Lactoperoxidase (LPO) belongs to the mammalian peroxidase family and catalyzes the oxidation of halides, pseudo-halides and a number of aromatic substrates (such as phenols, catechol(amine)s, arylamines) at the expense of hydrogen peroxide [1, 2]. It is part of a natural host defence system against bacterial infections found in milk, tears, saliva and other biological fluids, and is involved in carcinogenic mechanisms, inflammatory processes and in the pathogenesis of cystic fibrosis [3]. LPO is structurally complex as it is characterised by a bulky heme cofactor, covalently bound to the protein through two ester bonds; six disulfide linkages that keep the helices tightly packed around the heme group; a highly positively charged surface (pI 9) and several glycosidic residues, that makes the enzyme prone to intermolecular interactions crucial for its function. Despite the complex physiological role of LPO, very little data were available on the structural stability and folding of this protein. In order to get such information, we investigated the thermal and chemical denaturation pathways of LPO as well as the effects of pH on the tertiary and secondary structures [4, 5], by applying a pool of complementary biophysical techniques (UV-Vis absorption, Electron Paramagnetic Resonance, Circular Dichroism, Trp fluorescence, ANS-enhanced emission spectroscopy and DSC) and biochemical assays. The correlations existing between enzyme folding and catalytic proficiency have also been explored. Our experimental findings indicate that LPO is a particularly stable protein, capable of maintaining catalysis and structural integrity up to high temperature, high denaturant concentrations or extreme pH values. In addition, we found that LPO is characterized by a high degree of peripheral structural plasticity without perturbation of the heme-containing protein core even in the presence of non-optimal conditions. The possible physiological meaning of such features will be discussed.

A comprehensive unfolding study on bovine lactoperoxidase: a focus on the molecular determinants of its structural stability

GHIBAUDI, Elena Maria;BOSCOLO, BARBARA;
2009

Abstract

Lactoperoxidase (LPO) belongs to the mammalian peroxidase family and catalyzes the oxidation of halides, pseudo-halides and a number of aromatic substrates (such as phenols, catechol(amine)s, arylamines) at the expense of hydrogen peroxide [1, 2]. It is part of a natural host defence system against bacterial infections found in milk, tears, saliva and other biological fluids, and is involved in carcinogenic mechanisms, inflammatory processes and in the pathogenesis of cystic fibrosis [3]. LPO is structurally complex as it is characterised by a bulky heme cofactor, covalently bound to the protein through two ester bonds; six disulfide linkages that keep the helices tightly packed around the heme group; a highly positively charged surface (pI 9) and several glycosidic residues, that makes the enzyme prone to intermolecular interactions crucial for its function. Despite the complex physiological role of LPO, very little data were available on the structural stability and folding of this protein. In order to get such information, we investigated the thermal and chemical denaturation pathways of LPO as well as the effects of pH on the tertiary and secondary structures [4, 5], by applying a pool of complementary biophysical techniques (UV-Vis absorption, Electron Paramagnetic Resonance, Circular Dichroism, Trp fluorescence, ANS-enhanced emission spectroscopy and DSC) and biochemical assays. The correlations existing between enzyme folding and catalytic proficiency have also been explored. Our experimental findings indicate that LPO is a particularly stable protein, capable of maintaining catalysis and structural integrity up to high temperature, high denaturant concentrations or extreme pH values. In addition, we found that LPO is characterized by a high degree of peripheral structural plasticity without perturbation of the heme-containing protein core even in the presence of non-optimal conditions. The possible physiological meaning of such features will be discussed.
VIII European Symposium of the Protein Society of the Protein Society
Zurich ( Switzerland)
June 14-18 2009
Book of abstract of the VIII European Symposium of the Protein Society
The Protein Society
P298
P298
http://www.proteinsociety.org/
Protein folding; protein stability; ESR spectroscopy; CD spectroscopy; fluorescence spectroscopy
E. M. Ghibaudi; B. Boscolo; S. S. Leal; C. M. Gomes
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2318/96505
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