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.

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.

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][2][3] . Like many 'difficult' eukaryotic membrane proteins, expression in a fastgrowing 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][5][6][7][8][9] . Proteins involved in the processing of recombinant CFTR in yeast have been described [6][7][8][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 constructcontaining 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.

Background: Cystic fibrosis is a disease where mutations in the cftr gene lead to loss of a chloride channel.Results: The CFTR transmembrane domains show an outward facing configuration.Conclusion: The map shows regions that probably represent the channel's gate and its regulatory region.Significance: Residues associated with changes in channel function and disease are adjacent to the gate.

No abstract