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Deep sea biology is known to thrive at pressures up to ≈1 kbar, which motivates fundamental biophysical studies of biomolecules under such extreme environments. In this work, the conformational equilibrium of the lysine riboswitch has been systematically investigated by single molecule FRET (smFRET) microscopy at pressures up to 1500 bar. The lysine riboswitch preferentially unfolds with increasing pressure, which signals an increase in free volume (Δ V 0 > 0) upon folding of the biopolymer. Indeed, the effective lysine binding constant increases quasi-exponentially with pressure rise, which implies a significant weakening of the riboswitch–ligand interaction in a high-pressure environment. The effects of monovalent/divalent cations and osmolytes on folding are also explored to acquire additional insights into cellular mechanisms for adapting to high pressures. For example, we find that although Mg 2+ greatly stabilizes folding of the lysine riboswitch (ΔΔ G 0 < 0), there is negligible impact on changes in free volume (ΔΔ V 0 ≈ 0) and thus any pressure induced denaturation effects. Conversely, osmolytes (commonly at high concentrations in deep sea marine species) such as the trimethylamine N -oxide (TMAO) significantly reduce free volumes (ΔΔ V 0 < 0) and thereby diminish pressure-induced denaturation. We speculate that, besides stabilizing RNA structure, enhanced levels of TMAO in cells might increase the dynamic range for competent riboswitch folding by suppressing the pressure-induced denaturation response. This in turn could offer biological advantage for vertical migration of deep-sea species, with impacts on food searching in a resource limited environment.
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Single-molecule kinetic studies of DNA hybridization under extreme pressures
Hydrostatic pressure can perturb biomolecular function by altering equilibrium structures and folding dynamics. Its influences are particularly important to deep sea organisms, as maximum pressures reach ≈1100 bar at the bottom of the ocean as a result of the rapid increase in hydraulic pressure (1 bar every 10 meters) under water. In this work, DNA hybridization kinetics has been studied at the single molecule level with external, tunable pressure control ( P max ≈ 1500 bar), realized by incorporating a mechanical hydraulic capillary sample cell into a confocal fluorescence microscope. We find that the DNA hairpin construct promotes unfolding (“denatures”) with increasing pressure by simultaneously decelerating and accelerating the unimolecular rate constants for folding and unfolding, respectively. The single molecule kinetics is then investigated via pressure dependent van’t Hoff analysis to infer changes in the thermodynamic molar volume, which unambiguously reveals that the effective DNA plus solvent volume increases (Δ V 0 > 0) along the folding coordinate. Cation effects on the pressure dependent kinetics are also explored as a function of monovalent [Na + ]. In addition to stabilizing the overall DNA secondary structure, sodium ions at low concentrations are also found to weaken any pressure dependence for the folding kinetics, but with these effects quickly saturating at physiologically relevant levels of [Na + ]. In particular, the magnitudes of the activation volumes for the DNA dehybridization (Δ V ‡unfold) are significantly reduced with increasing [Na + ], suggesting that sodium cations help DNA adopt a more fold-like transition state configuration.
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- Award ID(s):
- 1734006
- PAR ID:
- 10231678
- Date Published:
- Journal Name:
- Physical Chemistry Chemical Physics
- Volume:
- 22
- Issue:
- 41
- ISSN:
- 1463-9076
- Page Range / eLocation ID:
- 23491 to 23501
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/D0CP04035E
