EfeO-cupredoxins: major new members of the cupredoxin superfamily with roles in bacterial iron transport

The EfeUOB system of Escherichia coli is a tripartite, low pH, ferrous iron transporter. It resembles the high-affinity iron transporter (Ftr1p-Fet3p) of yeast in that EfeU is homologous to Ftr1p, an integral-membrane iron-permease. However, EfeUOB lacks an equivalent of the Fet3p component—the multicopper oxidase with three cupredoxin-like domains. EfeO and EfeB are periplasmic but their precise roles are unclear. EfeO consists primarily of a C-terminal peptidase-M75 domain with a conserved ‘HxxE’ motif potentially involved in metal binding. The smaller N-terminal domain (EfeO-N) is predicted to be cupredoxin (Cup) like, suggesting a previously unrecognised similarity between EfeO and Fet3p. Our structural modelling of the E. coli EfeO Cup domain identifies two potential metal-binding sites. Site I is predicted to bind Cu2+ using three conserved residues (C41 and 103, and E66) and M101. Of these, only one (C103) is conserved in classical cupredoxins where it also acts as a Cu ligand. Site II most probably binds Fe3+ and consists of four well conserved surface Glu residues. Phylogenetic analysis indicates that the EfeO-Cup domains form a novel Cup family, designated the ‘EfeO-Cup’ family. Structural modelling of two other representative EfeO-Cup domains indicates that different subfamilies employ distinct ligand sets at their proposed metal-binding sites. The ~100 efeO homologues in the bacterial sequence databases are all associated with various iron-transport related genes indicating a common role for EfeO-Cup proteins in iron transport, supporting a new copper-iron connection in biology

Chiral and Racemic Tetramorphs of 2,6-Di-<i>t</i>-Butylditolylfuchsone

The title molecule 4-(α,α-ditolylmethylene)-2,6-di-<i>t</i>-butyl-1,4-benzoquinone (abbreviated as di-<i>t</i>-butylditolylfuchsone and numbered 2-<i>t</i>-Bu) serendipitously afforded four concomitant polymorphs during routine purification by column chromatography in the same solvent elution fraction. Polymorph I crystallized in chiral space group <i>P</i>2₁. Polymorphs II, III, and IV crystallized in centrosymmetric space groups <i>P</i>2₁/<i>n</i>, <i>Pbca</i>, and <i>C</i>2/<i>c</i>, respectively. The role of bulky <i>t</i>-Bu groups for crystallization in the chiral space group is discussed for 2,6-ditolyl and 2,6-diphenyl fuchsones. α,α-Diphenylmethylene-2,6-di-<i>t</i>-butyl-1,4-benzoquinone (di-<i>t</i>-butyldiphenylfuchsone, 1-<i>t</i>-Bu) crystallized in <i>P</i>2₁ (one polymorph) and <i>P</i>2₁/<i>c</i> (two polymorphs) space groups. Unfavorable steric repulsions due to bulky <i>t</i>-Bu groups result in voids in the crystal structures of centrosymmetric polymorphs II and III. Phase transformation of racemic structure II to III and finally to chiral polymorph I was monitored by thermal microscopy and differential scanning calorimetry. X-ray diffraction confirmed the phase transformation to be a single-crystal-to-single-crystal event. The chiral polymorph I is the stable modification in the tetramorphic system. Several randomly picked single crystals of 2-<i>t</i>-Bu polymorph I had the same absolute chirality by circular dichroism spectroscopy. A new molecule capable of exhibiting conformational chirality via atropisomerism is identified

A global benchmark study using affinity-based biosensors

International audienceTo explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used. (C) 2008 Elsevier Inc. All rights reserved