A BioLayer interferometry (BLI) derived association and dissociation traces of monomeric FICDL258D-H363A (mFICDH363A) or dimeric FICDH363A (dFICDH363A) from immobilised biotinylated BiPT229A-V461F in absence or presence of nucleotides. Unless indicated (*) BiP was saturated with ATP before exposure to FICD variants. A representative experiment of three independent repetitions is shown. B BLI dissociation traces of proteins as in (A). At t = 0 a pre-assembled complex of immobilised, ATP-saturated BiP and the indicated FICD proteins (associated without ATP) were transferred into a solution without or with ATP, as indicated. A representative experiment is shown and the biphasic dissociation kinetics are quantified in (C) and (D). C Graph of the slow dissociation rates (koff,slow) of monomeric FICD from BiP:ATP as shown in (B). Bars represent mean values ± SD of three independent experiments. D The ATP-induced fold change in the percentage of the dissociation phase attributed to a fast dissociation (%Fast), koff,fast, and koff,slow derived from the data represented in (B). Bars represent mean values ± SD of three independent experiments. 