Putting the Piezolyte Hypothesis under Pressure

Abstract

A group of small molecules that stabilize proteins against high hydrostatic pressure has been classified as piezolytes, a subset of stabilizing cosolutes. This distinction would imply that piezolytes counteract the effects of high hydrostatic pressure through effects on the volumetric properties of the protein. The purpose of this study was to determine if cosolutes proposed to be piezolytes have an effect on the volumetric properties of proteins through direct experimental measurements of volume changes upon unfolding of model proteins lysozyme and ribonuclease A, in solutions containing varying cosolute concentrations. Solutions containing the proposed piezolytes glutamate, sarcosine, and betaine were used, as well as solutions containing the denaturants guanidinium hydrochloride and urea. Changes in thermostability were monitored using differential scanning calorimetry whereas changes in volume were monitored using pressure perturbation calorimetry. Our findings indicate that increasing stabilizing cosolute concentration increases the stability and transition temperature of the protein, but does not change the temperature dependence of volume changes upon unfolding. The results suggest that the pressure stability of a protein in solution is not directly affected by the presence of these proposed piezolytes, and so they cannot be granted this distinction.

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

“…Stabilizing osmolytes, such as trimethylamine oxide (TMAO), glutamate, betaine, alanine, β‐hydroxybutyrate (β‐HB), mannosylglycerate, di‐myo‐inositol phosphate and N‐Acetyl‐β‐lysine, are present in high concentrations in many piezophilic organisms . These molecules were shown to increase protein stability (see, eg, References ) and this increase in stability is not accompanied by volume changes . Such increase in stability may have a physiological role in piezophiles.…”

“…B, Extant thioredoxins: Trx‐SB—green; Trx‐EC—black; Trx‐TT—gray; C, Experimentally derived P‐T diagrams for RNAseA in solution containing various concentrations of osmolytes; 2 M betaine—cyan; 2 M sarcosine—pink; 2 M glutamate—orange, or no osmolyte—black. P‐T diagrams on (A) and (B) were calculated using Equation ((8) from the experimental data shown in Figure S1 Source : (C) is adopted with modifications from Reference [Color figure can be viewed at wileyonlinelibrary.com]…”

“…9,51,52 These molecules were shown to increase protein stability (see, eg, References 53-60) and this increase in stability is not accompanied by volume changes. 39 Such increase in stability may have a physiological role in piezophiles. For example, Photobacterium profundum is a well-studied facultative piezophile that grows optimally at 200 to 300 bars of pressure but is also able to grow at atmospheric pressures.…”

“…Thermodynamic parameters obtained from DSC and PPC experiments were used to compute the stability of a protein using the following equation: where ΔH is the enthalpy of unfolding at Tm, Δ V is the volume change upon unfolding at T m , and p o is the reference pressure (0.1 MPa). The Δ c p is the change in heat capacity (calculated from the temperature dependence of enthalpy of unfolding see Figure S1), Δ α is the change in thermal expansivity (calculated from the temperature dependence of the volume changes upon unfolding see Figure S1), the Δ β is the change in compressibility upon unfolding, taken from Reference . A pressure‐temperature (P‐T) stability phase diagram can be generated by setting the Δ G ( p , T ) = 0 and defines the boundaries of stability for the protein (Figure ) The area under the line corresponds to the pressure and temperature ranges in which the protein will be largely in the native state.…”

The Genetics, Biochemistry, and Biophysics of Carbon Cycling by Deep Life

“…there are no specific volumetric effects associated with low-molecular-weight osmolytes). Rather, their effect is simply to stabilize folded proteins against all manner of perturbations, not specifically high hydrostatic pressure (172). It is not well understood whether there are inherent, specific physical attributes of proteins and nucleic acids that allow them to tolerate high-pressure environments.…”