(669) Conformational Dynamics of CFTR Revealed by Single-Molecule FRET

Background: Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is an anion transport membrane protein that plays a vital role in fluid homeostasis in epithelial tissues. Mutations in the CFTR gene cause defective CFTR protein, which leads to compromised anion conductance across the apical membrane of secretory epithelia resulting a life threating genetic disorder, cystic fibrosis (CF). The topology of CFTR shows two transmembrane domains (TMs), two intracellular nucleotide binding domains (NBDs) and an unstructured regulatory domain (R). CFTR channel activity is initiated by phosphorylation of the R-domain, following which, channel gating is controlled by dimerization of NBDs once bound to ATP. Experimental evidence suggests that induced conformational changes during NBD dimerization are transmitted through the intracellular loops of CFTR towards the transmembrane region, resulting in the opening of the channel pore. As a channel protein, CFTR undergoes dynamic global and local conformational changes during the gating cycle. The major conformational changes upon ATP binding/hydrolysis to NBDs and phosphorylation of the “R” domain regulate the CFTR gating mechanism in a complex manner. Presumably, these structural dynamics are altered in mutant CFTR. Our objective is to apply a Förster Resonance Energy Transfer (FRET) based method to study CFTR conformational dynamics during the gating cycle at a single molecule level in real-time.

Methods: Single-molecule FRET (smFRET) is a well-established biophysical technique for studying macromolecular conformations and real-time dynamics. To conduct smFRET experiments, several two-cysteine CFTR constructs (2C-CFTR) were generated and labeled with a FRET-pair (Alexa fluor 555-donor) and Alexa fluor 647-acceptor) in the cys-less CFTR background. HEK 293 cells were transfected with 2C-CFTR, and the protein was overexpressed, confirmed to be functional, and purified using affinity chromatography. The labeled 2C-CFTR was incorporated into liposomes (Figure 1) and immobilized on a functionalized quartz slide surface for single-molecule imaging. Total Internal Reflection Fluorescence (TIRF) microscope was used to accomplish fluorescent detection at the single-molecule level. smFRET experiments are conducted in a microfluidic chamber made on a quartz slide.

Results: The conformational dynamics of “R” domain were identified with respect to NBD1 and NBD2 during phosphorylation and ATP binding/hydrolyzing of CFTR.

Conclusions: We have successfully applied the smFRET technique to study CFTR conformational dynamics, which can be attributed to understand the relative local and global structural movements of NBDs and R-domain during the gating cycle.

Acknowledgements: Supported by Cystic Fibrosis Foundation (LIYANA19FO)

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