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

FEBS Letters
C RandakW Machleidt

Abstract

CFTR-NBF-2 was expressed in Escherichia coli in fusion with glutathione-S-transferase, the soluble portion was purified and identified as a structured 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 occurring as a result of binding to NBF-2. Best-fit monophasic binding curves to the fluorescence data indicated Kd values of 22 microM for TNP-ATP, 39 microM for TNP- ADP and 2.1 microM for TNP-AMP. The corrected Kd values for unlabelled adenine nucleotides competing with the fluorophores were determined to be 37 microM for ATP, 92 microM for ADP and 12 microM 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 closing and give the first experimental evidence that the channel inhibitor AMP could act via NBF-2.

References

Jan 1, 1992·The Journal of Clinical Investigation·G M DenningM J Welsh
Jan 1, 1992·The American Journal of Physiology·C E Bear, E F Reyes
Mar 1, 1992·Proceedings of the National Academy of Sciences of the United States of America·N ArispeH B Pollard
Jan 1, 1988·Methods in Enzymology·J G Nørby
Jan 6, 1981·Biochemistry·S W Provencher, J Glöckner
Jul 1, 1994·The Journal of Clinical Investigation·J LoganE J Sorscher
Nov 1, 1993·Proceedings of the National Academy of Sciences of the United States of America·L S SmitD C Dawson

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Citations

Oct 6, 2006·Pflügers Archiv : European journal of physiology·Andrei A AleksandrovJohn R Riordan
Oct 30, 2007·Journal of Bioenergetics and Biomembranes·Christoph O Randak, Michael J Welsh
Apr 29, 1998·Annual Review of Physiology·J K Foskett
Feb 12, 2014·The International Journal of Biochemistry & Cell Biology·Oscar Moran
Aug 16, 2005·The Journal of Biological Chemistry·Christoph O Randak, Michael J Welsh

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