A recombinant polypeptide model of the second predicted nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator is a GTP-binding protein

FEBS Letters
C RandakW Machleidt

Abstract

Association reactions of a recombinant CFTR-NBF-2 polypeptide fused to glutathione S-transferase with guanine nucleotides were monitored quantitatively by recording the fluorescence enhancement of excited trinitrophenol (TNP)-labelled GTP after binding to NBF-2. Binding of TNP-GTP to the recombinant NBF-2 polypeptide was characterized by a Kd value of 3.9 microM. The corrected Kd values for unlabelled guanine nucleotides were determined to be 33 microM for GTP, 92 microM for GDP and 217 microM for GMP. TNP-ATP bound to NBF-2 was competitively displaced by GTP indicating a common binding site for both nucleotides. The recombinant NBF-2 did not show an intrinsic GTPase activity above a detection limit of 0.007 min(-1). Our findings provide the first experimental evidence that NBF-2 can act as a GTP-binding subunit that would favor the release of GDP after GTP hydrolysis.

References

Nov 15, 1992·Proceedings of the National Academy of Sciences of the United States of America·E M SchwiebertB A Stanton
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
Aug 16, 2003·European Journal of Biochemistry·Toshiaki Hiratsuka
Apr 29, 1998·Annual Review of Physiology·J K Foskett
Apr 20, 2002·Biophysical Journal·Aneta KirilenkoJoanna Bandorowicz-Pikula

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