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Updated: Feb 14, 2026
09:59
Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein
Published on: March 9, 2015
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Structural stability of purified human CFTR is systematically improved by mutations in nucleotide binding domain 1
Zhengrong Yang1, Ellen Hildebrandt2, Fan Jiang3
1Department of Chemistry, University of Alabama at Birmingham, Birmingham, AL, USA.
Biochimica Et Biophysica Acta. Biomembranes
|February 10, 2018
Summary
Researchers engineered stable Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein variants by introducing specific mutations in its nucleotide-binding domains. These stabilized CFTR proteins retain essential functions, advancing cystic fibrosis drug development.
Area of Science:
- Biochemistry
- Structural Biology
- Molecular Biophysics
Background:
- The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a crucial chloride ion channel protein.
- Obtaining stable, monodisperse CFTR for research has been a significant challenge.
- CFTR comprises transmembrane and nucleotide-binding domains (NBD1 and NBD2).
Purpose of the Study:
- To investigate the impact of NBD1 mutations on the stability of both the NBD1 domain and full-length CFTR.
- To develop strategies for enhancing CFTR protein stability for biophysical and structural studies.
- To assess the functional consequences of stabilized CFTR variants.
Main Methods:
- Systematic introduction of single and multiple mutations into the NBD1 domain of CFTR.
- Calorimetric analysis (Tm) to determine protein thermal stability.
- Replica exchange molecular dynamics simulations to understand stability mechanisms.
- Functional assays (ATPase, channel activity) and cell surface expression studies.
Main Results:
- Combinations of NBD1 mutations (e.g., 6SS-NBD1) significantly increased NBD1 domain stability (Tm > 70°C).
- Stabilized NBD1 correlated with enhanced full-length CFTR structural stability (Tm up to 67.4°C).
- An NBD2 mutation (H1402S) further improved CFTR thermal stability.
- Stabilized CFTR variants showed normal cell surface expression, ATPase, and channel activity at elevated temperatures.
Conclusions:
- Strategic NBD1 mutations can dramatically enhance CFTR protein stability.
- Improved CFTR stability facilitates biophysical and structural investigations.
- These findings are crucial for advancing mechanistic understanding and drug development for cystic fibrosis.
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