Proteolysis of hen egg-white lysozyme by thermolysin in 50% aqueous trifluoroethanol for 6-24 h at 40-52 degrees C produces a 'nicked' protein species which was purified to homogeneity by reverse-phase HPLC and characterized. Protein chemistry analytical methods were used to establish that thermolysin cleaves the 129-residue chain of lysozyme at peptide bond Lys97-Ile98. Nicked lysozyme, which is therefore constituted by fragments 1-97 and 98-129 cross-linked by disulfide bonds, was approximately 20% and 60% active towards Micrococcus luteus cells in respect to native intact lysozyme when assayed at 25 degrees C or 5 degrees C, respectively. Circular dichroic measurements provided evidence that nicked lysozyme in aqueous buffer at low temperature maintains the secondary structure content of native lysozyme, whereas the microenvironment of the aromatic chromophores, in particular of tryptophan residue(s), was somewhat perturbed. The stability to heat and urea denaturation of nicked lysozyme was dramatically reduced with respect to that of the intact protein. For example, the tm of the nicked species was 28 degrees C in comparison with 73 degrees C for the unmodified enzyme, both at pH 7.0. Inspection of the X-ray structure of hen lysozyme reveals that thermolysin cleaves at the C-terminus of alpha-helix C (residues 88-98) located at the interface of the two structural domains of the protein, thus destabilizing the helix dipole and disrupting important tertiary interactions of the native enzyme. These results were interpreted considering that lysozyme in 50% aqueous trifluoroethanol is an expanded and flexible protein species largely maintaining native-like secondary structure, but lacking tertiary interactions [Buck, M., Radford, S. E. & Dobson, C. M. (1993) Biochemistry 32, 669-678]. Thus, whereas native lysozyme in its well-packed and rigid structure is quite resistant to proteolysis and only upon thermal unfolding is degraded to many small peptides in an all-or-none process, lysozyme in the trifluoroethanol state is sufficiently flexible to act as a substrate for the protease, but maintains significant secondary structure (helix) precluding extensive proteolytic degradation.

Limited Proteolysis of Lysozyme in Trifluoroethanol. Isolation and Characterization of a Partially Active Enzyme Derivative

POLVERINO DE LAURETO, PATRIZIA;DE FILIPPIS, VINCENZO;FONTANA, ANGELO
1995

Abstract

Proteolysis of hen egg-white lysozyme by thermolysin in 50% aqueous trifluoroethanol for 6-24 h at 40-52 degrees C produces a 'nicked' protein species which was purified to homogeneity by reverse-phase HPLC and characterized. Protein chemistry analytical methods were used to establish that thermolysin cleaves the 129-residue chain of lysozyme at peptide bond Lys97-Ile98. Nicked lysozyme, which is therefore constituted by fragments 1-97 and 98-129 cross-linked by disulfide bonds, was approximately 20% and 60% active towards Micrococcus luteus cells in respect to native intact lysozyme when assayed at 25 degrees C or 5 degrees C, respectively. Circular dichroic measurements provided evidence that nicked lysozyme in aqueous buffer at low temperature maintains the secondary structure content of native lysozyme, whereas the microenvironment of the aromatic chromophores, in particular of tryptophan residue(s), was somewhat perturbed. The stability to heat and urea denaturation of nicked lysozyme was dramatically reduced with respect to that of the intact protein. For example, the tm of the nicked species was 28 degrees C in comparison with 73 degrees C for the unmodified enzyme, both at pH 7.0. Inspection of the X-ray structure of hen lysozyme reveals that thermolysin cleaves at the C-terminus of alpha-helix C (residues 88-98) located at the interface of the two structural domains of the protein, thus destabilizing the helix dipole and disrupting important tertiary interactions of the native enzyme. These results were interpreted considering that lysozyme in 50% aqueous trifluoroethanol is an expanded and flexible protein species largely maintaining native-like secondary structure, but lacking tertiary interactions [Buck, M., Radford, S. E. & Dobson, C. M. (1993) Biochemistry 32, 669-678]. Thus, whereas native lysozyme in its well-packed and rigid structure is quite resistant to proteolysis and only upon thermal unfolding is degraded to many small peptides in an all-or-none process, lysozyme in the trifluoroethanol state is sufficiently flexible to act as a substrate for the protease, but maintains significant secondary structure (helix) precluding extensive proteolytic degradation.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11577/128191
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