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

• Published in: The journal of physical chemistry. B 2020-08, Vol.124 (34), p.7348-7360
• Main Authors: Sung, Hsuan-Lei, Nesbitt, David J
• Format: Article
• Language: English
• Subjects: B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules; Cobalt; Kinetics; Ligands; Nickel; Nucleic Acid Conformation; Riboswitch; RNA Folding; Thermodynamics
• ISSN: 1520-6106; 1520-5207
• DOI: 10.1021/acs.jpcb.0c05625

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.