D135–L14 ribozyme and its folding pathway. (A) Scheme of the secondary structure of D135–L14. The individual domains (D1, blue; D2, gray; D3, green; D4, light gray; D5, orange) and the fluorophore labeled DNA-oligonucleotides (black; Cy3, green; Cy5, red) are shown. (B) Folding pathway of D135–L14. Compaction of D1 from the unfolded state U to the extended intermediate I is slow. The remaining domains pack onto D1 to give the folded species F, which itself rearranges to the native state N occurring only in small amounts. k₁, k₋₁, k₂, and k₋₂ refer to smFRET results at 100 mM Mg²⁺, and k_U ↔ I was obtained from bulk FRET experiments and corresponds to earlier values of ≈1 min⁻¹ (14, 18, 19). B is partially adapted from ref. 19. Folding rates are given in seconds⁻¹.

smFRET analysis of D135–L14 ribozymes. (A) Principle of total internal reflection fluorescence spectroscopy (TIRF). Molecules are immobilized on a quartz slide and placed in the microscope. The fluorophores are excited by the laser's evanescent wave above the critical angle, giving emission signals collected through the objective with a high-quantum yield camera. (B) smFRET time trace and histogram at 10 mM Mg²⁺ showing two conformational states at 0.25 and 0.4. (C) smFRET time trace and histogram at 100 mM Mg²⁺ sampling the three FRET states 0.25, 0.4, and 0.6. (D) (Left) smFRET time trace shows a dynamic behavior only upon increase in MgCl₂ concentration from 10 to 100 mM at 50–58 s. (Right) Distributions of the states before and after the addition of Mg²⁺.

Effect of substrate binding on the dynamics and native state stabilization. (A) Histograms of FRET efficiency of fully labeled D135–L14 alone (Top), in the presence of WT substrate 17/7 (Middle), and in the presence of the slower cleaving substrate 17/7-dC (Bottom). The 0.6 state is clearly stabilized by substrate binding. (B) (Left) Typical smFRET time trace in the presence of 17/7-dC. The 0.6 state occurs at high frequency (compare with Fig. 3). The drop to 0.25 at 35 s might indicate cleavage. (Right) Distribution.

Bulk fluorescence measurements of the fully labeled D135–L14 in dependence of Mg²⁺ concentration. (A) Emission spectrum of the ribozyme in the absence (0 mM, solid line) and presence (100 mM, dotted line) of MgCl₂ showing correlating shifts in maxima at 565 nm (Cy3) and 665 nm (Cy5) on FRET. (B) FRET change upon addition of 100 mM Mg²⁺. The arrow indicates the addition of Mg²⁺. The single exponential fit of the experimental data is shown as a black line. (C) Maximal change in FRET upon increasing amounts of Mg²⁺ and fit to the Hill equation (S2) giving K_Mg = 42.3 ± 1.3 mM.

Histograms of FRET efficiency values from single-molecules traces in dependence of Mg²⁺ concentration. All traces (static and dynamic) were used to build the histogram at 1 mM, whereas all other histograms were created from traces showing dynamics only. Three distributions centered at FRET ≈0.25, ≈0.4, and ≈0.6. The peaks of the 0.25 and 0.4 states show a slight shift to lower FRET with higher Mg²⁺ concentration. The 0.6 distribution arises above 15–20 mM and is only minimally but steadily populated. Gaussian fits for the individual and the sum of all distributions are shown as solid lines. The dashed bars at 0.12 FRET correspond to the distribution in absence of acceptor (Top).

Mg²⁺ dependence of single-molecule dynamics and the observed rates k_obs. (A) The percentage of molecules on a slide showing dynamics are plotted versus the Mg²⁺ concentration. The ratio of dynamic molecules increases from 7 to 49% with K_Mg = 41.4 ± 1.4 mM. (B) Representative histogram of dwell times in the 0.25 FRET state at 50 mM Mg²⁺ showing the number of events at a particular dwell time range. The solid line corresponds to the double exponential decay fit characterizing the rate. (C) The number of dwell times in low FRET state N(t) shorter than time t, obtained by integration of the dwell time histogram in A. The double-exponential growth fit is shown as a solid line. (D) Effect of Mg²⁺ concentration on the observed single-molecule folding rates k₁ (●) and k₋₁ (○).