Expression and functional properties of the second predicted nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator fused to

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Abstract
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 μM for TNP-ATP, 39 μM for TNP- ADP and 2.1 μM for TNP-AMP. The corrected K values for unlabelled adenine nucleotides competing with the fluorophores were determined to be 37 μM for ATP, 92 μM for ADP and 12 μM for AMP. The recombinant NBF-2 did not show any hydrolytic activity on ATP (detection limit 0.001 s'). 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 closing and give the first experimental evidence that the channel inhibitor AMP could act via NBF-2.