Be Thou Exalted, Volume 3: With Firm Resolve I Held My Peace: Instrumental

Full conductor score including parts for organ, trumpets, flutes, oboes, clarinets, bass clarinet, contrabass clarinet, bassoon, alto saxophones, tenor saxophone, baritone saxophone, French horns, trombones, baritone, string bass, timpani and tuba

The Methylococcus capsulatus (Bath) Secreted Protein, MopE*, Binds Both Reduced and Oxidized Copper

Under copper limiting growth conditions the methanotrophic bacterium Methylococcus capsulatus (Bath) secrets essentially only one protein, MopE*, to the medium. MopE* is a copper-binding protein whose structure has been determined by X-ray crystallography. The structure of MopE* revealed a unique high affinity copper binding site consisting of two histidine imidazoles and one kynurenine, the latter an oxidation product of Trp130. In this study, we demonstrate that the copper ion coordinated by this strong binding site is in the Cu(I) state when MopE* is isolated from the growth medium of M. capsulatus. The conclusion is based on X-ray Near Edge Absorption spectroscopy (XANES), and Electron Paramagnetic Resonance (EPR) studies. EPR analyses demonstrated that MopE*, in addition to the strong copper-binding site, also binds Cu(II) at two weaker binding sites. Both Cu(II) binding sites have properties typical of non-blue type II Cu (II) centres, and the strongest of the two Cu(II) sites is characterised by a relative high hyperfine coupling of copper (

The Surface-Associated and Secreted MopE Protein of Methylococcus capsulatus (Bath) Responds to Changes in the Concentration of Copper in the Growth Medium

Expression of surface-associated and secreted protein MopE of the methanotrophic bacterium Methylococcus capsulatus (Bath) in response to the concentration of copper ions in the growth medium was investigated. The level of protein associated with the cells and secreted to the medium changed when the copper concentration in the medium varied and was highest in cells exposed to copper stress

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An oxidized tryptophan facilitates copper binding in Methylococcus capsulatus-secreted protein MopE

Proteins can coordinate metal ions with endogenous nitrogen and oxygen ligands through backbone amino and carbonyl groups, but the amino acid side chains coordinating metals do not include tryptophan. Here we show for the first time the involvement of the tryptophan metabolite kynurenine in a protein metal-binding site. The crystal structure to 1.35 angstrom of MopE* from the methane-oxidizing Methylococcus capsulatus (Bath) provided detailed information about its structure and mononuclear copper-binding site. MopE* contains a novel protein fold of which only one-third of the structure displays similarities to other known folds. The geometry around the copper ion is distorted tetrahedral with one oxygen ligand from a water molecule, two histidine imidazoles (His-132 and His-203), and at the fourth distorted tetrahedral position, the N1 atom of the kynurenine, an oxidation product of Trp-130. Trp-130 was not oxidized to kynurenine in MopE* heterologously expressed in Escherichia coli, nor did this protein bind copper. Our findings indicate that the modification of tryptophan to kynurenine and its involvement in copper binding is an innate property of M. capsulatus MopE*

The Methylococcus capsulatus (Bath) Secreted Protein, MopE*, Binds Both Reduced and Oxidized Copper

Under copper limiting growth conditions the methanotrophic bacterium Methylococcus capsulatus (Bath) secrets essentially only one protein, MopE*, to the medium. MopE* is a copper-binding protein whose structure has been determined by X-ray crystallography. The structure of MopE* revealed a unique high affinity copper binding site consisting of two histidine imidazoles and one kynurenine, the latter an oxidation product of Trp130. In this study, we demonstrate that the copper ion coordinated by this strong binding site is in the Cu(I) state when MopE* is isolated from the growth medium of M. capsulatus. The conclusion is based on X-ray Near Edge Absorption spectroscopy (XANES), and Electron Paramagnetic Resonance (EPR) studies. EPR analyses demonstrated that MopE*, in addition to the strong copper-binding site, also binds Cu(II) at two weaker binding sites. Both Cu(II) binding sites have properties typical of non-blue type II Cu (II) centres, and the strongest of the two Cu(II) sites is characterised by a relative high hyperfine coupling of copper (A(parallel to) = 20 mT). Immobilized metal affinity chromatography binding studies suggests that residues in the N-terminal part of MopE* are involved in forming binding site(s) for Cu(II) ions. Our results support the hypothesis that MopE plays an important role in copper uptake, possibly making use of both its high (Cu(I) and low Cu(II) affinity properties

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