skip to main content

Explore Research Products in the NSF-PAR

  • NSF Public Access
  • Search Results
  • High pressure single-molecule FRET studies of the lysine riboswitch: cationic and osmolytic effects on pressure induced denaturation
Title: High pressure single-molecule FRET studies of the lysine riboswitch: cationic and osmolytic effects on pressure induced denaturation
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.  more » « less
Award ID(s):
1665271 1734006
NSF-PAR ID:
10174492
Author(s) / Creator(s):
;
Date Published:
Journal Name:
Physical Chemistry Chemical Physics
ISSN:
1463-9076
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ( , Physical Chemistry Chemical Physics)
    null (Ed.)
    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. 
    more » « less
  2. ; ; ; ; ; ( , The FEBS Journal)

    We explore the principles of pressure tolerance in enzymes of deep‐sea fishes using lactate dehydrogenases (LDH) as a case study. We compared the effects of pressure on the activities of LDH from hadal snailfishesNotoliparis kermadecensisandPseudoliparis swireiwith those from a shallow‐adaptedLiparis floraeand an abyssal grenadierCoryphaenoides armatus. We then quantified the LDH content in muscle homogenates using mass‐spectrometric determination of the LDH‐specific conserved peptide LNLVQR. Existing theory suggests that adaptation to high pressure requires a decrease in volume changes in enzymatic catalysis. Accordingly, evolved pressure tolerance must be accompanied with an important reduction in the volume change associated with pressure‐promoted alteration of enzymatic activity (). Our results suggest an important revision to this paradigm. Here, we describe an opposite effect of pressure adaptation—a substantial increase in the absolute value ofin deep‐living species compared to shallow‐water counterparts. With this change, the enzyme activities in abyssal and hadal species do not substantially decrease their activity with pressure increasing up to 1–2 kbar, well beyond full‐ocean depth pressures. In contrast, the activity of the enzyme from the tidepool snailfish,L. florae, decreases nearly linearly from 1 to 2500 bar. The increased tolerance of LDH activity to pressure comes at the expense of decreased catalytic efficiency, which is compensated with increased enzyme contents in high‐pressure‐adapted species. The newly discovered strategy is presumably used when the enzyme mechanism involves the formation of potentially unstable excited transient states associated with substantial changes in enzyme–solvent interactions.

     
    more » « less
  3. ; ( , Physical Chemistry Chemical Physics)
    Correction for ‘High pressure single-molecule FRET studies of the lysine riboswitch: cationic and osmolytic effects on pressure induced denaturation’ by Hsuan-Lei Sung et al. , Phys. Chem. Chem. Phys. , 2020, DOI: 10.1039/d0cp01921f. 
    more » « less
  4. ; ( , Proceedings of the National Academy of Sciences)

    Single amino acid mutations provide quantitative insight into the energetics that underlie the dynamics and folding of membrane proteins. Chemical denaturation is the most widely used assay and yields the change in unfolding free energy (ΔΔG). It has been applied to >80 different residues of bacteriorhodopsin (bR), a model membrane protein. However, such experiments have several key limitations: 1) a nonnative lipid environment, 2) a denatured state with significant secondary structure, 3) error introduced by extrapolation to zero denaturant, and 4) the requirement of globally reversible refolding. We overcame these limitations by reversibly unfolding local regions of an individual protein with mechanical force using an atomic-force-microscope assay optimized for 2 μs time resolution and 1 pN force stability. In this assay, bR was unfolded from its native bilayer into a well-defined, stretched state. To measure ΔΔG, we introduced two alanine point mutations into an 8-amino-acid region at the C-terminal end of bR’s G helix. For each, we reversibly unfolded and refolded this region hundreds of times while the rest of the protein remained folded. Our single-molecule–derived ΔΔGfor mutant L223A (−2.3 ± 0.6 kcal/mol) quantitatively agreed with past chemical denaturation results while our ΔΔGfor mutant V217A was 2.2-fold larger (−2.4 ± 0.6 kcal/mol). We attribute the latter result, in part, to contact between Val217and a natively bound squalene lipid, highlighting the contribution of membrane protein–lipid contacts not present in chemical denaturation assays. More generally, we established a platform for determining ΔΔGfor a fully folded membrane protein embedded in its native bilayer.

     
    more » « less
  5. ; ; ( , Proteins: Structure, Function, and Bioinformatics)
    Abstract

    Hydrostatic pressure has a vital role in the biological adaptation of the piezophiles, organisms that live under high hydrostatic pressure. However, the mechanisms by which piezophiles are able to adapt their proteins to high hydrostatic pressure is not well understood. One proposed hypothesis is that the volume changes of unfolding (ΔVTot) for proteins from piezophiles is distinct from those of nonpiezophilic organisms. Since ΔVTotdefines pressure dependence of stability, we performed a comprehensive computational analysis of this property for proteins from piezophilic and nonpiezophilic organisms. In addition, we experimentally measured the ΔVTotof acylphosphatases and thioredoxins belonging to piezophilic and nonpiezophilic organisms. Based on this analysis we concluded that there is no difference in ΔVTotfor proteins from piezophilic and nonpiezophilic organisms. Finally, we put forward the hypothesis that increased concentrations of osmolytes can provide a systemic increase in pressure stability of proteins from piezophilic organisms and provide experimental thermodynamic evidence in support of this hypothesis.

     
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email