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1. Single-molecule kinetic studies of DNA hybridization under extreme pressures

   https://doi.org/10.1039/D0CP04035E  

   Sung, Hsuan-Lei; Nesbitt, David J. (October 2020, 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. 

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2. Differential responses in the core, active site and peripheral regions of cytochrome c peroxidase to extreme pressure and temperature

   https://doi.org/10.1101/2024.07.24.604936  

   Zawistowski, Rebecca K; Crane, Brian R (July 2024, bioRxiv)

   In consideration of life in extreme environments, the effects of hydrostatic pressure on proteins at the atomic level have drawn substantial interest. Large deviations of temperature and pressure from ambient conditions can shift the free energy landscape of proteins to reveal otherwise lowly populated structural states and even promote unfolding. We report the crystal structure of the heme-containing peroxidase, cytochrome c peroxidase (CcP) at 1.5 and 3.0 kbar and make comparisons to structures determined at 1.0 bar and cryo-temperatures (100 K). Compressibility plateaus after 1.5 kbar and pressure produces anisotropic changes in CcP. CcP responds to pressure with volume declines at the periphery of the protein where B-factors are relatively high but maintains nearly intransient core structure and active site channels. Compression at the surface affects neither alternate side-chain conformers nor B-factors. Thus, packing in the core, which resembles a crystalline solid, limits motion and protects the active site, whereas looser packing at the surface preserves side-chain dynamics. Changes in active-site solvation and heme ligation reveal pressure sensitivity to protein-ligand interactions and reveal a potential docking site for the substrate peroxide. 

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3. Protein adaptation to high hydrostatic pressure: Computational analysis of the structural proteome

   https://doi.org/10.1002/prot.25839  

   Avagyan, Samvel; Vasilchuk, Daniel; Makhatadze, George I. (October 2019, 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 (ΔV_Tot) for proteins from piezophiles is distinct from those of nonpiezophilic organisms. Since ΔV_Totdefines 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 ΔV_Totof acylphosphatases and thioredoxins belonging to piezophilic and nonpiezophilic organisms. Based on this analysis we concluded that there is no difference in ΔV_Totfor 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. 

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4. Chetaev Instability Framework for Kinetostatic Compliance-Based Protein Unfolding

   https://doi.org/10.1109/LCSYS.2022.3176433  

   Mohammadi, A; Spong, Mark W. (January 2022, IEEE Control Systems Letters)

   Understanding the process of protein unfolding plays a crucial role in various applications such as design of folding-based protein engines. Using the well-established kinetostatic compliance (KCM)-based method for modeling of protein conformation dynamics and a recent nonlinear control theoretic approach to KCM-based protein folding, this letter formulates protein unfolding as a destabilizing control analysis/synthesis problem. In light of this formulation, it is shown that the Chetaev instability framework can be used to investigate the KCM-based unfolding dynamics. In particular, a Chetaev function for analysis of unfolding dynamics under the effect of optical tweezers and a class of control Chetaev functions for synthesizing control inputs that elongate protein strands from their folded conformations are presented. Based on the presented control Chetaev function, an unfolding input is derived from the Artstein-Sontag universal formula and the results are compared against optical tweezer-based unfolding. 

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5. Compliant mechanical response of the ultrafast folding protein EnHD under force

   https://doi.org/10.1038/s42005-022-01125-5  

   Reifs, Antonio; Ruiz Ortiz, Irene; Saa, Amaia Ochandorena; Schönfelder, Jörg; De Sancho, David; Muñoz, Victor; Perez-Jimenez, Raul (December 2023, Communications Physics)

   Abstract Ultrafast folding proteins have become an important paradigm in the study of protein folding dynamics. Due to their low energetic barriers and fast kinetics, they are amenable for study by both experiment and simulation. However, single molecule force spectroscopy experiments on these systems are challenging as these proteins do not provide the mechanical fingerprints characteristic of more mechanically stable proteins, which makes it difficult to extract information about the folding dynamics of the molecule. Here, we investigate the unfolding of the ultrafast protein Engrailed Homeodomain (EnHD) by single-molecule atomic force microscopy experiments. Constant speed experiments on EnHD result in featureless transitions typical of compliant proteins. However, in the force-ramp mode we recover sigmoidal curves that we interpret as a very compliant protein that folds and unfolds many times over a marginal barrier. This is supported by a simple theoretical model and coarse-grained molecular simulations. Our results show the ability of force to modulate the unfolding dynamics of ultrafast folding proteins. 

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