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

Abstract

Conformational dynamics in riboswitches involves ligand binding and folding of RNA, each of which can be influenced by excluded volume effects under "crowded" in vivo cellular conditions and thus incompletely characterized by in vitro studies under dilute buffer conditions. In this work, temperature-dependent single-molecule fluorescence resonance energy transfer (FRET) spectroscopy is used to characterize the thermodynamics of (i) cognate ligand and (ii) molecular crowders (PEG, polyethylene glycol) on folding of the B. subtilis LysC lysine riboswitch. With the help of detailed kinetic analysis, we isolate and study the effects of PEG on lysine binding and riboswitch folding steps individually, from which we find that PEG crowding facilitates riboswitch folding primarily via a surprising increase in affinity for the cognate ligand. This is furthermore confirmed by temperature-dependent studies, which reveal that PEG crowding is not purely entropic and instead significantly impacts both enthalpic and entropic contributions to the free energy landscape for folding. The results indicate that PEG molecular crowding/stabilization of the lysine riboswitch is more mechanistically complex and requires extension beyond the conventional picture of purely repulsive solvent-solute steric interactions arising from excluded volume and entropy. Instead, the current experimental FRET data support an alternative multistep mechanism, whereby PEG first entropically crowds the unfolded riboswitch into a "pre-folded" conformation, which in turn greatly increases the ligand binding affinity and thereby enhances the overall equilibrium for riboswitch folding.