Figure 3. Rare conformational states of ligand-free SBPs.

(A) Schematic of the experimental strategy to study the conformational dynamics of ligand-free SBPs. Representative fluorescence trajectories of OpuAC(V360C/N423C) (B), PsaA(V76C/K237C) (C), MalE(T36C/S352C) (D), SBD1(T159C/G87C) (E), OppA(A209C/S441C) (F) and SBD2(T369C/S451) (G) in the absence of substrate. 10–20 μM of unlabeled protein or 1 mM EDTA (for PsaA) was added to scavenge any ligand contaminations. In all fluorescence trajectories presented in the figure: top panel shows calculated apparent FRET efficiency (blue) from the donor (green) and acceptor (red) photon counts as shown in the bottom panels. Orange lines indicate average apparent FRET efficiency value or most probable state-trajectory of the Hidden Markov Model (HMM). Statistics in Supplementary file 4. (H) Percentage of time a SBP is in the high FRET state. Statistics in Supplementary file 4.

Figure 3—figure supplement 1. Conformational dynamics of ligand-free and ligand-bound SBPs.

Representative fluorescence trajectories of OpuAC(V360C/N423C) (A), PsaA(V76C/K237C) (B), MalE(T36C/S352C) (C), SBD1(T159C/G87C) (D), OppA(A209C/S441C) (E) and SBD2(T369C/S451) (F) in the absence of substrate and under saturating conditions of ligand, as indicated. In the absence of ligand, 10–20 μM of unlabeled protein or 1 mM EDTA (for PsaA) was added to scavenge any ligand contaminations. The top panels show the calculated apparent FRET efficiency (blue) from the donor (green) and acceptor (red) photon counts as presented in bottom panels. The orange line indicates the average apparent FRET efficiency value or most probable state-trajectory of the HMM. Statistics in Supplementary file 4.

Figure 3—figure supplement 2. Intrinsic conformational dynamics in the presence of unlabeled protein.

Closing rate (A) and average lifetime of the closed conformation (B) for OppA, SBD1 and SBD2 in the absence of ligand and in the presence of different concentrations of unlabeled protein to scavenge potential ligand contaminations. Examples of the high FRET transitions are shown in Figure 3 and Figure 3—figure supplement 1. Error bars correspond to s.e.m. The closing rate was determined by dividing the total observation time of all molecules by the number of observed high FRET transitions. The statistical significance of the average closed state lifetime was determined by a two-tailed unpaired t-tests. The statistical significance of the closing rate was determined by testing for the difference in the proportion of time-bins in which a low to high FRET transition is made and using the z-test. Statistics in Supplementary file 4.