Sylviane Chevreux

Publications (3)6.33 Total impact

• Article: Coupling of native IEF and extended X-ray absorption fine structure to characterize zinc-binding sites from pI isoforms of SOD1 and A4V pathogenic mutant.

  Sylviane Chevreux, Isabelle Llorens, Pier Lorenzo Solari, Stéphane Roudeau, Guillaume Devès, Asuncion Carmona, Denis Testemale, Jean-Louis Hazemann, Richard Ortega
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  ABSTRACT: Extended X-ray absorption fine structure (EXAFS) has already provided high-resolution structures of metal-binding sites in a wide variety of metalloproteins. Usually, EXAFS is performed on purified metalloproteins either in solution or crystallized form but purification steps are prone to modify the metallation state of the protein. We developed a protocol to couple EXAFS analysis to metalloprotein separation using native gel electrophoresis. This coupling opens a large field of applications as metalloproteins can be characterized in their native state avoiding purification steps. Using native isoelectric focusing, the method enables the EXAFS analysis of metalloprotein pI isoforms. We applied this methodology to SOD1, wild-type, and Ala4Val mutant (A4V), a mutation found in amyotrophic lateral sclerosis (ALS) because decreased Zn affinity to SOD1 mutants is suggested to be involved in the pathogenesis of this neurodegenerative disease. We observed similar coordination structures for Zn in wild-type and mutant proteins, in all measured pI isoforms, demonstrating the feasibility of EXAFS on electrophoresis gels and suggesting that the Zn-binding site is not structurally modified in A4V SOD1 mutant.
  Electrophoresis 04/2012; 33(8):1276-81. · 3.30 Impact Factor
• Article: EXAFS analysis of a human Cu,Zn SOD isoform focused using non-denaturing gel electrophoresis

  Sylviane Chevreux, Pier Lorenzo Solari, Stéphane Roudeau, Guillaume Deves, Isabelle Alliot, Denis Testemale, Jean Louis Hazemann, Richard Ortega
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  ABSTRACT: Isoelectric point isoforms of a metalloprotein, copper-zinc superoxide dismutase (CuZnSOD), separated on electrophoresis gels were analyzed using X-ray Absorption Spectroscopy. Mutations of this protein are involved in familial cases of amyotrophic lateral sclerosis. The toxicity of mutants could be relied to defects in the metallation state. Our purpose is to establish analytical protocols to study metallation state of protein isoforms such as those from CuZnSOD. We previously highlighted differences in the copper oxidation state between CuZnSOD isoforms using XANES. Here, we present the first results for EXAFS analyses performed at Cu and Zn K-edge on the majoritary expressed isoform of human CuZnSOD separated on electrophoresis gels.
  Journal of Physics Conference Series 11/2009; 190(1):012205.
• Article: Multimodal analysis of metals in copper-zinc superoxide dismutase isoforms separated on electrophoresis gels.

  Sylviane Chevreux, Stéphane Roudeau, Aurélien Fraysse, Asunción Carmona, Guillaume Devès, Pier Lorenzo Solari, Sandra Mounicou, Ryszard Lobinski, Richard Ortega
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  ABSTRACT: It has been suggested that copper-zinc superoxide dismutase (CuZnSOD) isoforms of distinct isoelectric point (pI) could result from differences in their metallation state. Our aim was then to develop and validate analytical methods for the determination and understanding of metallation states in human CuZnSOD isoforms. To avoid metal losses during sample preparation steps, CuZnSOD isoforms were separated according to their pI using non-denaturing isoelectric focusing (IEF) gel electrophoresis. Metal quantification was directly performed in-gel. Cu/Zn ratios of CuZnSOD isoforms were quantified by Particle-Induced X-ray Emission (PIXE) and Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry (LA-ICP-MS). Cu/Zn ratios were measured close to the value of 1 as expected from the known stoichiometry of CuZnSOD with slight, but statistically significant, differences between acidic and basic isoforms. Overall, this study demonstrates that metal quantification can be performed directly on metalloproteins separated on electrophoresis gels.
  Biochimie 07/2009; 91(10):1324-7. · 3.02 Impact Factor