Cystic fibrosis transmembrane conductance regulator: Solution structures of peptides based on the Phe508 region, the most common site of disease-causing Delta F508 mutation

Most cases of cystic fibrosis (CF), a common inherited disease of epithelia l cell origin, are caused by the deletion of Phe508 located in the first nu cleotide-binding domain (NBF1) of the protein called CFTR (cystic fibrosis transmembrane conductance regulator). To gain greater insight into the stru cture within the Phe508 region of the wild-type protein and the change in s tructure that occurs when this residue is deleted, we conducted nuclear mag netic resonance (NMR) studies on representative synthetic 26 and 25 amino a cid peptide segments. 2D H-1 NMR studies at 600 MHz of the 26-residue pepti de consisting of Met498 to Ala523 in 10% DMSO, pH 4.0, at 25 degrees C show a continuous but labile helix from Gly500 to Lys522, based on both NH-NH(i ,i+1) and alpha H-NH(i,i+1) NOEs. Phe508 within this helix shows only short -range (i, less than or equal to i + 2) NOEs. The corresponding 25-residue peptide lacking Phe508 also forms a labile helix from Gly500 to Lys522. How ever, the relative intensities of the NH-NH(i,i+1)/alpha H-NH(i,i+1) NOEs, fewer intermediate-range NOEs, and downfield alpha H and NH chemical shifts indicate a lower helical propensity of the 25-mer between residues 505 and 517, surrounding the missing residue, Phe508. 2D H-1 NMR studies of both p eptides in saturating (43%) TFE reveal stable a-helices from Gly500 to Lys5 22, based on NH-NH(i,i+1,2,3), alpha H-NH(i,i+2,3,4), alpha H-beta H-(i,H-i +3), and weak alpha H-NH(i,i+1) NOEs. However, downfield shifts of the alph a H resonances from residues Gly500 to Ile507 and fewer intermediate-range NOEs suggest a less stable ex-helix in the 25-mer even in saturating TFE. T hese findings show that the Phe508 containing region of CFTR has a propensi ty to form an ct-helix, which is destabilized by the Delta F508 mutation fo und in most patients with CF, These studies have direct relevance to better understanding the CFTR misfolding problem associated with CF and to identi fying chemical agents, which correct this problem.