Cystic fibrosis transmembrane conductance regulator: expression and helicity of a double membrane-spanning segment

Abstract

The gene responsible for cystic fibrosis encodes a membrane protein – the 1480-residue cystic fibrosis transmembrane conductance regulator (CFTR) – in which membrane-based CF-phenotypic mutants alter pore structure and/or impair ion transport. We report the preparation in milligram quantities and conformational characterization of a polypeptide comprised of CFTR transmembrane (TM) segments 3–4, a putative `helical hairpin' portion of the CFTR TM1–6 domain. The TM segment 3–4 of CFTR was expressed in E. coli as a fusion protein linked to the C-terminus of His-tagged thioredoxin. Nickel chelate affinity chromatography, followed by release from the carrier by digestion with thrombin protease, gave free CFTR(TM3–4). Monitoring of the folding properties and conformational state(s) of the TM3–4 polypeptide using circular dichroism spectroscopy indicated a partial α-helical conformation in aqueous buffer, with up to 30% increase in α-helical content observed in membrane-mimetic environments.