Stoichiometry of the ΔF508 CFTR interaction with core chaperones at physiological and corrective temperatures.

<p>Table depicting the absolute amounts of CFTR, Hsp90, Hsc70 and Hsp40, expressed in pmol. Also shown are the molar ratios of chaperones to total ΔF508-CFTR at both 37°C and 30°C. The fold change in the absolute amounts of CFTR, Hsp90 and Hsc70, expressed in pmol, relative to ΔF508-CFTR at 37°C is shown in the final column.</p

Structural mapping of the Interaction of NBD1 with Hsp90 using cross-linking.

<p><b>A</b>. Ribbon diagram of NBD1 depicting Hsp90 interacting peptides. <b>B–C</b> Ribbon diagram of ΔF508-NBD1 (<b>B</b>) and WT-NBD1 (<b>C</b>) with associated Hsp90 interacting peptides shown as electrostatic map. <b>D–E.</b> Ribbon diagram of Hsp90 with associated ΔF508-NBD1 (<b>D</b>) and WT-NBD1 (<b>E</b>) interacting peptides shown as electrostatic map. Data shown is conserved peptides from 3 independent experiments.</p

Stoichiometry of the WT and ΔF508 CFTR interaction with core chaperones.

<p>Table depicting the absolute amounts of CFTR, Hsp90 and Hsc70, expressed in pmol. Also shown are the molar ratios of chaperones to total ΔF508- or WT-CFTR. The fold change in the absolute amounts of CFTR, Hsp90 and Hsc70, expressed in pmol, relative to ΔF508-CFTR is shown in the final column.</p

Quantification of CFTR, Hsc70 and Hsp90 in CFTR-containing complexes.

<p><b>A.</b> Absolute abundance (ng/µl) of Hsp90 calculated by ¹⁵N protein labeling, AQUA labeling and Western blotting (WB). <b>B.</b> Absolute abundance (ng/µl) of Hsc70 calculated by ¹⁵N protein labeling, AQUA labeling and Western blotting (WB). In all panels, data is shown as mean ± SD, n≥3.</p

Minimal sequential ordering of intra- and inter-domain folding events responsible for CFTR folding and trafficking.

<p>Intra-domain folding of NBD1 is dictated by the Hsp90 system (step 1). A structural rearrangement occurs in response to the binding of cytoplasmic loop 4 (CL4) to the F508 containing hydrophobic pocket present WT NBD1 (step 2). The binding of CL4 provides a stabilizing effect on NBD1, releasing Hsp90 and promoting H8–H9 helix-coil transition. This H8–H9 transition would expose the NBD2-binding interface of NBD1 and allow NBD1 to ‘chaperone’ <i>in trans</i> the folding of NBD2 (step 3).</p

Quantification of ΔF508-CFTR interaction with core chaperones following temperature shift

<p>. <b>A.</b> Western blot analysis of HEK293 cells stably expressing ΔF508-CFTR cultured at 37°C or 30°C in the presence of 50 μM cyclohexamide (CHX) or vehicle control for the indicated time. <b>B.</b> Absolute quantification of ΔF508 CFTR and interacting chaperones at 37°C (black) or 30°C for 16 h (white). Absolute protein abundance of CFTR, Hsp90, Hsc70, and Hsp40 in CFTR-containing complexes is shown and expressed in pmols. <b>C.</b> Immunoblot and densitometric analysis for CFTR, Hsp90, Hsc/p70 and Hsp40 in CFTR-containing immunoprecipitates. In the densitometric analysis, the relative protein amount is shown in arbitrary units (a.u.). In all panels, data is shown as mean ± SD, n = 3 and asterisks represent p value <0.05 as determined by two-tailed t-test using the ΔF508-CFTR at 37°C sample as the reference.</p

Quantification of WT and ΔF508 CFTR interactions with core chaperones.

<p><b>A.</b> The absolute levels of CFTR, Hsp90 and Hsc70, expressed in pmol, in CFTR-containing complexes were determined using the absolute quantification strategy from HEK293 ΔF508-CFTR (white) and WT-CFTR (black) producing cells. <b>B.</b> Immunoblot and densitometric analysis for CFTR, Hsp90, Hsc/p70 and Hsp40 from CFTR-containing immunoprecipitates. A representative blot is shown. In the densitometric analysis, the relative protein amount is shown in arbitrary units (a.u.). In all panels, data is shown as mean ± SD, n = 3 and asterisks represent p value <0.05 as determined by two-tailed t-test using the WT sample as the reference.</p