Integrated structural biology provides new clues for cysticfibrosis treatment nature
). However, establishment of this model revealed notable quantitative discrepancies that are worthy of consideration. First, our simulation predicts a modestly greater steady-state ATP hydrolysis rate than is experimentally estimated. We speculate that this may reflect either inadequacies of the simplified model or the presence of an inactive fraction in the bulk measurement that lowers the apparent turnover rate.
. Human CFTR with a C-terminal PreScission Protease-cleavable GFP tag was cloned into the BacMam vector. For single-molecule FRET, the following substitutions were introduced: C76L, C128S, C225S, C276S, C343S, T388C, C491S, C592M, C647S, C832S, C866S, C1344S, C1355S, C1395S, C1400S, C1410S, S1435C and C1458S. A deca-His tag was inserted C-terminally to CFTR and before the PreScission Protease cleavage site to allow for surface immobilization.
Recombinant baculovirus was generated using Sf9 cells cultured in sf-900 SFM medium , supplemented with 5% heat-inactivated fetal bovine serum and 1% antibiotic–antimycotic as described previously suspension cells were cultured in FreeStyle 293 medium supplemented with 2% heat-inactivated fetal bovine serum and 1% antibiotic–antimycotic , shaking at 37 °C with 8% COcells were authenticated by Gibco and ATCC, respectively and confirmed negative for mycoplasma contamination.
For protein purification, cells were solubilized for 75 min at 4 °C in extraction buffer containing 1.25% lauryl maltose neopentyl glycol , 0.25% cholesteryl hemisuccinate , 200 mM NaCl, 20 mM HEPES , 2 mM MgCl
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