MUTATION OF POTENTIAL PHOSPHORYLATION SITES IN THE RECOMBINANT R-DOMAIN OF THE CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR HAS SIGNIFICANT EFFECTS ON DOMAIN CONFORMATION

Mutation of potential cAMP dependent protein kinase sites in the R dom ain of the cystic fibrosis transmembrane conductance regulator has sig nificant effects on protein function. Mutation of the potential phosph orylation sites from serine to alanine, to abolish the site, reduced s ensitivity to activation, or to glutamic acid, to mimic phosphorylatio n, caused some constitutive activity. To explore the structural effect s of these mutations, recombinant R domain peptides were studied: the wild type, a mutant with nine serine residues changed to alanine, and a mutant with eight serine residues changed to glutamic acid. As asses sed by C.D. spectroscopy, the mutants have substantially different sec ondary structure than the wild type, in agreement with the predictive algorithm of Gascuel and Golmard. The results show that mutagenesis of residues alters the polypeptide structurally as well as preventing it from serving as a phosphorylation substrate. Hence, the functional co nsequences of the mutations may not be entirely due to effects on phos phorylation. (C) 1995 Academic Press, Inc.