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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.


Plot of the change in partial molar volume ΔV on unfolding against temperature. Best-fit to eq. 7 gives a change in expansivity Δα on unfolding (260~320 K, pH 2) to be 1.07 ml mol−1 deg−1.
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f4-5_1: Plot of the change in partial molar volume ΔV on unfolding against temperature. Best-fit to eq. 7 gives a change in expansivity Δα on unfolding (260~320 K, pH 2) to be 1.07 ml mol−1 deg−1.

Mentions: Figure 4 gives the plot of ΔV=VU–VN, as obtained from the fit in Figure 3 (right) against temperature, which are all negative within the temperature range studied (−10°C~ 50°C), but with a significant temperature dependence. From the slope, we obtain as the expansivity change upon unfolding Δα=1.07 ml mol−1 deg−1 in eq. 7. This value is comparable to those in staphylococcal nuclease (1.33 ml mol−1 deg−1)28 as well as in metmyoglobin (1.8 ml mol−1 deg−1)4 and in ribonuclease A (1.32 ml mol−1 deg−1)29. The positive value of Δα is taken to indicate the increased thermal volume due to the increased exposure of the polypeptide chain upon unfolding22.


The pressure-temperature phase diagram of hen lysozyme at low pH
Plot of the change in partial molar volume ΔV on unfolding against temperature. Best-fit to eq. 7 gives a change in expansivity Δα on unfolding (260~320 K, pH 2) to be 1.07 ml mol−1 deg−1.
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Related In: Results  -  Collection

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

f4-5_1: Plot of the change in partial molar volume ΔV on unfolding against temperature. Best-fit to eq. 7 gives a change in expansivity Δα on unfolding (260~320 K, pH 2) to be 1.07 ml mol−1 deg−1.
Mentions: Figure 4 gives the plot of ΔV=VU–VN, as obtained from the fit in Figure 3 (right) against temperature, which are all negative within the temperature range studied (−10°C~ 50°C), but with a significant temperature dependence. From the slope, we obtain as the expansivity change upon unfolding Δα=1.07 ml mol−1 deg−1 in eq. 7. This value is comparable to those in staphylococcal nuclease (1.33 ml mol−1 deg−1)28 as well as in metmyoglobin (1.8 ml mol−1 deg−1)4 and in ribonuclease A (1.32 ml mol−1 deg−1)29. The positive value of Δα is taken to indicate the increased thermal volume due to the increased exposure of the polypeptide chain upon unfolding22.

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.