A magnesium-induced triplex pre-organizes the SAM-II riboswitch
Fig 2
Detailed agreement between 13C CEST, smFRET, SAXS, and SEC elution profiles with the generalized Manning model corroborates pre-organization by Mg2+.
(a)-(d) Comparison between CEST-NMR experiments and Manning model simulations. (a) 13C CEST profiles for C43-C6 of SAM-II riboswitch in the ligand-free situation in the presence of two different concentrations of Mg2+ (black dots: 0.25 mM, green dots: 2.0 mM) at B1 field of 17.5 Hz. The population distribution involving two major states was obtained by fitting each 13C CEST profile (solid lines) into a two-state model. (b) The fraction of native contacts (Q) dynamics extracted from simulations indicate the transition between two major states at 0.25 mM. (c) Same as (b) for 2.0 mM Mg2+. (d) The fraction of native contact population histograms at these two concentrations. (e)-(f) Steady state smFRET analysis from fluorescently labeled experiments and theoretical prediction from simulations. (e) Experimental smFRET occupancy histogram in the presence of 2.0 mM Mg2+ (adapted from ref. 22) [22]. (f) Theoretical prediction of FRET occupancy in the presence of 2.0 mM Mg2+. (g) Superposition of a set of different conformations at 2.0 mM Mg2+ depicts the prevalence of two distinct sets of ensembles. (h) Comparison of experimental scattering profiles of SAM-II collected from four different buffer conditions. Theoretical SAXS predictions are depicted by solid lines and the experimental data by dots (experimental data adapted from ref. 40). (i) Compaction of SAM-II as a function of [Mg2+] obtained from SEC elution profiles is consistent with a decrease in hydrodynamic radius (experimental data adapted from ref. 40), [40] in qualitative agreement with the observed folding transition in the average radius of gyration (Rg) obtained from equilibrium simulations at Mg1/2≈6.0 mM.