Structural biology of Cystic Fibrosis Transmembrane Conductance Regulator, an ATP-binding cassette protein of medical importance

UoM administered thesis: Unknown

  • Authors:
  • Ateeq Ahmed Hassan Alzahrani

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is a transmembrane protein that functions as an ion channel. Mutations in this protein cause Cystic Fibrosis. For this reason, it is important to study the structure and function of CFTR. In this study, constructs of CFTR (C-terminii), a CFTR-interacting protein and full-length CFTR were cloned, expressed and purified for structural and functional studies. The purified C-terminal polypeptides of CFTR were soluble and shown to interact with NHERF1 PDZ 1 (a CFTR-interacting protein). The CFTR C-terminus and NHERF1 PDZ 1 domain were co-expressed and co-purified. The purified complex showed a strong interaction that might induces a conformational change. Site-directed mutation of the C-terminus of CFTR was performed in order to examine the effect of removing a potentially flexible amino acid (Arginine) on protein crystallization. Pull-down assay experiments with full-length CFTR demonstrated an interaction between CFTR (in DDM detergent) and NHERF1 PDZ 1(+). No interaction was observed for CFTR in LPG (a relatively denaturing detergent) and NHERF1, implying that the interaction between the PDZ motive of CFTR and NHERF1 requires a stable folded structure for both proteins. In addition, full-length CFTR in DDM has been studied by electron microscopy and Single Particle Analysis in the presence of NHERF1 PDZ 1(+). A 3D structure was generated for the CFTR-NHERF1 PDZ 1(+) complex at a resolution of ~ 18 A. This 3D structure showed a new open conformation of CFTR (V shape). In comparable studies with CFTR alone, a 3D structure was generated at a resolution of 27 A and this structure showed a closed state as previously reported. This new data suggest a possible role for NHERF1 in terms of CFTR channel gating or activation.

Details

Original languageEnglish
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Award date31 Dec 2012