Molecular-crowding effects on single-molecule RNA folding/unfolding thermodynamics and kinetics

Abstract

The effects of "molecular crowding" on elementary biochemical processes due to high solute concentrations are poorly understood and yet clearly essential to the folding of nucleic acids and proteins into correct, native structures. The present work presents, to our knowledge, first results on the single-molecule kinetics of solute molecular crowding, specifically focusing on GAAA tetraloop-receptor folding to isolate a single RNA tertiary interaction using time-correlated single-photon counting and confocal single-molecule FRET microscopy. The impact of crowding by high-molecular-weight polyethylene glycol on the RNA folding thermodynamics is dramatic, with up to ΔΔG° ∼ -2.5 kcal/mol changes in free energy and thus >60-fold increase in the folding equilibrium constant (Keq) for excluded volume fractions of 15%. Most importantly, time-correlated single-molecule methods permit crowding effects on the kinetics of RNA folding/unfolding to be explored for the first time (to our knowledge), which reveal that this large jump in Keq is dominated by a 35-fold increase in tetraloop-receptor folding rate, with only a modest decrease in the corresponding unfolding rate. This is further explored with temperature-dependent single-molecule RNA folding measurements, which identify that crowding effects are dominated by entropic rather than enthalpic contributions to the overall free energy change. Finally, a simple "hard-sphere" treatment of the solute excluded volume is invoked to model the observed kinetic trends, and which predict ΔΔG° ∼ -5 kcal/mol free-energy stabilization at excluded volume fractions of 30%.

Keywords: PEG; fluorescence; scaled particle theory.

Fig. 1.

(A) The single-molecule GAAA tetraloop–receptor construct is shown. The shaded circles represent the approximate effective volumes occupied by the folded and unfolded states of the tetraloop and receptor. The GAAA tetraloop is connected to the receptor helix via a PEG₆ linker. (B) A thermodynamic cycle describes the differential change in folding free energy between dilute and crowded solution conditions.

Fig. 2.

E_FRET trajectories comparing 0% and 4% PEG 8000 reveal a significant increase in the frequency of excursions to the high E_FRET state. Histograms (Left) show an increase in K_eq by more than sixfold, even for only 4% PEG 8000 solutions.

Fig. 3.

(A) K_eq is plot as a function of the PEG 8000 solution content. (B) The individual rate constants are shown in a logarithmic plot, highlighting the dramatic increase and much smaller decrease in k_fold and k_unfold, respectively.

Fig. 4.

Van’t Hoff plot for the tetraloop–receptor equilibrium in aqueous buffer and 8% PEG 8000, where the parallel shift upwards in y intercept reveals that crowding-induced stabilization by PEG 8000 occurs predominantly through reduction in the entropic cost of folding. Left-hand column shows enthalpy changes consistent with ΔΔH° = 0 within experimental error. The second and third columns (*) reflect fits with ΔΔH° = 0 to break parameter correlation and generate a more reliable prediction for entropic lowering of the free-energy difference, i.e., −TΔΔS°.

Fig. 5.

(A) Stabilization plots for the GAAA tetraloop–receptor folding equilibrium with respect to dilute buffer solution, revealing a nonlinear dependence of ΔΔG° on excluded volume. (B) Fits to the equilibrium and kinetic data reflect the effective radii of the tetraloop–receptor unfolded/folded (UF, F) and transition states (TS), respectively, as well as reveal a large loss of free volume between UF and TS states followed by a much smaller loss between the TS and F states.