Stability Analysis of CFTR via Tryptic Digestion

Abstract

Cystic fibrosis (CF) is caused by malfunctioning cystic fibrosis transmembrane conductance regulator (CFTR). When the flow of chloride ions through the CFTR channel becomes hindered or ceases, mucus begins to accumulate within the body causing a plethora of respiratory, digestive, and reproductive complications. From the 1,500 different CFTR mutations that exist, a genetic mutation resulting from the deletion of a phenylalanine residue at the 508 position (ΔF508) has been identified as the most common. For that reason, the ΔF508 mutation has become a target for initial treatment options. Due to the transmembrane nature of CFTR and a length of 1480 residues, the crystal structure and consequently the functionality of CFTR, are still vastly unknown. The objective of this research is to more thoroughly understand the relative stability of CFTR, the ΔF508 mutant and the treated ΔF508 mutant via limited tryptic digestion and the quantification of western blots. The ΔF508 mutant was treated with an energy source in which it typically binds (ATP), a non-hydrolysable ATP analogue (AMP-PNP), and with an available cystic fibrosis treatment option (lumacaftor/VX-809). To adequately represent both of the nucleotide binding domains (NBDs) in CFTR, antibodies 660 (IGg1) and 769 (IGg2b) were used. The quantification of results indicated that the induced stability of the AMP-PNP treated and ATP treated ΔF508 CFTR were significantly greater than the ΔF508 mutant alone, but not significantly greater than the VX-809 treated CFTR.