Overexpression, purification and biophysical characterisation of E. coli MerT.

Mercury resistance is the most widespread of all anti-microbial resistance occurring in a wide variety of Gram-negative and Gram-positive bacterial genera. The systems that are most studied and best understood are those encoded in mercury resistance (Mer) operons in Gram-negative bacteria. The mercury detoxification functions by the importation of highly toxic Hg(2+) into cytoplasm and enzymic reduction to volatile Hg(0). MerT is a small (13kDa) inner membrane protein involved in mercuric ion transport system. We have overexpressed recombinant 6His-tagged MerT from Escherichia coli in a native folded form and purified it to homogeneity in n-dodecyl-ß-d-maltopyranoside (DDM) by immobilized metal affinity chromatography (IMAC). Circular dichroism showed that the protein is largely α-helical. Size-exclusion chromatography (SEC) in a variety of detergents showed that the protein exists in a multiple of oligomeric states as also confirmed by SEC coupled with multiple-angle light scattering.

The cystic fibrosis transmembrane conductance regulator (CFTR): three-dimensional structure and localization of a channel gate.

Cystic fibrosis affects about 1 in 2500 live births and involves loss of transmembrane chloride flux due to a lack of a membrane protein channel termed the cystic fibrosis transmembrane conductance regulator (CFTR). We have studied CFTR structure by electron crystallography. The data were compared with existing structures of other ATP-binding cassette transporters. The protein was crystallized in the outward facing state and resembled the well characterized Sav1866 transporter. We identified regions in the CFTR map, not accounted for by Sav1866, which were potential locations for the regulatory region as well as the channel gate. In this analysis, we were aided by the fact that the unit cell was composed of two molecules not related by crystallographic symmetry. We also identified regions in the fitted Sav1866 model that were missing from the map, hence regions that were either disordered in CFTR or differently organized compared with Sav1866. Apart from the N and C termini, this indicated that in CFTR, the cytoplasmic end of transmembrane helix 5/11 and its associated loop could be partly disordered (or alternatively located).

Self-directed assembly and clustering of the cytoplasmic domains of inwardly rectifying Kir2.1 potassium channels on association with PSD-95.

The interaction of the extra-membranous domain of tetrameric inwardly rectifying Kir2.1 ion channels (Kir2.1NC(4)) with the membrane associated guanylate kinase protein PSD-95 has been studied using Transmission Electron Microscopy in negative stain. Three types of complexes were observed in electron micrographs corresponding to a 1:1 complex, a large self-enclosed tetrad complex and extended chains of linked channel domains. Using models derived from small angle X-ray scattering experiments in which high resolution structures from X-ray crystallographic and Nuclear Magnetic Resonance studies are positioned, the envelopes from single particle analysis can be resolved as a Kir2.1NC(4):PSD-95 complex and a tetrad of this unit (Kir2.1NC(4):PSD-95)(4). The tetrad complex shows the close association of the Kir2.1 cytoplasmic domains and the influence of PSD-95 mediated self-assembly on the clustering of these channels.

Characterization of a Pixelated Cadmium Telluride Detector System Using a Polychromatic X-Ray Source and Gold Nanoparticle-Loaded Phantoms for Benchtop X-Ray Fluorescence Imaging.

Pixelated semi-conductor detectors providing high energy resolution enable parallel acquisition of x-ray fluorescence (XRF) signals, potentially leading to performance enhancement of benchtop XRF imaging or computed tomography (XFCT) systems utilizing ordinary polychromatic x-ray sources. However, little is currently known about the characteristics of such detectors under typical operating conditions of benchtop XRF imaging/XFCT. In this work, a commercially available pixelated cadmium telluride (CdTe) detector system, HEXITEC (High Energy X-ray Imaging Technology), was characterized to address this issue. Specifically, HEXITEC was deployed into our benchtop cone-beam XFCT system, and used to detect gold Kα XRF photons from gold nanoparticle (GNP)-loaded phantoms. To facilitate the detection of XRF photons, various parallel-hole stainless steel collimators were fabricated and coupled with HEXITEC. A pixel-by-pixel spectrum merging algorithm was introduced to obtain well-defined XRF + scatter spectra with parallel-hole collimators. The effect of charge sharing addition (CSA) and discrimination (CSD) algorithms was also investigated for pixel-level CS correction. Finally, the detector energy resolution, in terms of the full-width at half-maximum (FWHM) values at two gold Kα XRF peaks (~68 keV), was also determined. Under the current experimental conditions, CSD provided the best energy resolution of HEXITEC (~1.05 keV FWHM), compared with CSA and no CS correction. This FWHM value was larger (by up to ~0.35 keV) than those reported previously for HEXITEC (at ~60 keV Am-241 peak) and single-crystal CdTe detectors (at two gold Kα XRF peaks). This investigation highlighted characteristics of HEXITEC as well as the necessity for application-specific detector characterization.

Characterization of a CorA Mg2+ transport channel from Methanococcus jannaschii using a Thermofluor-based stability assay.

The Thermofluor assay has been a valuable asset in structural genomics, providing a high-throughput method for assessing the crystallizability of proteins. The technique has been well characterized for soluble proteins but has been less extensively described for membrane proteins. Here we show the successful application of a Thermofluor-based stability assay to an ion channel, CorA from Methanococcus jannaschii. Optimization of the concentration of free detergent within the assay was important, as excessive concentrations mask the fluorescence change associated with thermal unfolding of the protein. CorA was shown to be stabilized by low pH, but relatively insensitive to salt concentration. Divalent metal cations were also capable of stabilizing the protein, in the order Co2+>Ni2+>Mn2+>Mg2+>Ca2+. Finally, removal of the oligohistidine tag was also shown to improve the thermal stability of CorA. Conclusions are drawn from this detailed study about the general applicability of this technique to other membrane proteins.

Expression and purification of the cystic fibrosis transmembrane conductance regulator protein in Saccharomyces cerevisiae.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein(1-3). Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed (4-9). Proteins involved in the processing of recombinant CFTR in yeast have been described(6-9) .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)(10). The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be >90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.