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. 2014 Nov 17;136(48):16700–16703. doi: 10.1021/ja5073146

Figure 1.

Figure 1

Molecular crowding stabilizes the docked conformation of the hairpin ribozyme. (a) Schematic diagram of single molecule FRET experiments. Fluorophore labeled ribozyme is surface immobilized on a quartz slide via a biotin–streptavidin linkage. The donor fluorophore (D) is excited in a prism-based total internal reflection microscope. Arrow indicates cleavage site. (b) Characteristic single molecule FRET time trajectory shows the ribozyme dynamically switching between the docked (0.8 FRET) and the undocked (0.2 FRET) conformations. (c) Time binned FRET histograms reveal the distribution in the docked and undocked states. The docked fraction increases with increasing amounts of PEG. (d) Fraction of docked molecules as a function of %PEG and fit to a binding isotherm (solid line). (e) Change in docking Gibbs free energy change (ΔΔG°) as a function of %PEG. PEG stabilizes the docked conformation. (f) Docking rate constant increases with %PEG. (g) A scatter plot showing the folding heterogeneity in the presence (red) and absence (blue) of crowding agents. In the presence of 25% PEG, the distribution narrows and its average increases (dashed lines).