Fig. 5: A monomeric proIGF2 client construct.
From: Mechanism of client loading from BiP to Grp94 and its disruption by select inhibitors
A ProIGF2 client fragments: E-peptide (tan), mIGF2 (red), and site 1 (purple). B SEC retention time versus molecular weight of standard proteins (black circles). Positions of a proIGF225-120 monomer, BiP, and Grp94 dimer are indicated in blue squares. C Upper panel shows retention profile of BiP and FITC-labeled proIGF225-120 (*ProIGF2) under ADP conditions as measured by absorption at 280 nm (blue) and 495 nm (green). Lower panel shows the same experiment under ATP conditions, as well as the retention of BiP in the absence of client (dashed blue line). D BiP SBD bulk FRET assay measuring SBD lid closure kinetics with and without client. Solid lines are a fit to a single exponential (closure rate with 5 μM proIGF225-120: 0.30 ± 0.01 min-1; 5 μM mIGF2: 0.06 ± 0.01 min-1; without client: 0.05 ± 0.01 min-1). Uncertainty on closure rates is the fitting error. Closure is initiated by first equilibrating BiP with 0.1 mM ATP and flushing in 1 mM of ADP with hexokinase and glucose (HK-ADP, see Methods) and client. Cartoon shows schematic of BiP conformations and fluorophore positions. Source data are provided as a Source Data file.
