Sequential Folding of the Nickel/Cobalt Riboswitch Is Facilitated by a Conformational Intermediate: Insights from Single-Molecule Kinetics and Thermodynamics

Abstract

The present work presents the first single-molecule fluorescence resonant energy transfer (smFRET) studies of the nickel/cobalt (NiCo) riboswitch, with temperature-dependent, single-molecule confocal microscopy to provide comprehensive kinetic and thermodynamic information on folding into a biochemically competent structure. The results indicate that the NiCo riboswitch first folds into a more compact “prefolded” conformation, with a preorganized binding pocket partially stabilized under physiological conditions by noncognate monovalent/divalent cations. Such a prefolded intermediate then has opportunity to fold further into a tightly ligand-bound structure, in response to the cognate ligands, Ni2+ or Co2+, with submicromolar affinities. Such stepwise ligand-induced folding represents a particularly clean example of a conformational selection (“fold-then-bind”) mechanism, whereby a configuration dynamically accessible by thermal fluctuation is stabilized into the final folded state by ligand association. In addition, we observe a strong positive cooperativity in the ligand-induced folding kinetics with respect to both Ni2+ and Co2+ ligands. This provides maximal sensitivity in the riboswitch conformational response near [Ni2+] or [Co2+] ≈ K d, which facilitates more accurate biochemical probing of the cell environment and therefore bioregulation of gene expression. Temperature-dependent kinetics at the single-molecule level has also been explored, which permits free energies to be deconstructed into enthalpic and entropic components along the folding coordinate. In the absence of the cognate ligand, a predominantly enthalpic barrier between the unfolded riboswitch (U) and the prefolded intermediate (I) suggests a rearrangement of the hydrogen bonding network, whereas in the presence of the cognate ligand, a large entropic penalty (−TΔS 0 > 0) in forming the folded riboswitch conformation (F) is almost perfectly counterbalanced by an equivalent enthalpic gain (ΔH 0 < 0) to yield ΔG 0 ≈ 0. The thermodynamic results are therefore consistent with a simple physical picture of riboswitch folding, whereby association of the cognate ligand is strongly stabilized by Coulombic attraction while forming an entropically more ordered structure around the binding site.

Synergism in the Molecular Crowding of Ligand-Induced Riboswitch Folding: Kinetic/Thermodynamic Insights from Single-Molecule Spectroscopy

“…The folding equilibrium constant K fold can be readily extracted as the ratio of total folding and unfolding dwell times ( K fold = T fold / T unfold ), from which the folded fraction F fold can be calculated ( F fold = T fold /( T fold + T unfold )). The folding ( k fold ) and unfolding ( k unfold ) rate constants themselves can be obtained from the analysis of the corresponding dwell time distributions for t unfold and t fold (see sample data in Figure C, D), respectively . Cumulative distributions for both t fold and t unfold are well fit to single exponential decay functions, consistent with folding and unfolding of the lysine riboswitch well described by simple first order kinetics.…”

Modelling the impact of magnesium ions concentration on the folding of the SAM-II riboswitch

“…Based on a multistep transition, the model overcome limitations of the classical two state transitions, that had been used to explain magnesium induced RNA structural changes [50,55]. Further, unlike several analysis methods used in the literature [3,65], equilibrium constants and standard free energies extracted here are based on a bimolecular interaction and not a unimolecular transition. As a result, the free energies and equilibrium constants therefore do not vary with concentrations.…”

Reconsidering the czcD (NiCo) Riboswitch as an Iron Riboswitch

“…Nevertheless, all three czcD riboswitches bind at least two metal ions�suggesting that the two interconnected metal sites, C1 and C2 at the four-way junction (Figure 1B), may be the most critical for metal responsiveness and accounting for the cooperative binding observed in previous studies. 43,44,54 These results highlight how the rules for iron ligation by RNA are not yet understood, motivating future structural biology studies of iron-bound riboswitches.…”