Helical wheel representation of helix 1 of five frataxin structure.

<p>(A) Human frataxin; residues (94–114, PDB entry 1EKG) (B) Yeast frataxin Yfh1 (residues 76–88; PDB entry 3OEQ) (C) <i>E</i>. <i>coli</i> CyaY; (residues 3–22; PDB entry 1EW4) (D) <i>Psychromonas ingrahamii</i> CyaY (residues 3–24; PDB entry 4HS5) (E) <i>Burkholderia cenocepacia</i> CyaY (residues 4–26; PDB entry 4JPD). Acidic residues are shown as red filled circles, basic residues as blue, polar in magenta and hydrophobic in yellow. The letters represent the amino acids in one-letter code. Figure prepared using the HeliQuest server [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184961#pone.0184961.ref060" target="_blank">60</a>].</p

Masses of protein species detected by native mass spectrometry.

<p>Masses of protein species detected by native mass spectrometry.</p

Rigid-body modeling for iron-induced CyaY dimer.

<p>The crystallographic structure of monomeric CyaY (PDB entry: 1ew4) was used to generate the CyaY dimer structure using SASREF. Two models were obtained with <b>A)</b> <i>head-to-tail</i> and <b>B)</b> <i>head-to-head</i> arrangement of monomers. <b>C)</b> SASREF modeling fit of the <i>head-to-tail</i> dimer to the SEC-SAXS data. <b>D)</b> The <i>head-to-tail</i> model docked into the SAXS-filtered <i>ab initio</i> model with the corresponding P(r) plot shown in the insert.</p

Nano-DSF analysis of iron-induced CyaY oligomer samples.

<p><b>A)</b> The F_350nm/F_330nm is plotted against the temperature gradient. <b>B)</b> The first derivative for the F_350nm/F_330nm curve against the temperature gradient from which the T_m for each sample was derived.</p

OLIGOMER fitting for the 1:2 iron-to-protein ratio.

<p>The experimental SAXS data are represented by circles, while the corresponding fit of OLIGOMER is shown as a red line. <b>A)</b> 1:2 iron-to-protein ratio (1 mg/ml of CyaY) and <b>B)</b> 1:2 iron-to-protein ratio (3 mg/ml CyaY) as performed in an earlier study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184961#pone.0184961.ref022" target="_blank">22</a>].</p

SEC-SAXS for a 7:1 iron-to-CyaY ratio.

<p><b>A)</b> Elution profile (normalized absorbance at 280 nm) for the sample with 8 mg/ml CyaY concentration loaded on a Superdex 75 gel filtration column after the elimination of high oligomeric species through 100 kDa concentrators optimized for SEC-SAXS. <b>B)</b> SEC-SAXS results for the same sample at protein concentration of 56 mg/ml. Each frame corresponds to 1-second exposure time and is plotted against I_sum (blue) and R_g (red). Frames that showed higher stability in R_g for the first peak were averaged, and the buffer was subtracted for data processing. The second peak was for monomeric CyaY.</p

Radius of gyration (R_g), maximum size (D_max), Porod volume (V_p), and the corresponding molecular weight M_wt calculated from the SAXS profiles for CyaY incubated with different iron concentrations.

<p>Radius of gyration (R_g), maximum size (D_max), Porod volume (V_p), and the corresponding molecular weight M_wt calculated from the SAXS profiles for CyaY incubated with different iron concentrations.</p

<i>Burkholderia cenocepacia</i> CyaY monomer assembly in the crystals (PDB entry: 4JPD).

<p><b>A)</b> The hexamer is built up by two trimers packed against each other around the threefold symmetry axis of the crystal. One trimer layer is shown in yellow and the other in red. The N- and C-termini of the subunits are labeled in blue. <b>B)</b> One of the trimer layers of the hexamer showing the arrangement of the monomers.</p

Alignment of CyaY sequences.

<p>The acidic residues in the sequence are boxed in red for clarity. CYAY_ECOLI–<i>E</i>. <i>coli</i>; CYAY_PSYIN—<i>Psychromonas ingrahamii</i>; CYAY_BURCJ—<i>Burkholderia cenocepacia</i>.</p

DLS studies of iron-dependent oligomerization of human frataxin FXN^81-210.

<p><b>A</b>) Measurements after 30 min of incubation with iron at 2:1 equivalents of iron-to-protein (magenta), 4:1 (green), and 10:1 (blue). <b>B</b>) Measurements after 60 min of incubation showing buildup of oligomers. In black is monomeric human FXN^81-210 without the addition of iron. On the x-axis is the hydrodynamic radius of the particles and on the y-axis is the volume percentage of particles.</p