PURIFICATION, CHARACTERIZATION, AND EXPRESSION OF CFTR NUCLEOTIDE-BINDING DOMAINS

The nucleotide binding domains (NBDs) within CFTR were initially predi cted to lie in the cell cytoplasm, and to gate anion permeability thro ugh a pore that was present in membrane spanning a helices of the over all polypeptide. Our studies designed to characterize CFTR suggest sev eral important features of the isolated nucleotide binding domain. NBD -1 appears to bind nucleotides with similar affinity to the full-lengt h CFTR protein. In solution, the domain contains a high beta sheet con tent and self-associates into ordered polymers with molecular mass gre ater than 300,000 Daltons. The domain is very lipophilic, disrupts lip osomes, and readily enters the planar lipid bilayer. Clinically import ant mutations in the domain may disrupt the nucleotide binding capabil ities of the protein, either through a direct effect on the nucleotide binding site, or through effects that influence the overall folding o f the domain in vitro. Finally, after expression in human epithelial c ells (including epithelial cells from a CF patient), the first nucleot ide binding domain targets the plasma membrane even in the absence of other constituents of full-length CFTR and mediates anion permeability in these cells.