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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.

No MeSH data available.


The phase diagram of hen lysozyme at pH 2 as obtained by fluorescence measurements. Plot of pm (pressure at the midpoint of transition between N and U or half-denaturation) obtained from eq. 9 against Tm. The solid curve represents the best-fit of eq. 5 for ΔG=0 to the experimental points.
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f6-5_1: The phase diagram of hen lysozyme at pH 2 as obtained by fluorescence measurements. Plot of pm (pressure at the midpoint of transition between N and U or half-denaturation) obtained from eq. 9 against Tm. The solid curve represents the best-fit of eq. 5 for ΔG=0 to the experimental points.

Mentions: Figure 6 shows the phase diagram (for ΔG=0) of hen lysozyme for the first time in aqueous environment on the pressure-temperature plane. So far, the thermodynamic stability on the temperature-pressure plane in aqueous environment has been reported for a limited number of proteins, including metmyoglobin4 chymotrypsinogen30, ribonuclease A29 and Staphylococcal nuclease28,31–32. In these proteins, except for chymotrypsinogen which clearly gives a region of pressure-induced folding at elevated temperature30, an ellipsoid type pattern like that in Figure 6 with no regions of pressure-induced folding has been commonly observed, although the individual pattern is characteristic of each protein33.


The pressure-temperature phase diagram of hen lysozyme at low pH
The phase diagram of hen lysozyme at pH 2 as obtained by fluorescence measurements. Plot of pm (pressure at the midpoint of transition between N and U or half-denaturation) obtained from eq. 9 against Tm. The solid curve represents the best-fit of eq. 5 for ΔG=0 to the experimental points.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036640&req=5

f6-5_1: The phase diagram of hen lysozyme at pH 2 as obtained by fluorescence measurements. Plot of pm (pressure at the midpoint of transition between N and U or half-denaturation) obtained from eq. 9 against Tm. The solid curve represents the best-fit of eq. 5 for ΔG=0 to the experimental points.
Mentions: Figure 6 shows the phase diagram (for ΔG=0) of hen lysozyme for the first time in aqueous environment on the pressure-temperature plane. So far, the thermodynamic stability on the temperature-pressure plane in aqueous environment has been reported for a limited number of proteins, including metmyoglobin4 chymotrypsinogen30, ribonuclease A29 and Staphylococcal nuclease28,31–32. In these proteins, except for chymotrypsinogen which clearly gives a region of pressure-induced folding at elevated temperature30, an ellipsoid type pattern like that in Figure 6 with no regions of pressure-induced folding has been commonly observed, although the individual pattern is characteristic of each protein33.

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.