Mechanism of Amyloid‑β Fibril Elongation

Amyloid-β is an intrinsically disordered protein that forms fibrils in the brains of patients with Alzheimer’s disease. To explore factors that affect the process of fibril growth, we computed the free energy associated with disordered amyloid-β monomers being added to growing amyloid fibrils using extensive molecular dynamics simulations coupled with umbrella sampling. We find that the mechanisms of Aβ40 and Aβ42 fibril elongation have many features in common, including the formation of an obligate on-pathway β-hairpin intermediate that hydrogen bonds to the fibril core. In addition, our data lead to new hypotheses for how fibrils may serve as secondary nucleation sites that can catalyze the formation of soluble oligomers, a finding in agreement with recent experimental observations. These data provide a detailed mechanistic description of amyloid-β fibril elongation and a structural link between the disordered free monomer and the growth of amyloid fibrils and soluble oligomers

Relation between and Ras density <i>λ</i> for immunoEM data of gold labeled GFP-tH (black symbols) and RFP-tH (gray symbols), simulation averages and 99% confidence intervals (red), as well as a linear least-squares fit to simulation averages (red line).

<p> data points were extracted from Ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006148#pone.0006148-Plowman1" target="_blank">[11]</a> with IMAGE J. (Left inset) (black) and (green) as a function of <i>λ</i>.(Right inset) as a function of <i>λ</i> without long-range repulsion (). Error bars represent standard deviations. For simulation details, including calculation of confidence intervals, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006148#s4" target="_blank"><i>Methods</i></a>.</p

Representative Ras densities with corresponding numbers of Ras molecules on discretized lattice membrane as well as gold densities.

<p>Shown are the four Ras densities used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006148#pone-0006148-g004" target="_blank">Figs. 4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006148#pone-0006148-g005" target="_blank">5</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006148#pone-0006148-g006" target="_blank">6</a>. For lattice parameters, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006148#s4" target="_blank"><i>Methods</i></a>.</p

Monte Carlo simulations and point-pattern analysis.

<p>Snapshots of equilibrated Ras molecules on lattice membrane (left column; active Ras in red and inactive Ras in blue) and corresponding plots (right column) after gold labeling for the four densities from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006148#pone-0006148-t001" target="_blank">Table 1</a> (density of Ras molecules increases from top to bottom). Shown in the plots are individual simulations (cyan curves), their averages (thick black curves), as well as 68.3%, 95.4%, and 99.0% confidence intervals (red, green, and blue dashed lines, respectively). For simulation details, including calculation of confidence intervals, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006148#s4" target="_blank"><i>Methods</i></a>.</p

Model ingredients.

<p>(A) Short-range attraction (red) and long-range repulsion (blue) as a function of distance between two Ras molecules for the parameters given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006148#s4" target="_blank"><i>Methods</i></a>. Also shown is the cut-off beyond which the repulsive energy is set to zero (blue dashed line). (Inset) Representative part of lattice membrane showing three active Ras molecules (red) and one inactive Ras molecule (blue). Neighboring active Ras molecules interact via the attractive short-range interaction (green bar). The cut-off used for the long-range repulsion is representatively shown for the central Ras (blue dashed circle). (B) Schematic of a gold-labeled antibody associated with a GFP-Ras molecule in the inner leaflet of the plasma membrane.</p

Signaling properties of Ras clusters.

<p>(A) Cluster activity as a function of input (parameter ). Cluster activity is defined as fraction of active Ras in clusters from simulations (bar chart), where a cluster contains two or more contacting Ras molecules. Also shown is approximate cluster activity , which assumes that all <i>N</i> Ras molecules in a cluster (here chose ) are tightly coupled and hence are either all on (active) or all off (inactive) together (black line). Black error bars show standard deviation and represent intrinsic noise. Green error bars represent extrinsic noise, calculated with noise propagation formula for . (B) Total activity of all Ras molecules in the membrane, normalized by the total number of Ras molecules (grey bar chart, left axis), and number of Ras clusters (white bar chart, right axis). Also shown are linear fits. Error bars represent standard deviations. To enlarge black error bars for better visualization in <i>B</i>, we used the square-root of the total variance from pooled simulations of inputs , , and .</p

Signaling properties of non-interacting Ras molecules.

<p>(A) Activity of single Ras molecule (dashed line; calculated with Eq. 1 for ) as a function of input (parameter ). Black error bars represent intrinsic noise, calculated from the square-root of the binomial variance . Green error bars are approximately 0.01 in magnitude and represent extrinsic noise, calculated with the noise propagation formula and . (B) Total activity of all Ras molecules in the membrane, normalized by the total number of Ras molecules (bar chart) and linear fit (dashed line). Error bars represent standard deviation, calculated from the square-root of the total variance from pooled simulations of inputs , , and .</p

Experimental immunoEM data and statistical clustering analysis.

<p>(A) Electron micrograph of immunogold-labeled Ras domain (GFP-tH where tH is minimal plasma membrane targeting motifs of H-Ras) in an <i>in vitro</i> plasma membrane sheet. Scale bar is 100 nm. (B) Corresponding point-pattern analysis (red) and 99% confidence interval (black). ©Prior <i>et al.</i> (2003), originally published in <i>The Journal of Cell Biology</i>. doi:10.1083/jcb.200209091 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006148#pone.0006148-Prior1" target="_blank">[7]</a>.</p

Monte Carlo simulations and point-pattern analysis for conventional clustering model without long-range repulsion ().

<p>For a description of symbols and lines, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006148#pone-0006148-g004" target="_blank">Fig. 4</a>.</p

The Dynamic Structure of α‑Synuclein Multimers

α-Synuclein, a protein that forms ordered aggregates in the brains of patients with Parkinson’s disease, is intrinsically disordered in the monomeric state. Several studies, however, suggest that it can form soluble multimers <i>in vivo</i> that have significant secondary structure content. A number of studies demonstrate that α-synuclein can form β-strand-rich oligomers that are neurotoxic, and recent observations argue for the existence of soluble helical tetrameric species <i>in cellulo</i> that do not form toxic aggregates. To gain further insight into the different types of multimeric states that this protein can adopt, we generated an ensemble for an α-synuclein construct that contains a 10-residue N-terminal extension, which forms multimers when isolated from <i>Escherichia coli</i>. Data from NMR chemical shifts and residual dipolar couplings were used to guide the construction of the ensemble. Our data suggest that the dominant state of this ensemble is a disordered monomer, complemented by a small fraction of helical trimers and tetramers. Interestingly, the ensemble also contains trimeric and tetrameric oligomers that are rich in β-strand content. These data help to reconcile seemingly contradictory observations that indicate the presence of a helical tetramer <i>in cellulo</i> on the one hand, and a disordered monomer on the other. Furthermore, our findings are consistent with the notion that the helical tetrameric state provides a mechanism for storing α-synuclein when the protein concentration is high, thereby preventing non-membrane-bound monomers from aggregating