β–strand content and solvent accessible area.

<p>Probability that each residue is in β-strand conformation. Data are averaged over three time windows (A) t* = 2153~6727, (B) 19648~24266 and (C) 38120~42736. (D) Solvent accessible area for each residue. Data are averaged over time window (t* = 38120~42736).</p

Snapshots for the 5^th run.

<p>The time evolution of the structure for the 5^th run at T* = 0.20 in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004258#pcbi.1004258.g001" target="_blank">Fig 1C and 1D</a>. Snapshots are taken at (A) t* = 5, (B) 1244, (C) 2608, (D) 3656, (E) 4233, (F) 5442, (G) 6086, (H) 10454, (I) 11063. See <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004258#pcbi.1004258.s018" target="_blank">S1 Video</a>. The β-strand contents measured by the STRIDE program are (A) 0%, (B) 12%, (C) 26%, (D) 50%, (E) 48%, (F) 66%, (G) 64%, (H) 74%, (I) 75%. The α-helix content is insignificant in these structures and the remaining portions are coil and turns.</p

Structural Conversion of Aβ_17–42 Peptides from Disordered Oligomers to U-Shape Protofilaments via Multiple Kinetic Pathways

<div><p>Discovering the mechanisms by which proteins aggregate into fibrils is an essential first step in understanding the molecular level processes underlying neurodegenerative diseases such as Alzheimer’s and Parkinson's. The goal of this work is to provide insights into the structural changes that characterize the kinetic pathways by which amyloid-β peptides convert from monomers to oligomers to fibrils. By applying discontinuous molecular dynamics simulations to PRIME20, a force field designed to capture the chemical and physical aspects of protein aggregation, we have been able to trace out the entire aggregation process for a system containing 8 Aβ17–42 peptides. We uncovered two fibrillization mechanisms that govern the structural conversion of Aβ17–42 peptides from disordered oligomers into protofilaments. The first mechanism is monomeric conversion templated by a U-shape oligomeric nucleus into U-shape protofilament. The second mechanism involves a long-lived and on-pathway metastable oligomer with S-shape chains, having a C-terminal turn, en route to the final U-shape protofilament. Oligomers with this C-terminal turn have been regarded in recent experiments as a major contributing element to cell toxicity in Alzheimer’s disease. The internal structures of the U-shape protofilaments from our PRIME20/DMD simulation agree well with those from solid state NMR experiments. The approach presented here offers a simple molecular-level framework to describe protein aggregation in general and to visualize the kinetic evolution of a putative toxic element in Alzheimer’s disease in particular.</p></div

Time evolution of the interaction energy.

<p>The total interaction energy in units of ɛ_HB for (A) 1st, 2nd, 3rd, (B) 4th, 5th, 6th, (C) 7th, 8th, 9th, 10th trajectories. The 3rd (green), 5th (red), 10th (blue) trajectories show lower energies than the others. (D) P_max (max population) within each Δt* = 5000 interval which is defined in text.</p

Salt-bridge and hydrophobic interactions for the 10^th run.

<p>(A) Structure at 568 billion collision (t*≈52,000) for the 10^th run. (B)(C) Fibril axis view with ribbon diagram or with side-chain spheres. (D)~(K) Fibril axis views for each chain showing side-chain spheres; F19(purple), D23(red), K28(cyan), I32(green) and L34(pink sphere). Figs (D)~(H) have salt-bridge pairs (D23-K28) and hydrophobic interactions between I32, L34 and F19; the rest do not.</p

Residue-specific characteristics of MTH1880 unfolding.

<p>(A) Chemical shift perturbation (CSP) analysis to detect residues susceptible to urea denaturation. The average chemical-shift changes were calculated using the following formula: Δδ_AV = [(Δδ_1H)²+(Δδ_15N/5)²]^1/2, where Δ<i>δ</i>_AV, Δ<i>δ</i>_1H, and Δ<i>δ</i>_15N are the average, proton, and ¹⁵N chemical-shift changes, respectively. (B) Structure of the MTH1880 represented by a ribbon diagram. K13-D36 and K13-D38 form salt bridges. Dashed lines indicate the salt bridges. K13-D36 and K13-D38 salt bridges contributed to the stability of the folding structure of MTH1880. Red and blue atoms mean oxygen and nitrogen, respectively. Hydrophobic core is formed by side-chain connectivity of hydrophobic residues. It is represented by sphere and stick, respectively. (C) Thermal-induced denaturation curves of wild type MTH1880 and mutants. The fraction of unfolding extracted from far-UV CD spectra at 222 nm with a constant heating rate of 10°C/h and 25μM protein concentrations. MTH1880 wt (filled circle), mutants in the salt bridge; K13A (triangle), D36A (square), D38A (circle) and mutants in the hydrophobic pocket; V23A (diamond), V53A(filled square). (D) Thermal stabilities were investigated for MTH1880 mutants. Tm values of mutant proteins are indicated as black bars.</p

GdnHCl denaturation monitored by circular dichroism and fluorescence spectroscopy.

