The Cystic fibrosis transmembrane conductance regulator (CFTR) is the membrane protein product of the gene known to be defective in cystic fibrosis patients. CFTR functions by transporting chloride ions through a transmembrane (TM) domain believed to consist of two sets of six tightly packed and assembled membrane spanning helical segments (TM1-6, TM7- 12, ca, 20 amino acids each). However, channel remain to be deduced in molecular terms. (1) Since packing of TM domains appears to be directed by specific recognition elements contained within the individual TM segments, and the N-terminal portion of CFTR (TM1-6) is known to form a functional channel, we propose to prepare a series of peptides based on the native sequences of the CFTR TM domain segments 1-6 by solid-phase synthesis, and to determine the affinities of the individual TM segments for each other. Individual TM segments will be combined in combinations (e.g., TM5-6, TM6-1, TM1-2, etc.).Affinities would be quantitated by fluorescence energy transfer, circular dichroism spectroscopy, and gel electrophoresis. These experiments will be supported by molecular modeling to identify favorably- packed helical dimers of CFTR helices, and by channel conductance measurements performed on CFTR TM peptides individually and in combinations. Pairs found to have affinity of dimerization would likely be in direct 3-D contact in the native channel. (2) An increasing number o missense mutations within the CFTR TM domain are now being associated with CF disease. We reason that certain 'high-information' residues impair channel function by interfering with helix-helix packing. Peptides prioritized to key TM-domain mutations observed in genotyped CF patients from the in-house database at the Hospital for Sick Children (e.g., G85E, R347H, R347W) ill be prepared and studied as above in combinations with wild type partners. From the combined information thus obtained, we will construct a working model for the molecular geometry of the CFTR channel, and use the epidemiological data to generate hypotheses concerning possible relationships between severity of CF disease and molecular defects in the corresponding mutant CFTR's.
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