Radical stress affecting proteins is a matter of interest in life sciences. Because of its enhanced gene expression in organisms exposed to reactive oxygen species and related processes such as inflammation, tumours or apoptosis, metallothioneins (MTs), low molecular weight metalloproteins (6-7 kDa), characterized by a high content in Cys and high heavy metal binding capacity, are of upmost importance in this field. Some of these MTs have shown to have the capacity to release sulfur-centered radicals capable of damaging lipids in a biomimetic model of the cell membrane when attacked by radical reductive [1,2]. Overall, the general structural properties that characterize all MTs are the formation of metal-thiolate clusters involving terminal and bridging cysteinyl thiolate groups. Recently, it has been shown that also exogenous ligands such as inorganic ions (i.e. sulfide or chloride ions) can participate in the coordination sphere of metals in MTs. Some Zn-complexed MTs, representative of different MT families, enclosing plant MTs, have been analysed by Raman Spectroscopy [3]. Raman spectroscopy has resulted to be very useful in shedding light on the secondary structures eventually present in MTs and the ligands involved in metal coordination. The oxidation state of Cys residues and their participation in the metal chelation can be clearly defined (Figure 1), as well as the eventual involvement of His residues. Some MTs were undergone to free radical stress and the protein degradation due to radical exposure was evaluated by Raman spectroscopy [1,2]. In fact, Raman spectrum can provide valuable information on both amino acid side chains (i.e. S-S, Tyr, Trp, Cys-Metal) and conformational changes (as example Figure 1). Experiments were carried out in both aqueous solutions and vesicle suspensions. Free radical generation, mimicking an endogenous radical stress, was obtained by gamma-irradiation of aqueous solutions. By changing the appropriate conditions of irradiation, a selection of the reacting radical species was carried out. Protein structure and amino acid content resulted to play an important role in blocking the ready access of free radicals both to the sulfur-containing residues and the active site, so strongly affect both the radio-sensitivity of proteins. Thus, Raman can be an useful tool for providing information on the favourite sites of the radical attack and radical-induced modification in protein folding. In conclusion, Raman spectroscopy, in coupling with analytical techniques, can be one of a very promising experimental strategies in the research on new hints on MTs. References [1] Torreggiani A, Chatgilialoglu C., Ferreri C, Melchiorre M., Atrian S, Capdevila M J Proteomics 92, 204 (2013) [2] Torreggiani A, Domènech J, Orihuela R, Ferreri C, Atrian S, Capdevila M, Chatgilialoglu C. Chem Eur J 15 6015 (2009)
Metalloproteins under radical stress: an help from Raman spectroscopy
TINTI, ANNA
2014
Abstract
Radical stress affecting proteins is a matter of interest in life sciences. Because of its enhanced gene expression in organisms exposed to reactive oxygen species and related processes such as inflammation, tumours or apoptosis, metallothioneins (MTs), low molecular weight metalloproteins (6-7 kDa), characterized by a high content in Cys and high heavy metal binding capacity, are of upmost importance in this field. Some of these MTs have shown to have the capacity to release sulfur-centered radicals capable of damaging lipids in a biomimetic model of the cell membrane when attacked by radical reductive [1,2]. Overall, the general structural properties that characterize all MTs are the formation of metal-thiolate clusters involving terminal and bridging cysteinyl thiolate groups. Recently, it has been shown that also exogenous ligands such as inorganic ions (i.e. sulfide or chloride ions) can participate in the coordination sphere of metals in MTs. Some Zn-complexed MTs, representative of different MT families, enclosing plant MTs, have been analysed by Raman Spectroscopy [3]. Raman spectroscopy has resulted to be very useful in shedding light on the secondary structures eventually present in MTs and the ligands involved in metal coordination. The oxidation state of Cys residues and their participation in the metal chelation can be clearly defined (Figure 1), as well as the eventual involvement of His residues. Some MTs were undergone to free radical stress and the protein degradation due to radical exposure was evaluated by Raman spectroscopy [1,2]. In fact, Raman spectrum can provide valuable information on both amino acid side chains (i.e. S-S, Tyr, Trp, Cys-Metal) and conformational changes (as example Figure 1). Experiments were carried out in both aqueous solutions and vesicle suspensions. Free radical generation, mimicking an endogenous radical stress, was obtained by gamma-irradiation of aqueous solutions. By changing the appropriate conditions of irradiation, a selection of the reacting radical species was carried out. Protein structure and amino acid content resulted to play an important role in blocking the ready access of free radicals both to the sulfur-containing residues and the active site, so strongly affect both the radio-sensitivity of proteins. Thus, Raman can be an useful tool for providing information on the favourite sites of the radical attack and radical-induced modification in protein folding. In conclusion, Raman spectroscopy, in coupling with analytical techniques, can be one of a very promising experimental strategies in the research on new hints on MTs. References [1] Torreggiani A, Chatgilialoglu C., Ferreri C, Melchiorre M., Atrian S, Capdevila M J Proteomics 92, 204 (2013) [2] Torreggiani A, Domènech J, Orihuela R, Ferreri C, Atrian S, Capdevila M, Chatgilialoglu C. Chem Eur J 15 6015 (2009)I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
