CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR - EXPRESSION AND HELICITY OF A DOUBLE MEMBRANE-SPANNING SEGMENT

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 struc ture and/or impair ion transport. We report the preparation in milligr am quantities and conformational characterization of a polypeptide com prised of CFTR transmembrane (TM) segments 3-4, a putative 'helical ha irpin' portion of the CFTR TM1-6 domain, The TM segment 3-4 of CFTR wa s expressed in E. coli as a fusion protein linked to the C-terminus of His-tagged thioredoxin. Nickel chelate affinity chromatography, follo wed by release from the carrier by digestion with thrombin protease, g ave free CFTR (TM3-4). Monitoring of the folding properties and confor mational state(s) of the TM3-4 polypeptide using circular dichroism sp ectroscopy indicated a partial a-helical conformation in aqueous buffe r, with up to 30% increase in a-helical content observed in membrane-m imetic environments. (C) 1998 Federation of European Biochemical Socie ties.