Fig. 4: Detection and characterization of conformational dynamics on the submillisecond timescale in MalE and U2AF2.
a,b, Schematic representations of BVA (a) and E–τ (b) plots. The ds is defined as the excess standard deviation compared to the static line (shown in black). Dynamic FRET lines are indicated in red. c, BVA of MalE-2 labeled with Alexa546–Alexa647 without maltose (apo, left) and U2AF2 labeled with Atto532–Atto643 without RNA (apo, right). Here, the BVA is based on a photon binning of five photons. Red diamonds indicate the average standard deviation of all bursts within a FRET efficiency range of 0.05. The mean positions of the populations (cyan crosses) were determined by fitting a two-dimensional Gaussian distribution to the data (Supplementary Note 5). d, The plots of the FRET efficiency E versus intensity-weighted average donor lifetime \(\left\langle {\tau _{{\mathrm{D(A)}}}} \right\rangle _{\mathrm{F}}\) of the same measurement as in c. The donor-only population was excluded from the plot. For MalE-2, the population falls on the static FRET line, while a clear ds is observed for U2AF2. The endpoints of the dynamic FRET line for U2AF2 were determined from a subensemble analysis of the fluorescence decay. e,f, The apparent ds of the peak of the population was determined graphically from BVA (eight laboratories for MalE and seven laboratories for U2AF2, respectively) (e) and E–τ (five laboratories) (f) plots (Methods). For U2AF2 in the holo state, the ds was assessed only for the low-FRET RNA-bound population. Boxes indicate the median and 25/75% quartiles of the data. Whiskers extend to the lowest or highest data point within 1.5 times the interquartile range. The gray area indicates the ds obtained for the dsDNA used in a previous study18 based on measurements performed in laboratory 1 for BVA (dsDNA = 0.0033 ± 0.0033) and laboratory 2 for the E–τ plot (dsDNA = 0.0026 ± 0.0044). The horizontal red lines indicate the expected ds for a potential conformational exchange between the apo and holo states. We computed the expected change in FRET efficiency using their structural models in the PDB (Supplementary Note 6 and Supplementary Table 9).
