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

Structural organization of the MopE protein.

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

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

Retention of MopE* on Cu(II) charged IMAC columns.

<p>10% SDS-PA gels containing samples from Cu(II) affinity chromatography columns; application at pH 6.8, elution performed with pH 4.0 Each eluted fraction (corresponding to one column volume = 0.5 ml) was analyzed The lanes show the load to the column (Load), the flow-through (FT), the pH 6.8 washes (Wash), the pH 6.0 elutions (pH 6.0), the pH 4.0 elutions (pH 4.0), and finally the EDTA-stripping of the column (50 mM EDTA). Application and washing was performed using 20 mM sodium phosphate buffer pH 6.8, containing 0.5 M NaCl. Elution was performed using 20 mM sodium phosphate buffer containing 0.5 M NaCl and 1 mM CaCl₂, pH 6.0 and 4.0, respectively. The protein band indicated with an arrow represents MopE*⁻²⁴ (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043146#pone-0043146-g001" target="_blank">Fig. 1</a>).</p