Cystic fibrosis (CF) is a genetic disease caused by mutations in the DNA coding for a chloride ion channel, the CF transmembrane conductance regulator (CFTR). Comparison of the three-dimensional structures of wild-type and mutant CFTR is crucial for an understanding of the molecular basis of the pathology of CF. A realistic approach to a structural understanding of this large membrane protein is to build up the structure of CFTR from the individual domains. The structure of soluble intracellular domains can be determined using solution NMR techniques. Isolated fragments of the N-terminal nucleotide binding fold (NBF1) and the regulatory (R) domains will be the focus of the pilot project. As preliminary data support, some NBF1 and R domain constructs are expressed as insoluble aggregates in bacteria and cannot be resolubilized in high yields or are susceptible to proteolysis. Our research will first identify suitable regions of NBF1 and R domain that allow soluble and stable expression. Since milligram quantities of isotopically enriched protein is required for many NMR studies, overexpression in bacterial systems will be utilized. A variety of constructs and culture will be explored. NBF1 regions to be tested range form short peptides that encompass the site of the most common deltaF508 mutation in the NBF1 (mimicking the previously studied synthetic P-67 peptide) up to 180 residues which covers the full region of sequence identity for the N-terminal ATP-binding domain among members of the ATP- binding cassette membrane protein family to which CFTR belongs. Constructs with N- and C-terminal flanking charged and polar residues added to aid in solubilization, such as Lys-Ser repeats, will also be investigated. R domain regions will include the full 240 residue protein product of exon 13, as well as the N-terminal 100 residues and the C- terminal 140 residues which may represent independent folding units. Plasmid expression vectors will include the simple pET system, as well as fusion proteins to aid in solubilization and purification, such as the pGEX glutathione-S-transferase system, the FLAG secretion system, and the histidine tag approach which enables nickel affinity column purification. Expression of protein will be induced at lower temperatures, from 30degreesC to 5degreesC, with the aim of reducing the formation of insoluble aggregates. Once milligram amounts of purified, soluble material are obtained, multidimensional multinuclear NMR experiments will be utilized to confirm the feasibility of NMR structural studies and to then assign the resonances of the domains. Detailed atomic level structures of both wild type and mutant fragments of NBF1 and of the R domain will be the ultimate goal of these studies.
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