EXPRESSION AND FUNCTIONAL-PROPERTIES OF THE 2ND PREDICTED NUCLEOTIDE-BINDING FOLD OF THE CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR FUSED TO GLUTATHIONE-S-TRANSFERASE

CFTR-NBF-2 was expressed in Escherichia coli in fusion with glutathione-S-transferase, the soluble portion was purified and identified as a stuctured protein by its CD spectrum. Association reactions of the recombinant NBF-2 with adenine nucleotides were monitored qualitatively by demonstrating its ability to bind specifically to ATP-, ADP- and AMP-affinity agarose and quantitatively by recording the fluorescence enhancement of excited trinitrophenol (TNP)-labelled adenine nucleotides occuring as a result of binding to NBF-2. Best-fit monophasic binding curves to the fluorescence data indicated K-d values of 22 mu M for TNP-ATP, 39 mu M for TNP- ADP and 2.1 mu M for TNP-AMP. The corrected K-d values for unlabelled adenine nucleotides competing with the fluorophores mere determined to be 37 mu M for ATP, 92 mu M for ADP and 12 mu M for AMP. The recombinant NBF-2 did not show any hydrolytic activity on ATP (detection limit 0.001 s(-1)). Our findings support the concept of a central role of NBF-2 in CFTR activity regulation acting as an allosteric switch between channel opening and dosing and give the first experimental evidence that the channel inhibitor AMP could act via NBF-2.