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 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 alpha-helical conformation in aqueous buffer, with up to 30% increase in alpha-helical content observed in membrane-mimetic environments.
Division of Structural Biology and Biochemistry, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
S
S
Peng
L P
LP
Liu
A Q
AQ
Emili
C M
CM
Deber
D002462:Cell Membrane / Q000737:chemistry; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D013879:Thioredoxins / Q000096:biosynthesis / Q000235:genetics; D013917:Thrombin / Q000378:metabolism
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D013879:Thioredoxins; D013917:Thrombin