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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.


(A) 1H NMR spectra (800 MHz) of hen lysozyme recorded at various pressures from 100MPa to 400MPa at −5°C. The spectrum at 0.1MPa (bottom) was obtained at 25°C at 600MHz. The spectrum at 0.1MPa (top) was also obtained at 25°C, but in the presence of 8M urea. Chemical shifts are referenced to dioxane δ= 3.70 ppm. All the protein solutions were prepared in 50 mM maleate buffer, 90% 1H2O/10% 2H2O (pH 2). (B) The fraction of unfolding against pressure as estimated from the fractional decrease of the combined intensity of high-field shifted methyl 1H NMR signals of Leu17, Thr51, Ile56, Ile88 and Ile98 (δ=−0.3~0.8 ppm)26 (open circles) and from the red shift of the wavelength of maximum fluorescence emission (λmax) (closed circles). The solid curve is drawn by best-fitting the fluorescence data to eq. 8.
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f2-5_1: (A) 1H NMR spectra (800 MHz) of hen lysozyme recorded at various pressures from 100MPa to 400MPa at −5°C. The spectrum at 0.1MPa (bottom) was obtained at 25°C at 600MHz. The spectrum at 0.1MPa (top) was also obtained at 25°C, but in the presence of 8M urea. Chemical shifts are referenced to dioxane δ= 3.70 ppm. All the protein solutions were prepared in 50 mM maleate buffer, 90% 1H2O/10% 2H2O (pH 2). (B) The fraction of unfolding against pressure as estimated from the fractional decrease of the combined intensity of high-field shifted methyl 1H NMR signals of Leu17, Thr51, Ile56, Ile88 and Ile98 (δ=−0.3~0.8 ppm)26 (open circles) and from the red shift of the wavelength of maximum fluorescence emission (λmax) (closed circles). The solid curve is drawn by best-fitting the fluorescence data to eq. 8.

Mentions: 1H one-dimensional NMR measurements were carried out on hen lysozyme (pH 2) at varying pressures up to 400MPa, the highest pressure available in the current high pressure NMR system at 800MHz at various temperatures. Except at subzero temperatures, e.g. at −5°C, we could not attain full unfolding even at 400MPa. In Figure 2A, the 1H NMR spectra recorded at −5°C above 100MPa only to avoid freezing are shown. The spectrum at 0.1MPa was recorded at 25°C and is shown in Figure 2A (bottom). The spectrum for the “fully unfolded” structure was recorded also at 25°C at 0.1MPa in the presence of 8M urea and is also shown in Figure 2A (top).


The pressure-temperature phase diagram of hen lysozyme at low pH
(A) 1H NMR spectra (800 MHz) of hen lysozyme recorded at various pressures from 100MPa to 400MPa at −5°C. The spectrum at 0.1MPa (bottom) was obtained at 25°C at 600MHz. The spectrum at 0.1MPa (top) was also obtained at 25°C, but in the presence of 8M urea. Chemical shifts are referenced to dioxane δ= 3.70 ppm. All the protein solutions were prepared in 50 mM maleate buffer, 90% 1H2O/10% 2H2O (pH 2). (B) The fraction of unfolding against pressure as estimated from the fractional decrease of the combined intensity of high-field shifted methyl 1H NMR signals of Leu17, Thr51, Ile56, Ile88 and Ile98 (δ=−0.3~0.8 ppm)26 (open circles) and from the red shift of the wavelength of maximum fluorescence emission (λmax) (closed circles). The solid curve is drawn by best-fitting the fluorescence data to eq. 8.
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Related In: Results  -  Collection

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f2-5_1: (A) 1H NMR spectra (800 MHz) of hen lysozyme recorded at various pressures from 100MPa to 400MPa at −5°C. The spectrum at 0.1MPa (bottom) was obtained at 25°C at 600MHz. The spectrum at 0.1MPa (top) was also obtained at 25°C, but in the presence of 8M urea. Chemical shifts are referenced to dioxane δ= 3.70 ppm. All the protein solutions were prepared in 50 mM maleate buffer, 90% 1H2O/10% 2H2O (pH 2). (B) The fraction of unfolding against pressure as estimated from the fractional decrease of the combined intensity of high-field shifted methyl 1H NMR signals of Leu17, Thr51, Ile56, Ile88 and Ile98 (δ=−0.3~0.8 ppm)26 (open circles) and from the red shift of the wavelength of maximum fluorescence emission (λmax) (closed circles). The solid curve is drawn by best-fitting the fluorescence data to eq. 8.
Mentions: 1H one-dimensional NMR measurements were carried out on hen lysozyme (pH 2) at varying pressures up to 400MPa, the highest pressure available in the current high pressure NMR system at 800MHz at various temperatures. Except at subzero temperatures, e.g. at −5°C, we could not attain full unfolding even at 400MPa. In Figure 2A, the 1H NMR spectra recorded at −5°C above 100MPa only to avoid freezing are shown. The spectrum at 0.1MPa was recorded at 25°C and is shown in Figure 2A (bottom). The spectrum for the “fully unfolded” structure was recorded also at 25°C at 0.1MPa in the presence of 8M urea and is also shown in Figure 2A (top).

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.