Figure 6.

Extended model-free analysis of ¹⁵N relaxation data measured in E-SL1m in the absence (49) and presence of Mg²⁺. (A) Secondary structure of E-GC-SL1m and E-AU-SL1m. ¹³C/¹⁵N-labeled and -unlabeled residues are shown in color and gray, respectively. (B) 2D ¹H–¹⁵N HSQC spectra of the imino region of E-GC-SL1m + E-AU-SL1m (black) overlaid on non-elongated SL1m (red) in the absence and presence of Mg²⁺. Watson–Crick guanine signals that could not be directly assigned using NOESY data are labeled with ‘?’ and may correspond to either G26 or G7. (C and D) E-SL1m dynamics in the absence of Mg²⁺ (49). (C) R₂/R₁ values measured in guanine (filled circles) and uridine (filled diamonds) residues in stems I (red) and II (green) in the absence of Mg²⁺. Values predicted using hydrodynamic calculations are shown as black open circles. Error bars represent the measurement uncertainty. (D) Dynamical parameters for E-SL1m in the absence of Mg²⁺ obtained previously (49) from an extended model-free analysis (69,70). The time constants/amplitudes for fast (τ_f/mfcaret; S_fcaret;2) and slow (τ_s/mscaret; S_scaret;2) internal motions are shown. (E and F) Dynamics in E-SL1m in the presence of 10 mM Mg²⁺. (E) Measured R₂/R₁ values (filled color symbols) and those predicted hydrodynamically assuming a static conformation (black open circles) and a population-weighted average of free and Mg²⁺- bound states (see text) (filled gray circles). The error bar in the latter reflects different relative populations of free and bound states due to the spread of the observed K_ds (0.9–7.0 mM corresponding to a free population ranging between 20 and 60%, respectively). (F) Dynamical parameters obtained in the presence of 10 mM Mg²⁺ using extended model-free analysis of the ¹⁵N relaxation data. Results for loop residue G16 (open symbol) varied considerably when using different input E-SL1m + Mg²⁺ conformations that reflect the RDC uncertainty and is therefore considered to be unreliable.