Monitoring of the FIV capsid assembly by DLS with varying protein concentration.

<p>T = 0 indicates the addition of 1 M NaCl to the protein solution at 7 mg/ml (gray triangles) or 5 mg/ml (black squares) at 37°C.</p

DLS experiments showing the kinetics of spherical assembly for p24 FIV.

<p>A) The assembly kinetics of the FIV p24 particles were monitored by dynamic light scattering with an initial protein concentration of 7 mg/ml and 1 M of NaCl at 37°C. Arrows indicate the time points when samples were taken for TEM analysis. B) Size distribution profiles as a function of the volume of FIV p24-His at different time points during assembly by DLS. Sampling times are indicated above their respective peak.</p

Circular dichroism spectrum in the far-UV for the FIV p24-His protein.

<p>Experiments were performed at 20°C with 150 µl of protein at 10 µM in 50 mM sodium phosphate (pH 7.4).</p

Monitoring of FIV capsid assembly by DLS with varying salt concentration and incubation temperature.

<p>T = 0 indicates the addition of NaCl to the protein solution at 7 mg/ml. Two NaCl induction conditions were tested: A) 1 M and B) 0.5 M NaCl (final concentration), with three different incubation temperatures: 37°C (triangles), 30°C (squares), and 25°C (diamonds).</p

Kinetics of p24 assembly as shown by negatively stained TEM.

<p>Images taken after 4 min (A, B), 8 min (C, D), 12 min (E, F) and 60 min (G) of incubation, representative of 5 different assembly experiments. The samples were negatively stained with 2% ammonium molybdate. Magnification and bars, 45,000× and 50 nm.</p

Analysis of the oligomeric state of FIV p24 as a function of the protein concentration.

<p>A) Size distribution profiles as a function of the volume of FIV p24-His at 5 mg/ml (left) and 7 mg/ml (right), by Dynamic Light Scattering. The graph shows the superposition of three successive measurements from 12 runs at each concentration, which are representative for at least three independent experiments. B) Chemical cross-linking of recombinant FIV capsid protein with protein at 5 mg/ml or 7 mg/ml. Lane MW, protein molecular mass marker. A negative control without BS³ has been realized on the same proteins (−BS³). C) Dissociation of FIV p24 dimer as followed by ITC. The thermogram (top panel) and the plotted titration curve (bottom panel) were obtained with a Microcal ITC200. The solid line (bottom panel) represents the fitting of the data by the built-in dimer dissociation model.</p

Electron microscopy reveals that ECAM is an elongated, flexible molecule.

<p>(A) Comparison between raw images, which were low-path filtered at 25 Š(lines 1, 3) and re-projections of the obtained 3D reconstructions (lines 2, 4). (B) Isosurface representations of the 3D reconstruction obtained for the activated form of ECAM (gray) and comparison with the 15 Šfiltered structure of C3b (PDB 2I07, blue). For the isosurface representations, an averaged mass density of 0.84 Da/ų and a molecular weight value of 190 kDa were used. Black arrows represent top (1) and bottom (2) views, while the red arrows point to the regions of potentially greatest flexibility.</p

ECAM shows a change in gel mobility and overall structure upon activation.

<p>(A) Native PAGE showing the migration profile of ECAM. Lanes 1) native molecule; 2) methylamine-treated; 3) after reaction with chymotrypsin; 4) native molecule; 5) after reaction with porcine pancreatic elastase (B) Small-angle X-ray scattering (SAXS) results for native, methylamine-activated, elastase- and chymotrypsin-reacted ECAM. The radially averaged scattered X-ray intensity was plotted as a function of the momentum transfer <i>s</i>. Scattering patterns for ECAM in native form (black), after reaction with methylamine (green), elastase (blue) and chymotrypsin (red) were recorded in different concentrations (from 0.5 to 8 mg/mL) but only the curves relating to the highest concentration are shown. Inset, detail of differences in distinct side maxima. (C) Distance distributions <i>p</i>(r) of native, methylamine-reacted, elastase, and chymotrypsin of ECAM. All curves were normalized. Inset, detail of maxima of <i>p</i>(r) functions.</p

Schematic representations of human α-macroglobulin (α₂M), C3 convertase (C3), and <i>E. coli</i> α-macroglobulin (ECAM).

<p>Domain assignments for α₂M and C3 were based on their respective crystal structures <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035384#pone.0035384-Marrero1" target="_blank">[13]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035384#pone.0035384-Janssen1" target="_blank">[17]</a>. Assignments for ECAM were performed with the JPRED server, supported by the analysis performed by Doan and Gettins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035384#pone.0035384-Doan1" target="_blank">[27]</a>. Note the similarity in domain predictions, including MG and TED domains. The CLEQ sequence, a signature of the thioester bond, is present in all proteins. For simplicity, only a limited number of the domains identified or predicted for the different molecules are depicted, and only one monomer of α₂M is shown. The C-terminus of ECAM displays low sequence similarity to that of C3 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035384#pone.0035384.s006" target="_blank">Fig. S6</a>).</p

<i>Ab initio</i> models of ECAM generated by SAXS.

<p>Each model results from averaging 10 individual models calculated by the program GASBOR using: (A) native ECAM, (B) methylamine-treated, (C) chymotrypsin-treated, and (D) elastase-treated ECAM. Note the appearance of a central cavity in all of the treated forms of the molecule. GASBOR was used in “user" mode, following default options, except for the total number of residues, which corresponded to total ECAM (1653 residues). The envelopes are based on the <i>p</i>(r) funtions shown on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035384#pone-0035384-g003" target="_blank">Fig. 3</a>, and the GNOM files generated were used as input for GASBOR. The models are drawn to scale.</p