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Updated: May 20, 2026
09:59
Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein
Published on: March 9, 2015
A potentiator induces conformational changes on the recombinant CFTR nucleotide binding domains in solution
Elena Galfrè1, Lauretta Galeno, Oscar Moran
1Istituto di Biofisica, Consiglio Nazionale delle Ricerche, Via De Marini, 6, 16149 Genoa, Italy.
Cellular and Molecular Life Sciences : CMLS
|July 4, 2012
Summary
The drug 2-pyrimidin-7,8-benzoflavone (PBF) alters the structure of nucleotide binding domains (NBD1/NBD2) in cystic fibrosis transmembrane conductance regulator (CFTR). This conformational change, particularly in the presence of ATP, may facilitate the channel's open state.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein.
- The nucleotide binding domains (NBD1 and NBD2) of CFTR control chloride channel gating and are drug targets.
- 2-pyrimidin-7,8-benzoflavone (PBF) is a known potentiator of CFTR function.
Purpose of the Study:
- To investigate the effects of PBF on the conformational properties of CFTR's NBD1/NBD2 mixture.
- To understand how PBF influences ATP binding and hydrolysis in NBD1/NBD2.
- To elucidate the structural impact of PBF on NBD1/NBD2, with and without ATP.
Main Methods:
- Studied recombinant NBD1/NBD2 mixture in solution.
- Utilized intrinsic fluorescence and guanidinium denaturation assays.
- Performed small-angle X-ray scattering (SAXS) experiments.
Main Results:
- PBF induced significant conformational changes in the NBD1/NBD2 dimer.
- PBF did not alter ATP binding but reduced ATP hydrolysis activity.
- SAXS revealed PBF disrupted the dimer without ATP, but maintained dimerisation with 2 mM ATP, altering structure size and shape.
Conclusions:
- PBF binding induces distinct conformational changes in NBD1/NBD2, dependent on ATP presence.
- These structural modifications may enhance CFTR channel opening by altering NBDs-intracellular loop interactions.
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