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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 structure of hen lysozyme in crystal (PDB ID; 135 L), which consists of two domains, α and β. The six tryptophan residues are colored green and the water-accessible internal cavity is shown by navy-spheres.
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f1-5_1: The structure of hen lysozyme in crystal (PDB ID; 135 L), which consists of two domains, α and β. The six tryptophan residues are colored green and the water-accessible internal cavity is shown by navy-spheres.

Mentions: Figure 1 shows the three-dimensional structure of hen lysozyme (wild-type) in crystal (PDB ID: 135L)16, and a similar structure has been reported in solution17. The folded structure consists of two domains, α and β, the α domain having a large hydrophobic core with a large water-containing cavity17. There are in total six tryptophan residues in the molecule, two of which (Trp 62 and 63) are found in the β domain and the rest are found in the α domain, of which Trp 28, 108 and 111 are close to the large cavity (Fig. 1). The fluorescence from the six Trp residues will be used for the thermodynamic analysis of equilibrium unfolding, while the side chain signals of 1H NMR spectrum will be used to monitor the cooperative transition.


The pressure-temperature phase diagram of hen lysozyme at low pH
The structure of hen lysozyme in crystal (PDB ID; 135 L), which consists of two domains, α and β. The six tryptophan residues are colored green and the water-accessible internal cavity is shown by navy-spheres.
© Copyright Policy
Related In: Results  -  Collection

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

f1-5_1: The structure of hen lysozyme in crystal (PDB ID; 135 L), which consists of two domains, α and β. The six tryptophan residues are colored green and the water-accessible internal cavity is shown by navy-spheres.
Mentions: Figure 1 shows the three-dimensional structure of hen lysozyme (wild-type) in crystal (PDB ID: 135L)16, and a similar structure has been reported in solution17. The folded structure consists of two domains, α and β, the α domain having a large hydrophobic core with a large water-containing cavity17. There are in total six tryptophan residues in the molecule, two of which (Trp 62 and 63) are found in the β domain and the rest are found in the α domain, of which Trp 28, 108 and 111 are close to the large cavity (Fig. 1). The fluorescence from the six Trp residues will be used for the thermodynamic analysis of equilibrium unfolding, while the side chain signals of 1H NMR spectrum will be used to monitor the cooperative transition.

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.