Observation of long-range tertiary interactions during ligand binding by the TPP riboswitch aptamer
- Stanford University, United States
Figures
Figure 1
Single-molecule assay, crystal structure and schematic of the TPP aptamer.
(A) Experimental geometry of the dumbbell optical-trapping assay with simultaneous FRET monitoring, with key components labeled (not to scale). (B) Crystal structure of the truncated TPP riboswitch …
Figure 2
Schematic optical layout of the dual-beam optical trap and FRET.
Solid lines indicate lasers and light sources: 830 nm detection laser (orange), 1,064 nm trapping laser (red), 532 nm excitation laser (green), and 470 nm illuminating LED (blue). Filled bars are …
Figure 3 with 2 supplements
Representative FEC and FRET traces.
Representative traces of unfolding for the aptamer conformations (A) F, (B) F′•TPP, and (C) F″•TPP. Simultaneous force-extension curves (FECs; black lines) and FRET trajectories (black circles, gray …
Figure 3—figure supplement 1
Representative traces of various secondary state conformations and TPP concentrations.
Force curves (black lines) and FRET trajectories (black circles, gray lines) are shown parametrized by time for the following aptamer conformations: (A) F″•TPP in the presence of 5 μM TPP, (B) …
Figure 3—figure supplement 2
FRET changes occur throughout the refolding period and the force ramp, up until the first rip.
Representative traces show FRET signatures indicative of a particular helix arm configuration at the end of a refolding period that is maintained throughout the force ramp. Colored boxes, lines, …
Figure 4 with 1 supplement
Clustering analysis of opening FRET values, and global analysis of full-length FRET traces.
(A) k-means clustering for opening FRET and opening force values. Filled diamonds mark the mean opening force and opening FRET values for each of the k = 3 populations; dashed vertical and …
Figure 4—figure supplement 1
k-means cluster analysis of opening force and FRET.
k-means clustering results and summary tables shown for (A) k = 2, (B) k = 3, and (C) k = 4.
Figure 5
Distributions of segmented FRET data in the presence and absence of TPP and its analogs.
(A) Segmented FRET data in the presence of saturating TPP (2 mM). After segmenting, FRET records were categorized into APO (blue), WB (green), and SB (yellow) FRET states. (B) Table summarizing FRET …
Figure 6
HMM modeling of refolding FRET trajectories.
(A) Four-state HMM fit to a 30-second portion of concatenated refolding FRET trajectories in the presence of 5 µM TPP. (B) Top: Reaction diagram for the four-state, sequential HMM model, with states …
Figure 7
Correspondences among single-molecule state identifications, and model of aptamer binding to TPP.
(A) Cartoon summarizing the correspondence between analysis and data collection methodologies. The corresponding states and reaction schemes obtained from HMM analysis (top line; pink circles), …
Figure 8
Dye characterization using DNA hairpins.
(A) Opening distances (F1/2) and forces (Δx) of DNA hairpins SJ1 and SJ2 were measured at non-equilibrium and compared to previous measurements at equilibrium. (B) FRET trajectories and FECs are …
