A functional R domain from cystic fibrosis transmembrane conductance regulator is predominantly unstructured in solution

Phosphorylation of the regulatory (R) domain initiates cystic fibrosis tran smembrane conductance regulator (CFTR) Cl- channel activity. To discover ho w the function of this domain is determined by its structure, we produced a n R domain protein (R8) that spanned residues 708-831 of CFTR. Phosphorylat ed, but not unphosphorylated, R8 stimulated activity of CFTR channels lacki ng this domain, indicating that Rs is functional. Unexpectedly, this functi onal R8 was predominantly random coil, as revealed by CD and limited proteo lysis. The Co spectra of both phosphorylated and nonphosphorylated R8 were similar in aqueous buffer. The folding agent trimethylamine N-oxide induced only a small increase in the helical content of nonphosphorylated R8 and e ven less change in the helical content of phosphorylated R8. These data, in dicating that the R domain is predominantly random coil, may explain the se emingly complex way in which phosphorylation regulates CFTR channel activit y.