Supplementary Figure 3: Titration measurements to calculate the K d values.
From: Bap (Sil1) regulates the molecular chaperone BiP by coupling release of nucleotide and substrate
Analytical ultracentrifugation (AUC) measurements were performed in the presence of 1 mM nucleotide to detect the increase in size of the BiP-Bap complex depending on the Bap or Bap-C concentration. The presence of the Bip-Bap complex can be observed by a shift in the sedimentation coefficient with increasing Bap concentrations. (a-c) Titration performed with 1 mM AMP-PNP. (a) AUC sedimentation velocity (AUC-SV) curves for 0.4 μM ATTO 488-labeled BiP-167-638 (final concentration) titrated with different Bap concentrations. (b) AUC-SV curves for 0.4 μM ATTO 488-labeled BiP-167-638 (final concentration) titrated with different concentration of Bap-C. (c) AUC-SV curves for 0.5 μM ATTO 488-labelled BiP-NBD-167 (final concentration) titrated with Bap. The titrations are shown in Figure 2b. (d-g) AUC-SV curves for 0.4 μM ATTO 488-labeled BiP-167-638 (final concentration) titrated with different concentration of Bap (d, e) without nucleotide or (f, g) in presence of 1 mM ADP. (h-j) Interaction of BiP’s isolated NBD with Bap. Sedimentation velocity (SV) was determined by analytical ultracentrifugation (AUC). 0.5 μM Atto 488-labeled BiP NBD-167 was tested without Bap (black line) and in the presence of 5 μM Bap (red line). Measurements were performed (h) in the absence of a nucleotide (apo), (i) with 1 mM ATP or (j) with 1 mM ADP.
