Supplementary Figure 9: SpFRET experiments of BiP-Δlid.
From: Bap (Sil1) regulates the molecular chaperone BiP by coupling release of nucleotide and substrate
(a-c) SpFRET efficiency histograms were measured for the NBD-SBD BiP sensor (gray line), BiP-Δlid (black line), BiP-Δlid in the presence of 10 μM Bap (red line) or BiP-Δlid with 10 μM Bap-C (dark yellow line). Experiments were performed (a) in the absence of nucleotide (apo), (b) with 1 mM ATP or (c) 1 mM ADP included in the buffer. Sample preparation was performed by incubation of BiP (1 μM) with Bap, Bap-C and nucleotide at their final concentrations, which were kept constant during the dilution of BiP to spFRET concentrations (~20 pM). (d-f) SpFRET efficiency histograms for the SBD-lid BiP sensor (BiP 519 - 638), the NBD-SBD BiP sensor (BiP 167 - 519), and BiP- Δlid in 1 mM ADP without peptide or Bap (gray line), with 70 μM HTFPVAL (black line) and with 70 μM HTFPVAL and 10 μM Bap (red line)
