Helix-helix interactions between transmembrane α-helices 3 and 4 within the cystic fibrosis transmembrane conductance regulator protein

Abstract

Cystic fibrosis (CF) is an autosomal recessive disease for which the associated defective protein is the cystic fibrosis transmembrane conductance regulator (CFTR) [1]. While the three dimensional structure of CFTR is currently unknown, its main role has been shown to act as a chloride ion channel at the apical membrane of epithelial cells [2]. Amongst the many mutations that cause CF, some lie within the transmembrane (TM) which are thought to constitute the bulk of the chloride ion pore. In order to understand how these mutations result in CF disease, it is necessary to understand, at the molecular level, how the individual TM pack together within the TM domain. This information could be obtained by determining which helices have affinity for each other. We are currently undertaking this task by synthesizing the individual TM helices and using fluorescence resonance energy transfer (FRET) to detect helix–helix interactions within membrane–mimetic environments. Thus far, we have synthesized peptides TM-3 ( K K K M G L A L A H F V W I A P L Q V A L L M G L I W G K K K ) and T M – 4 (KKKLQASAFCGLGFLIVLALFQAGLGRMKKK). The two native tryptophans (Trp) in TM–3 can be used as FRET donors. During synthesis, an AEDANS group was added to the N–terminus of TM–4 to act as a FRET acceptor. Initial FRET results suggest that TM-3 and TM–4 have a helix–helix interaction.