X-ray Absorption Near Edge Structure analysis of endogenously expressed CorA.

<p>XANES spectrum of crystalline CorA, recorded in steps of 0.7-edge feature at 8984 eV, typical for Cu(I) compounds (marked with an arrowhead).</p

MALDI-TOF MS and MS/MS analyses of the PA-gel excised 26 kDa polypeptide.

<p>a) MALDI-TOF MS spectrum of trypsin-produced peptides derived from the 26 kDa polypeptide excised from the outer membrane fraction of low-copper grown <i>M</i>. <i>album</i> BG8. Monoistopic peaks are labeled with their respective <i>m/z</i> ratio. b) MALDI-TOF MS/MS spectrum of the <i>m/z</i> 2625.37 ion, indicating the observed fragmentation pattern and the sequence ion assignment.</p

Overall view and copper-binding site of CorA.

<p>a) Stereo plot of CorA, illustrating the 15 β-strands, Cu(I)(yellow sphere), Ca²⁺ (green sphere), and residues coordinating the metal ions (ball-and-stick models). b) Superimposition of CorA (red) and MopE* (blue). The copper and calcium in CorA are illustrated as yellow and green spheres, respectively. The metals in MopE* are coloured blue. Because of the superposition calcium appears as blue and copper as yellow. The 15 β-strands of CorA superimpose well on 15 of the 21 strands found in MopE*. Also the residues involved in copper and calcium binding superimpose well. c) Stereo plot illustrating the 2foDfc electron density, contoured at 1 σ in the copper binding site, demonstrating the oxidation of tryptophan to kynurenine at residue 62. The copper and the coordinating water are illustrated as yellow and red spheres, respectively. The polyethylene glycol molecule shown is found near the kynurenine residue in all CorA molecules in the crystal structure.</p

SDS-PAGE analyses of protein fractions obtained from NaCl extraction of a) the outer membrane and b) of whole fermentor-grown cells.

<p>12.5% PA-gels were used and stained with Coomassie Brilliant Blue R-250. a) Lane 1, <i>M. album</i> BG8 outer membrane fraction; lane 2, Tris-HCl wash of low ionic strength; lane 3, 1 M NaCl extract of the outer membrane fraction; lane 4, outer membrane fraction after treatment with 1 M NaCl. b) Lane 1, <i>M. album</i> BG8 cells resuspended in buffer of low ionic strength; lane 2, Tris-HCl wash of low ionic strength; lane 3, 1 M NaCl extract of whole cells; lane 4, whole cells after treatment with 1 M NaCl. CorA is indicated in both a) and b). Molecular mass markers are indicated.</p

Crystal Parameters, X-ray Data Collection, and Refinement Statistics.

a<p>The values in parentheses are for the highest resolution shell.</p>b<p><i>R</i>_merge = ∑<i>hkl</i> ∑<i>j</i> |<i>Ij</i>−<i>I</i> |/∑<i>hkl</i>∑<i>j Ij</i> where <i>Ij</i> is the intensity from an individual measurement of reflection <i>hkl</i> and <i>I</i> is the mean intensity of the same reflection.</p>c<p><i>R</i>_work = ∑<i>hkl</i> |<i>F</i>o−<i>F</i>c|/∑<i>hkl</i> |<i>F</i>o|; <i>R</i>_test is the same but for the 5% of the total reflections never used for refinement.</p>d<p>Cruickshanks DPI for coordinate error.</p><p>DPI = sqrt(N_atom/(N_refl−N_param)) R_factor D_max compl^−1/3. N_atom is the number of the atoms included in the refinement, N_refl is the number of reflections included in the refinement, R_factor is the overall R-factor, D_max is the maximum resolution of reflections included in the refinement, compl is the completeness of the observed data.</p

Binding of Cu²⁺ to MopE under equilibrium conditions.

<p>(A) MopE (10 µM, 500 µl) was dialysed overnight at 4°C against 100 ml of 20 mM Tris pH 7.5, 80 mM NaCl and 1 mM CaCl₂ containing from 0 to 100 µM CuCl₂. MopE bound Cu²⁺ was determined by ICP-MS (subtracting the Cu(II) concentrations inside and outside the dialysis cassette). The molar ratio (<i>r</i>) of bound Cu(II) to MopE* has been plotted against the concentration of CuCl₂ in the dialysis buffer. The data were adjusted for copper bound to MopE* at no addition of CuCl₂.</p

XAS analysis of MopE*.

<p>Normalised XANES of a) Cu(I) oxide (–), b) MopE* protein (–), and c) Cu(NH₄)₂(SO₄)₂⋅6H₂O (Cu(II) tutton salt) (–).</p

Structural organization of the MopE protein.

<p>Structural organization of the MopE protein.</p

Analysis of the two major EPR signals observed during titration of MopE* with CuCl₂.

<p>(A) The solid line shows the EPR spectrum of MopE* at 33 K with one molar equivalent of CuCl₂ (identical to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043146#pone-0043146-g002" target="_blank">Fig. 2B</a>, lane ii). The EPR parameters (g_⊥, g_||, A_||) were read directly from the line positions, and the inset shows the superhyperfine structure observed at 77 K with one molar equivalent of CuCl₂. Dashed line: The spectrum was simulated with the software SimFonia using Lorenzian/Gaussian ratio of 1, and line widths 6.8 mT, 7.2 mT and 5.2 mT with g = 2.197, 2.06 and 2.04, A<b>_||</b>_Cu = 20 mT (B) The solid line corresponds to the difference spectrum obtained when MopE* with one molar equivalent of CuCl₂ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043146#pone-0043146-g002" target="_blank">Fig. 2B</a>, lane ii ) was subtracted from MopE* with two molar equivalents of CuCl₂ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043146#pone-0043146-g002" target="_blank">Fig. 2B</a>, lane iii). The EPR parameters (g_⊥, g_||, A_||) were read directly from the line positions, and the spectrum was simulated (dashed line) using Lorenzian/Gaussian ratio of 1, and line widths 7.2 mT, 7.2 mT and 8.2 mT with g = 2.27, 2.06 and 2.06, A_||Cu = 16 mT.</p

EPR analysis of MopE*.

<p>A) EPR spectrum of Mops buffer with 1 mM CuCl₂. The spectrum was recorded at a temperature of 77 K with a modulation frequency of 100 kHz, a modulation amplitude of 1.0 mT; and a time constant of 164 ms. The microwave frequency was 9.57 GHz, and the microwave power was 1 mW. B) EPR spectra of MopE* (360 uM) as purified (i), and with 1, 2, 4, and 8 (ii–v) molar equivalents of Cu(II) respectively. Copper was added as CuCl₂ from a freshly prepared solution in water. The spectra were recorded at 33 K, a modulation frequency of 100 kHz, a modulation amplitude of 0.6 mT, a time constant of 41 ms, a microwave frequency of 9.37 GHz, and a microwave power of 0.1 mW. The inset shows EPR-detected Cu(II) as a function of added Cu(II), demonstrating near saturation after addition of 4 molar equivalents of Cu(II). C) EPR spectrum of MopE* after treatment with 2.5% nitric acid. The spectrum was recorded at a temperature of 27 K, a modulation amplitude of 0.6 mT, a time constant of 40,960 ms, a microwave frequency of 9.39 GHz, and a microwave power of 0.05 mW.</p