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The pressure-temperature phase diagram of hen lysozyme at low pH

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ABSTRACT

The equilibrium unfolding of hen lysozyme at pH 2 was studied as a function of pressure (0.1~700MPa) and temperature (−10°C~50°C) using Trp fluorescence as monitor supplemented by variable pressure 1H NMR spectroscopy (0.1~400MPa). The unfolding profiles monitored by the two methods allowed the two-state equilibrium analysis between the folded (N) and unfolded (U) conformers. The free energy differences ΔG (=GU–GN) were evaluated from changes in the wavelength of maximum fluorescence intensity (λmax) as a function of pressure and temperature. The dependence of ΔG on temperature exhibits concave curvatures against temperature, showing positive heat capacity changes (ΔCp=CpU–CpN= 1.8–1.9 kJ mol−1 deg−1) at all pressures studied (250~400 MPa), while the temperature TS for maximal ΔG increased from about 10°C at 250MPa to about 40°C at 550MPa. The dependence of ΔG on pressure gave negative volume changes (ΔV=VU–VN) upon unfolding at all temperatures studied (−86~−17 mlmol−1 for −10°C~50°C), which increase significantly with increasing temperature, giving a positive expansivity change (Δα~1.07mlmol−1 deg−1). A phase-diagram between N and U (for ΔG=0) is drawn of hen lysozyme at pH 2 on the pressure-temperature plane. Finally, a three-dimensional free energy landscape (ΔG) is presented on the p-T plane.

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Plots of the change in free energy ΔG on unfolding for hen lysozyme (pH 2) at various pressures. The solid lines show best-fit of eq. 10 to the experimental points, with melting temperature (Tm), enthalpy change at Tm (ΔHm) and heat capacity change on unfolding (ΔCp) as fitting parameters listed in Table 2.
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f5-5_1: Plots of the change in free energy ΔG on unfolding for hen lysozyme (pH 2) at various pressures. The solid lines show best-fit of eq. 10 to the experimental points, with melting temperature (Tm), enthalpy change at Tm (ΔHm) and heat capacity change on unfolding (ΔCp) as fitting parameters listed in Table 2.

Mentions: In Figure 5, we plotted the experimentally determined values of ΔG (eq. 4) against temperature at constant pressures, which depict concave features at all pressures studied. Data are limited to above 200MPa, as no significant fraction unfolds in the lower pressure range to give sufficiently reliable ΔG values below 200MPa. The plots were best-fitted with eq. 10, giving parameters of ΔCp, TS (the temperature for ΔS=0), Tm, ΔHm and ΔSm (for both heat and cold denaturations) as summarized in Table 2. The Gibbs free energy changes (ΔG) are fitted reasonably well with a single positive ΔCp value at each pressure, covering both the cold denaturation and heat denaturation ranges. In general, a positive ΔCp upon unfolding is accepted as due to the exposure of nonpolar amino acid groups into the solvent water2. In accordance with this, ΔHm for heat denaturation increases with increasing Tm. Interestingly, while the stability is found to decrease with pressure, TS, the temperature of maximum stability (the temperature for ΔS=0) increases with increasing pressure.


The pressure-temperature phase diagram of hen lysozyme at low pH
Plots of the change in free energy ΔG on unfolding for hen lysozyme (pH 2) at various pressures. The solid lines show best-fit of eq. 10 to the experimental points, with melting temperature (Tm), enthalpy change at Tm (ΔHm) and heat capacity change on unfolding (ΔCp) as fitting parameters listed in Table 2.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC5036640&req=5

f5-5_1: Plots of the change in free energy ΔG on unfolding for hen lysozyme (pH 2) at various pressures. The solid lines show best-fit of eq. 10 to the experimental points, with melting temperature (Tm), enthalpy change at Tm (ΔHm) and heat capacity change on unfolding (ΔCp) as fitting parameters listed in Table 2.
Mentions: In Figure 5, we plotted the experimentally determined values of ΔG (eq. 4) against temperature at constant pressures, which depict concave features at all pressures studied. Data are limited to above 200MPa, as no significant fraction unfolds in the lower pressure range to give sufficiently reliable ΔG values below 200MPa. The plots were best-fitted with eq. 10, giving parameters of ΔCp, TS (the temperature for ΔS=0), Tm, ΔHm and ΔSm (for both heat and cold denaturations) as summarized in Table 2. The Gibbs free energy changes (ΔG) are fitted reasonably well with a single positive ΔCp value at each pressure, covering both the cold denaturation and heat denaturation ranges. In general, a positive ΔCp upon unfolding is accepted as due to the exposure of nonpolar amino acid groups into the solvent water2. In accordance with this, ΔHm for heat denaturation increases with increasing Tm. Interestingly, while the stability is found to decrease with pressure, TS, the temperature of maximum stability (the temperature for ΔS=0) increases with increasing pressure.

View Article: PubMed Central - PubMed

ABSTRACT

The equilibrium unfolding of hen lysozyme at pH 2 was studied as a function of pressure (0.1~700MPa) and temperature (−10°C~50°C) using Trp fluorescence as monitor supplemented by variable pressure 1H NMR spectroscopy (0.1~400MPa). The unfolding profiles monitored by the two methods allowed the two-state equilibrium analysis between the folded (N) and unfolded (U) conformers. The free energy differences ΔG (=GU–GN) were evaluated from changes in the wavelength of maximum fluorescence intensity (λmax) as a function of pressure and temperature. The dependence of ΔG on temperature exhibits concave curvatures against temperature, showing positive heat capacity changes (ΔCp=CpU–CpN= 1.8–1.9 kJ mol−1 deg−1) at all pressures studied (250~400 MPa), while the temperature TS for maximal ΔG increased from about 10°C at 250MPa to about 40°C at 550MPa. The dependence of ΔG on pressure gave negative volume changes (ΔV=VU–VN) upon unfolding at all temperatures studied (−86~−17 mlmol−1 for −10°C~50°C), which increase significantly with increasing temperature, giving a positive expansivity change (Δα~1.07mlmol−1 deg−1). A phase-diagram between N and U (for ΔG=0) is drawn of hen lysozyme at pH 2 on the pressure-temperature plane. Finally, a three-dimensional free energy landscape (ΔG) is presented on the p-T plane.

No MeSH data available.


Related in: MedlinePlus