Figure 5. Mss116-mediated group II intron folding using smFRET.^124,162 (A) Experimental design. The fluorophore-labeled D135 ribozyme (Cy3, green cycle; Cy5, red cycle) is immobilized on a PEG-coated quartz slide via a biotin-streptavidin linkage (“B,” “S”). Structural interconversion under different folding conditions is monitored by following FRET efficiency over time. (B–F) Averaged FRET histograms, each built from over 100 single-molecule time traces. Imaging conditions: (B) 8 mM Mg²⁺, 500 mM K⁺. Three FRET distributions are observed, termed “intermediate” (“I”), “folded” (“F”) and “native” (“N”) based on earlier results.^162,163 (C)Eight mM Mg²⁺, 100 mM K⁺. Only the “I” FRET state is observed at near-physiological conditions. (D) Eight mM Mg²⁺, 100 mM K⁺, 25 nM Mss116, 1 mM ATP. Addition of Mss116 and ATP shifts the distribution of FRET states toward the folded intermediate and the native state. (E and F) Eight mM Mg²⁺, 100 mM K⁺, 25 nM Mss116 (and 1 mM AMPPNP). Effect of ATP hydrolysis on D135 folding. Prevalence of the native state is lowered in the absence of ATP (E) and in the presence of non-hydrolyzable AMPPNP (F). (G) Percentage of dynamic molecules at different imaging conditions. Red, 500 mM K⁺, 8 mM Mg²⁺; green, 8 mM Mg²⁺, 100 mM K⁺; blue, 8 mM Mg²⁺, 100 mM K⁺, 25 nM Mss116, 1 mM ATP; yellow, 8 mM Mg²⁺, 100 mM K⁺, 25 nM Mss116; purple, 8 mM Mg²⁺, 100 mM K⁺, 25 nM Mss116, 1 mM AMPPNP. (H) Proposed model of Mss116-mediated group II intron folding. D135 interconverts between four conformations referred to as “unfolded” (“U”), “intermediate” (“I”), “folded” (“F”) and “native” (“N”). Mss116 promotes the transition from U to I, even in the absence of ATP. It further catalyzes ATP-dependent conversion from F to N. Figure modified from reference 124.