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. 2022 Jul 1;11:e78982. doi: 10.7554/eLife.78982

Figure 4. Single-molecule fluorescence resonance energy transfer (smFRET) analysis of MNI-137 effects on cysteine-rich domain (CRD) conformational dynamics.

(A–C) smFRET population histograms of azi-CRD sensor in the presence of 0 μM (372 particles), 15 μM (560 particles), and 1 mM (479 particles) glutamate and 5 μM MNI-137. Histograms were fitted (black) to four Gaussian distributions centered around 0.24 (inactive; purple), 0.38 (intermediate 1; blue), 0.70 (intermediate 2; cyan), and 0.87 (active; red) FRET. Error bars represent SEM. (D) Mean occupancy of four conformational states of azi-CRD in varying ligand conditions. Values represent area under each FRET peak from smFRET histogram as a fraction of total area. Mean and SEM values are reported in Table 3. (E) Transition density plots of azi-CRD at 1 mM glutamate with and without MNI-137. Dashed lines represent four distinct FRET states. (F) Mean cross-correlation of donor and acceptor intensities in the presence of 0 μM, 15 μM, and 1 mM glutamate and 5 μM MNI-137. Data was acquired at 50 ms time resolution. Data represents mean from three independent experiments.

Figure 4—source data 1. Source data for Figure 4.

Figure 4.

Figure 4—figure supplement 1. Representative single-molecule fluorescence resonance energy transfer (smFRET) traces for 5 μM MNI-137 conditions.

Figure 4—figure supplement 1.

(A–C) Representative smFRET traces of azi-cysteine-rich domain (azi-CRD) in the presence of 5 μM MNI-137 and (A) 0 μM, (B) 15 μM, and (C) 1 mM glutamate showing donor (green) and acceptor (red) and corresponding FRET (blue). Dashed lines represent four distinct FRET states. Data was acquired at 50 ms time resolution.
Figure 4—figure supplement 2. Allosteric modulator effects on azi-cysteine-rich domain (azi-CRD) cross correlation.

Figure 4—figure supplement 2.

(A) Cross-correlation of azi-CRD donor and acceptor intensities in the presence of 0 μM glutamate alone and with 5 μM MNI-137 or 10 μM BINA. (B) Cross-correlation of azi-CRD donor and acceptor intensities in the presence of 1 mM glutamate alone and with 5 μM MNI-137 or 10 μM BINA. Data was acquired at 50 ms time resolution.