<p>(A) Data were acquired at 0 M (black triangle), 1 M (red square), 2 M (blue circle), 3 M (hkaki filled diamond),4.5 M (red triangle), 5 M (green square), 6 M (red circle), 7 M (hkaki diamond), and 8 M (cyan cross) urea. (B) The fraction of unfolding extracted from far-UV CD spectra (25 μM) at 222nm as a function of GdnHCl concentration was plotted and fit by a sigmoidal curve. The transition mid-concentration of GdnHCl (C_m) is 3.95 ± 0.1 M. (C) Fluorescence-emission spectra for different GdnHCl concentrations ranging from 0 to 6.0 M. (D) The fraction of unfolding extracted from fluorescence-emission spectra (25 μM) at 308 nm, as a function of urea concentration, was plotted and fit by a sigmoidal curve. The transition mid-concentration of urea is 4.1 ± 0.05 M.</p

Thermal folding-unfolding of MTH1880.

<p>(A) Data were acquired at 25°C (black triangle), 45°C (red square), 65°C (blue circle), 75°C (yellow square), 85°C (red triangle), 95°C (green square), and 105°C (red circle). Protein concentration was ~25 μM in a cell of 0.1 mm path length. (B) The fraction of unfolding extracted from far-UV CD spectra at 222 nm with a constant heating rate of 10°C/h as a function of temperature was plotted and fit by a sigmoidal curve. The transition mid-temperature (T_m) of MTH1880 was 76 ± 0.5°C. (C) Results from molecular dynamics (MD) simulations. (Left panel) Radius of gyrations (R_g) of MTH1880 as a function of time at temperatures from 300 K to 525 K. (Middle panel) R_g averaged over 400,000 snapshots in the time window from 100 ns to 500 ns. The error bar denotes one standard deviation. (Right panel) The probability distribution of R_g is plotted for each temperature and results in sharp distributions at low temperatures and broad distributions at high temperatures. (D) (Left panel) Atomic fluctuation (RMSF) of MTH1880 by residue in the same time window and same temperatures as (C). (Right panel) RMSF averaged over 88 residues. (C, D) This data were acquired at 300K(red filled circle), 325K(green filled triangle), 350K(blue filled triangle), 375K(pink filled square), 400K(cyan cross), 425K(black cross), 450K(red circle), 475K(green triangle), 500K(blue triangle), and 525K(pink square). (E) Secondary structures of MTH1880 are shown with residue numbers. A ladder diagram displays residue-residue pairwise contacts denoted by a semicircle line with the distance cut-off 6.5A to emphasize the major topology of pairwise residue-residue interactions based on three-dimensional structure of MTH1880. (F) Extended Munoz-Eaton (ME) model. Data were acquired at 0.7T_m(black reverse triangle), 0.8T_m(pink square), 0.9T_m(green cross), 1.0T_m(red circle), 1.1T_m(cyan cross), 1.2T_m(blue square), and 1.3T_m(black triangle). (Left panel) Free energy (ΔG) landscape of MTH1880 as a function of the reaction coordinate, where M = 0 denotes the fully denatured structure and M = 1 denotes the native structure. The free energy of folding ΔΔG_D-N (ΔG_D—ΔG_N, right top panel) and the fraction of unfolded protein as a function of the reduced temperature (T/T_m), where T_m is the transition mid-temperature (right bottom panel). At T = T_m, ΔΔG_D-N = 0, and the fraction of unfolding is 0.5. (G) Correlation matrix of MTH1880 at 300 K is calculated from 400,000 snapshots in the same time window as (C). The secondary structure of MTH1880, in the N to C direction, are depicted next to the axes. (H) Correlation matrix of MTH1880 at (Top left to right) 325K, 350K, 375K, (Middle left to right) 400K, 425K, 450K, (Bottom left to right) 475K, 500K and 525K.</p