Figure 7. Analysis of bimodally-distributed isotope clusters of IRE1^LD peptic peptides reveals active destabilisation of the IRE1^LD dimer by BiP.

(A) Intensity distributions of the isotope clusters of peptide 655.273⁺ (residues 297–302) from IRE1^LD, untreated or exposed to BiP, ERDj4 and ATP (30 min at 30°C) following different incubation times in D₂O, as indicated. Curves are fits of the sum of two Gaussian distributions (Prism GraphPad 7.0, see Equation 4 in Materials and methods). A representative plot of three independent experiments is shown. (see: Figure 7—source data 1) (B) Plot of time-dependent change in the fractional contribution of high mass species to the isotope clusters of peptide 655.273⁺ (from ‘A’) calculated as described in Figure 7—figure supplement 1A and B. Shown are data points from three independent samples of IRE1^LD in presence and absence of BiP, ERdj4 and ATP. The curves were fitted to a one-phase association model in Prism GraphPad 7.0. Data for a second informative peptide is shown in Figure 7—figure supplement 1C. (C) Bar diagram of the transition rate constant k_trans extracted by analysis of bimodal distributions in the isotope clusters of peptic fragments 636.380²⁺ and 655.273⁺ from IRE1^LD in presence and absence of BiP, ERdj4 and ATP (from Figure 7B and Figure 7—figure supplement 1C). All the data points from three independent experiments are shown and the mean ± standard deviation (**: p<0.01, ****: p<0.0001, one-way ANOVA with Sidak’s multiple comparison test).

Figure 7—figure supplement 1. Analysis of the bimodal distributions of the isotope clusters detected by HX-MS.

(A) Shown are original spectra of peptic peptide 655.273⁺ (residues 297–302) from monomeric IRE1^{LD P108A} (left panel) or wild-type IRE1^LD (right panel) at the indicated time point of incubation in D₂O. The contribution of the low (blue) and high (red) exchanging populations to the peaks was extracted by fitting the data to a two Gaussian distribution model (Equation 4). Unexchanged and 100% exchanged spectra are provided as references. (B) Plot of the two individual Gaussian distributions of the high (h) and low (l) exchanging populations deconvoluted computationally in Prism GraphPad 7.0 from the composite data set shown in Figure 7A for each time point of incubation in D₂O. (C) Plot of time-dependent change in the fractional contribution of high mass species to the isotope clusters of peptic peptide 636.380²⁺ (residues 96–106). Shown are data points from three independent samples of IRE1^LD in presence and absence of BiP, ERdj4 and ATP (as in Figure 7B). The curves were fitted to a one-phase association model in Prism GraphPad 7.0.