LOCALIZATION AND SUPPRESSION OF A KINETIC DEFECT IN CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR FOLDING

A growing body of evidence indicates that the most common cystic fibro sis-causing mutation, Delta F508, alters the ability of the cystic fib rosis transmembrane conductance regulator (CFTR) protein to fold and t ransit to the plasma membrane, Here we present evidence that the Delta F508 mutation affects a step on the folding pathway prior to formatio n of the ATP binding site in the nucleotide binding domain (NBD), Nota bly, stabilization of the native state with 4 mM ATP does not alter th e temperature-dependent folding yield of the mutant Delta F508 NBD1 in vitro. In contrast, glycerol, which promotes Delta F508-CFTR maturati on in vivo, increases the folding yield of NBD1 Delta F and reduces th e off pathway rate in vitro, although it does not significantly alter the free energy of stability, Likewise a second site mutation, R553M, which corrects the maturation defect in vivo, is a superfolder which c ounters the effects of Delta F508 on the temperature-dependent folding yield in vitro, but does not significantly alter the free energy of s tability, A disease-causing mutation, G551D, which does not alter the maturation of CFTR in vivo but rather its function as a chloride chann el, and the S549R maturation mutation have no discernible effect on th e folding of the domain, These results demonstrate that Delta F508 is a kinetic folding mutation that affects a step early in the process, a nd that there is a significant energy barrier between the native state and the step affected by the mutation precluding the use of native st ate ligands to promote folding, The implications for protein folding i n general are that the primary sequence may not necessarily simply def ine the most stable native structure, but rather a stable structure th at is kinetically accessible.