Figure 1.

The ES-stabilizing mutant TAR^UUCG-ES experiences back exchange with a low-abundance and short-lived ES that has conformational features similar to the GS of wild-type TAR. a. Chemical exchange between the GS and ES in wtTAR and TAR^UUCG-ES. Shown are the populations (p_GS and p_ES) of the GS and ES, respectively and the rate constants (k₁ and k₋₁) for inter-conversion deduced from 2-state analysis of the R_1ρ RD data. Residues are labeled according to the wtTAR GS secondary structure: stem (blue), bulge (orange), apical loop (red). Residues that differ between the two TAR variants are in gray. b. R_1ρ RD profiles measured for TAR^UUCG-ES. Off-resonance profiles show the dependence of R₂ + R_ex on spinlock power (ω2π⁻¹) and offset (Ω2π⁻¹). Shown are the global fits (solid line) to a two-state model using the Bloch-McConnell equations assuming a two-state exchange process (Methods). On-resonance profiles show the dependence of R_1ρ on spinlock power (ω2π⁻¹). Error bars represent experimental error determined by propagation of error determined by Monte Carlo analysis of monoexponential decay curves and experimental signal to noise. Data was collected on a 600 MHz (¹H frequency) spectrometer. Buffer conditions are 15 mM sodium phosphate, 25 mM sodium chloride, 0.1 mM EDTA, 100% D₂O at pH 6.4 and 25 °C. c. Comparison of the difference between chemical shifts measured for the ES and GS (Δω = ω_ES - ω_GS) in TAR^UUCG-ES (green) and the inverse (Δω = ω_GS - ω_ES) in wtTAR (black) [13] using R_1ρ RD NMR.