A recombinant polypeptide model of the second predicted nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator is a GTP‐binding protein
- 25 November 1996
- journal article
- Published by Wiley in FEBS Letters
- Vol. 398 (1) , 97-100
- https://doi.org/10.1016/s0014-5793(96)01217-3
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 K d value of 3.9 μM. The corrected K d values for unlabelled guanine nucleotides were determined to be 33 μM for GTP, 92 μM for GDP and 217 μM 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.Keywords
This publication has 23 references indexed in Scilit:
- A recombinant peptide model of the first nucleotide‐binding fold of the cystic fibrosis transmembrane conductance regulator: Comparison of wild‐type and ΔF508 mutant formsProtein Science, 1996
- Sequence homologies between nucleotide binding regions of CFTR and G-proteins suggest structural and functional similaritiesFEBS Letters, 1995
- Expression and functional properties of the second predicted nucleotide binding fold of the cystic fibrosis transmembrane conductance regulator fused toFEBS Letters, 1995
- GTPase mechanism of Gproteins from the 1.7-Å crystal structure of transducin α - GDP AIF−4Nature, 1994
- Cystic fibrosis transmembrane conductance regulator mutations that disrupt nucleotide binding.Journal of Clinical Investigation, 1994
- Regulation by ATP and ADP of CFTR Chloride Channels That Contain Mutant Nucleotide-Binding DomainsScience, 1992
- Demonstration That CFTR Is a Chloride Channel by Alteration of Its Anion SelectivityScience, 1991
- The GTPase superfamily: conserved structure and molecular mechanismNature, 1991
- Identification of the Cystic Fibrosis Gene: Cloning and Characterization of Complementary DNAScience, 1989
- Biological and biochemical properties of human rasH genes mutated at codon 61Cell, 1986