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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.


Trp fluorescence changes of hen lysozyme at pH 2 as a function of pressure at various temperatures. (A–G) (Left) Overlay of fluorescence spectra of hen lysozyme recorded as a function of pressure at various temperatures. The upward and downward arrows indicate whether the fluorescence intensity is increased or decreased with increasing pressure in the pressure range indicated. (A–G) (Right) Plots of the wavelength of maximum fluorescence intensity (λmax) as a function of pressure at various temperatures. The solid curves are the best-fit of eq. 8 to λmax, giving ΔG0 and ΔV (eq. 7) values at different temperatures, which are listed in Table 1.
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f3-5_1: Trp fluorescence changes of hen lysozyme at pH 2 as a function of pressure at various temperatures. (A–G) (Left) Overlay of fluorescence spectra of hen lysozyme recorded as a function of pressure at various temperatures. The upward and downward arrows indicate whether the fluorescence intensity is increased or decreased with increasing pressure in the pressure range indicated. (A–G) (Right) Plots of the wavelength of maximum fluorescence intensity (λmax) as a function of pressure at various temperatures. The solid curves are the best-fit of eq. 8 to λmax, giving ΔG0 and ΔV (eq. 7) values at different temperatures, which are listed in Table 1.

Mentions: Figure 3(A–G left) compiles the fluorescence spectral data from six Trp residues of hen lysozyme (35 μM in 50 mM maleate buffer, pH 2), measured as a function of pressure from 3MPa to 700 MPa at different temperatures (−5°C, −10°C, 5°C, 15°C, 25°C, 40°C and 50°C). In all cases, the fluorescence spectrum changes with pressure both in intensity and wavelength, which are fully reversible with pressure with respect to the maximum wavelength of emission (λmax) but less reversible (~80%) with respect to the intensity of emission. The lack of full reversibility is often encountered in high-pressure fluorescence experiments owing to some technical reasons. On the other hand, the shift in λmax of Trp fluorescence is considered to represent correctly the change in the microenvironment of the tryptophan ring22: The blue shift (λmax ~330 nm) indicates that the Trp ring is in the non-polar environment or buried in the hydrophobic core, while the red shift (λmax ~350–355 nm) indicates that the Trp ring is in the polar environment or exposed to the solvent water27. Although at 700MPa below ~5°C water is expected to go into ice VI, the smooth transitions in Figure 3A, B and C suggest that the solution went into the super-cooled state.


The pressure-temperature phase diagram of hen lysozyme at low pH
Trp fluorescence changes of hen lysozyme at pH 2 as a function of pressure at various temperatures. (A–G) (Left) Overlay of fluorescence spectra of hen lysozyme recorded as a function of pressure at various temperatures. The upward and downward arrows indicate whether the fluorescence intensity is increased or decreased with increasing pressure in the pressure range indicated. (A–G) (Right) Plots of the wavelength of maximum fluorescence intensity (λmax) as a function of pressure at various temperatures. The solid curves are the best-fit of eq. 8 to λmax, giving ΔG0 and ΔV (eq. 7) values at different temperatures, which are listed in Table 1.
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Related In: Results  -  Collection

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f3-5_1: Trp fluorescence changes of hen lysozyme at pH 2 as a function of pressure at various temperatures. (A–G) (Left) Overlay of fluorescence spectra of hen lysozyme recorded as a function of pressure at various temperatures. The upward and downward arrows indicate whether the fluorescence intensity is increased or decreased with increasing pressure in the pressure range indicated. (A–G) (Right) Plots of the wavelength of maximum fluorescence intensity (λmax) as a function of pressure at various temperatures. The solid curves are the best-fit of eq. 8 to λmax, giving ΔG0 and ΔV (eq. 7) values at different temperatures, which are listed in Table 1.
Mentions: Figure 3(A–G left) compiles the fluorescence spectral data from six Trp residues of hen lysozyme (35 μM in 50 mM maleate buffer, pH 2), measured as a function of pressure from 3MPa to 700 MPa at different temperatures (−5°C, −10°C, 5°C, 15°C, 25°C, 40°C and 50°C). In all cases, the fluorescence spectrum changes with pressure both in intensity and wavelength, which are fully reversible with pressure with respect to the maximum wavelength of emission (λmax) but less reversible (~80%) with respect to the intensity of emission. The lack of full reversibility is often encountered in high-pressure fluorescence experiments owing to some technical reasons. On the other hand, the shift in λmax of Trp fluorescence is considered to represent correctly the change in the microenvironment of the tryptophan ring22: The blue shift (λmax ~330 nm) indicates that the Trp ring is in the non-polar environment or buried in the hydrophobic core, while the red shift (λmax ~350–355 nm) indicates that the Trp ring is in the polar environment or exposed to the solvent water27. Although at 700MPa below ~5°C water is expected to go into ice VI, the smooth transitions in Figure 3A, B and C suggest that the solution went into the super-cooled state.

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.