Extended Data Fig. 8: Characterization of cystic fibrosis-associated variants.

a. Positions of cystic fibrosis-causing missense mutations. Mutated sites are shown as spheres. b–c. Representative smFRET traces of ATP delivery to and withdrawal from phosphorylated G551D (b) and L927P (c) CFTR_FRET (at the vertical dashed lines). Horizontal dashed lines indicate mean FRET efficiencies of the low and high FRET states. ATP concentration was 3 mM. d. Contour plots showing time-dependent changes in FRET after application and withdrawal of 3 mM ATP (at the dashed lines) to and from phosphorylated G551D and L927P variant CFTR_FRET. e. Dwell time distributions of the NBD-dimerized state for phosphorylated wild-type, L927P, and G551D CFTR_FRET at 3 mM ATP. Data represent means and standard errors for 8 (wild-type) or 3 (G551D and L927P) experiments. f. FRET responses of G551D and L927P variants to ATP withdrawal (3 mM ATP to nucleotide-free at the dashed line). Relaxation to low FRET was fit with monoexponential functions (solid lines). g. Open dwell time distribution of L927P variant CFTR. The distribution was fit with a monoexponential function (orange line). The dashed line is a monoexponential fit of the open dwell time distribution of wild-type CFTR. h. Mean open dwell times for wild-type and L927P variant CFTR. Whiskers represent minima and maxima and boxes represent 25^th, 50^th, and 75^th percentiles for 13 (wild-type) or 13 (L927P) bilayer recordings. Statistical significance was tested using two-tailed Student’s t-test (****p = 2 x 10⁻⁸). i. ATP dose-responses of mean FRET for G551D and L927P variants. Responses were fitted using the Hill equation with EC₅₀ values of 37 ± 7 µM for G551D and 52 ± 18 µM for L927P. j-k. Contour plots of phosphorylated G551D (j) and L927P (k) CFTR_FRET at the indicated ATP concentrations.