank">[90]</a>. Coefficients less than 0.25 were considered insignificant and values that were at least one or two standard deviations from zero are marked as significant (*) or highly significant (**) respectively.</p

Frequency-Based Analysis of Gramicidin A Nanopores Enabling Detection of Small Molecules with Picomolar Sensitivity

Methods to detect low concentrations of small molecules are useful for a wide range of analytical problems including the development of clinical assays, the study of complex biological systems, and the detection of biological warfare agents. This paper describes a semisynthetic ion channel platform capable of detecting small molecule analytes with picomolar sensitivity. Our methodology exploits the transient nature of ion channels formed from gramicidin A (gA) nanopores and the frequency of observed single channel events as a function of concentration of free gA molecules that reversibly dimerize in a bilayer membrane. We initially use a protein (here, a monoclonal antibody) to sequester the ion channel activity of a <i>C</i>-terminally modified gA derivative. When a small molecule analyte is introduced to the electrical recording medium, it competitively binds to the protein and liberates the gA derivative, restoring its single ion channel activity. We found that monitoring the frequency of gA channel events makes it possible to detect picomolar concentrations of small molecule in solution. In part, due to the digital on/off nature of frequency-based analysis, this approach is 10³ times more sensitive than measuring macroscopic membrane ion flux through gA channels as a basis for detection. This novel methodology, therefore, significantly improves the limit of detection of nanopore-based sensors for small molecule analytes, which has the potential for incorporation into miniaturized and low cost devices that could complement current established assays

Single-Particle Characterization of Aβ Oligomers in Solution

Determining the pathological role of amyloids in amyloid-associated diseases will require a method for characterizing the dynamic distributions in size and shape of amyloid oligomers with high resolution. Here, we explored the potential of resistive-pulse sensing through lipid bilayer-coated nanopores to measure the size of individual amyloid-β oligomers directly in solution and without chemical modification. This method classified individual amyloid-β aggregates as spherical oligomers, protofibrils, or mature fibers and made it possible to account for the large heterogeneity of amyloid-β aggregate sizes. The approach revealed the distribution of protofibrillar lengths (12- to 155 -mer) as well as the average cross-sectional area of protofibrils and fibers