Supplementary Figure 6: Representative PIE-MFD analysis shown for mutant BiP (167-638) in the presence of 1 mM ADP and 10 μM Bap-N.

(a) A 2D histogram of stoichiometry versus FRET efficiency after filtering for double-labeled molecules. A stoichiometry of ~0.5 indicates a ratio of 1:1 labeling between donor and acceptor fluorophores. (b) A 2D histogram of FRET efficiency versus donor lifetime \({\tau }_{D(A)}\). The relationship between FRET efficiency and donor lifetime for a static population (the static FRET line) is shown in black. No deviation from the static FRET line is observed indicating an absence of conformational dynamics during the ~1 ms long observation time. (c,d) 2D histograms of the burstwise anisotropy of (c) the donor \({r}_{D}\) versus the fluorescence lifetime of the donor \({\tau }_{D(A)}\) and (d) the acceptor \({r}_{A}\) versus the fluorescence lifetime of the acceptor fluorophore \({\tau }_{A}\). The black lines are given by the Perrin equation \(r={r}_{0}/\left(1+\frac{\tau }{\rho }\right)\), where \({r}_{0}\) is the fundamental anisotropy, \(\tau \) is the fluorescence lifetime and \(\rho \) is the rotational correlation time. \({r}_{0}\) is assumed to be 0.4. (e) The time-resolved anisotropy decay for the donor (blue) and acceptor (red) fluorophores are shown. Photons from all double-labelled molecules are pooled together to obtain the cumulative fluorescence decays, from which the anisotropy is determined. Fits to bi-exponential model functions are given by solid lines, accounting for the fast rotation of the fluorophore on the nanosecond timescale and the slow rotation of the protein on the timescale of tens of nanoseconds