Investigation of a multi-ligand approach to increase thermal stability of NBD1-?F508 domain of CFTR
- WCU Author/Contributor (non-WCU co-authors, if there are any, appear on document)
- Christopher Shawn Robinson (Creator)
- Institution
- Western Carolina University (WCU )
- Web Site: http://library.wcu.edu/
- Advisor
- Robert T. Youker
Abstract: Cystic Fibrosis (CF) is the most common monogenic hereditary disease in Caucasians affecting greater than 70,000 persons worldwide. Primary symptoms of CF patients are electrolyte imbalance in epithelial cells creating thick mucus layers in the lung, pancreas, and digestive track which hinders their function. The thick mucus buildup leads to CF patients having severe lung infections that eventually shorten their lifespan by decades in the most severe cases. This disease is caused by mutations in the 1480 amino acid long Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein which regulates the chloride and bicarbonate transport across the apical membrane of epithelial cells. The most common mutation, ?F508 in the NBD1 domain of CFTR, results in the misfolding and aggregation during protein synthesis. Such aggregates are marked for degradation preventing the protein from trafficking to the plasma membrane.Previous screening studies have identified small corrector molecules (ligands) that can increase ?F508 CFTR’s thermal stability. These compounds enhance folding, or activity of the channel, and appear to act through binding to NBD1. Several of these compounds have been approved by the FDA to treat multiple CFTR mutations, including the ?F508 mutation. However, the mechanism of action for these compounds is ill-defined, and there is scant information on the combinatorial effects of these compounds on the thermal stability of NBD1-?F508.Purified ?F508-NBD1 thermal stability was measured using Differential Scanning Fluorimetry (DSF) in the absence, or presence of several small molecules (BIA, Oleuropein, Hydroxy-Tyrosol) alone, or in combination. The thermal stability of ?F508-NBD1 was increased in the presence of 1 - 5 mM ATP, as previously reported. Interestingly, lower concentrations of ATP (1.5mM) combined with 1 mM BIA had a similar effect on the stability of NBD1-?F508 as the higher concentration of 5 mM ATP alone. There was no change observed in the stability of NBD1-?F508 in the present of Oleuropein or Hydroxy-Tyrosol. Molecular dynamic simulations were performed to provide insight into the results obtained from the DSF experiments.
Investigation of a multi-ligand approach to increase thermal stability of NBD1-?F508 domain of CFTR
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Created on 11/7/2019
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Additional Information
- Publication
- Thesis
- Language: English
- Date: 2019
- Keywords
- CFTR, DSF, Ligand, NBD1
- Subjects
- Cystic fibrosis gene
- Cystic fibrosis -- Genetic aspects
- Protein folding
- Fluorimetry
- Ligands (Biochemistry)