Molecular dissection of the CFTR gating cycle, combining mathematical tools with experimental biophysics. (360G-Wellcome-081298_Z_06_Z)

CFTR, whose dysfunction causes the disease cystic fibrosis, belongs to the superfamily of ABC proteins, which couple hydrolytic cycles at conserved nucleotide-binding domains (NBDs) to diverse cellular functions. CFTR is unique among ABC proteins in that its transmembrane domains comprise an ion channel. Opening and closing (gating) of the ion-permeation pathway is controlled by ATP binding and hydrolysis at its NBDs. But how are conformational signals, originating at the catalytic site, transmi tted to the channel gate? In alignments of homologous sequences one can discern sets of positions at which amino acid distribution has varied in a concerted way. This correlation in evolutionary space likely reflects conservation of a network of energetically coupled residues that mediates transmission of allosteric signals in individual ABC proteins. We will select coevolving positions as targets for mutagenesis and, using new mathematical tools to analyse single channel activity, we will foll ow how the energetic coupling between pairs of side-chains changes during the gating cycle. Together, these studies will compose a molecular description - in both space (within the protein s structure), and time (along the reaction coordinate of CFTR s functional cycle) of the allosteric coupling mechanism that links CFTR s hydrolytic and gating cycles.