Experimental approach for measuring interdomain dynamics in the SBDs of OpuA. (A) A cryo-EM structure of OpuA (PDB: 7AHH). Mutations K521C and N414C are highlighted as grey spheres. (B) Size-exclusion chromatography profiles of OpuA-nanodiscs that were purified according to the previously described protocol [6] (blue) or according to the new protocol that is described here (yellow). The numbers refer to the elution fractions that were loaded on an SDS-PAA gel. (C) SDS-PAA gel with the size exclusion fractions of the blue line in (B). (D) SDS-PAA gel with the size-exclusion fractions of the yellow line in (B). (E) A schematic representation of how confocal, solution-based smFRET was used to study different states of the SBDs. (F) A representation of a fluorescent burst time trace, displaying the photon counts in the donor (green) and acceptor (red) detection channel over time (left). The zoom-in is a representation of a photon time trace, in which single photons are represented as lines and the most likely state path from the Viterbi algorithm in mpH2MM as a blue line.

The SBDs of OpuA sample two dynamic FRET states. The proteins were analyzed in 50 mM HEPES-K pH 7.0, 600 mM KCl with the following additions: (A) OpuA-K521C without further additions. (B) OpuA-N414C without further additions. (C) OpuA-E190Q- K521C with 20 mM Mg-ATP plus 100 µM glycine betaine. (D) OpuA-K521C with 20 mM Mg-ATP. From top to bottom: (i) FRET histogram showing the corrected bursts that were selected after removing donor-only and acceptor-only bursts. (ii) 2D E-S histogram showing the same data as in (i). Black dots represent the average value of each state after mpH2MM and after application of the correction factors. (iii) Burst variance analysis of the same burst data as in (i). The standard deviation of FRET in each burst is plotted against its mean FRET. Black squares represent average values per FRET bin. The black dotted line shows the expected standard deviation in the absence of within-burst dynamics. (iv) E-S scatter plot of the corrected dwells. Dwells are colored on the basis of the assigned state of the chosen mpH2MM model. Black dots represent the average value of each state and the numbers at the arrows show transition rate constants (s-1) between the two FRET states. (v) Plot of the ICL-values for each final model. The model used in the analysis is shown as a red star.

The FRET (E) and Stoichiometry (S) of the FRET states of OpuA-K521C, their relative abundance and the transition rate constants between the states

The FRET (E) and Stoichiometry (S) of the FRET states of OpuA-N414C, their relative abundance, and the transition rate constants between the states

Reduction in SBD docking efficiency does not affect the two dynamic FRET states. (A) A cryo-EM structure of OpuA (PDB: 7AHH) highlighting Val-149 and the two most important residues in the vicinity of Val-149. Coloring of the domains is similar to that in Figure 1A. (B) Results of an enzyme-coupled ATPase assay for OpuA-WT (filled circles, grey) and OpuA-V149Q-K521C (open circles, yellow). Each sample contains 50 mM HEPES-K pH 7.0, 450 mM KCl, 10 mM Mg-ATP, 4 mM phosphoenolpyruvate, 600 μM NADH, 2.1 to 3.5 U of pyruvate kinase, and 3.2 to 4.9 U of lactate dehydrogenase. Standard deviation over at least two measurements with different protein purifications and membrane reconstitutions, each consisting of three technical replicates is represented as shaded areas. (C) Results of (B) represented as activity relative to the activity at 100 µM glycine betaine. (D) smFRET results for OpuA-V149Q-K521C in 50 mM HEPES-K pH 7.0, 600 mM KCl. From top to bottom: (i) FRET histogram showing the corrected bursts that were selected after removing donor-only and acceptor-only bursts. (ii) 2D E-S histogram showing the same data as in (i). Black dots depict the average value of each state after mpH2MM and after application of the correction factors. (iii) Burst variance analysis of the same burst data as in (i). The standard deviation of FRET in each burst is plotted against its mean FRET. Black squares represent average values per FRET bin. Black dotted line shows the expected standard deviation in the absence of within-burst dynamics. (iv) E-S scatter plot of the corrected dwells. Dwells are colored on the basis of the assigned state of the chosen mpH2MM model. Black dots represent the average value of each state and the numbers at the arrows show transition rate constants (s-1) between the two FRET states. (v) Plot of the ICL-values for each final model. The model used in the analysis is shown as a red star.

Shifts in FRET states under different conditions. (A,B) FRET (E) histograms of OpuA-K521C (A) and OpuA-N414C (B) corresponding to the corrected dwells of the two FRET states after mpH2MM. Black lines show the mean E of the two states. (C) A schematic representation of the diffusion freedom in 2D of the SBDs, shown as blue semicircles. The radius is defined as the sum of the radius of an SBD and the length of the linker region in a fully extended conformation. The sequence of the linker region was defined based on the occluded OpuA structure (PDB: 7AHD) and spans the N- and C-terminal end of the SBD and anchoring helix, respectively. (D) Lowpass-filtered cryo-EM density maps of OpuA-WT in three different ionic strength conditions (50 mM, 100 mM and 200 mM KCl). The top-views of each condition are horizontally aligned (left) and super-positioned (right) for better comparison. The densities corresponding to the SBDs are highlighted in teal, blue and brown ellipses, respectively.

Schematic of possible states of the SBDs of OpuA. (A) Both SBDs dock in a non- productive manner onto the TMDs. (B) The SBDs interact with each other in an upright orientation either back-to-back, front-to-front or front-to-back. (C) The SBDs interact sideways with each other.

Steady-state anisotropy values of free dyes and dyes which were attached to different proteins

Primers used in this study to generate mutations in OpuA