title: 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.
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.
authors.affiliation: CENBG, Centre d'Etudes Nucléaires de Bordeaux Gradignan, CNRS UMR5797, University Bordeaux 1, Gradignan, France.
authors.forename: Sylviane
authors.initials: S
authors.lastname: Chevreux
authors.forename: Isabelle
authors.initials: I
authors.lastname: Llorens
authors.forename: Pier Lorenzo
authors.initials: PL
authors.lastname: Solari
authors.forename: Stéphane
authors.initials: S
authors.lastname: Roudeau
authors.forename: Guillaume
authors.initials: G
authors.lastname: Devès
authors.forename: Asuncion
authors.initials: A
authors.lastname: Carmona
authors.forename: Denis
authors.initials: D
authors.lastname: Testemale
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Hazemann
authors.forename: Richard
authors.initials: R
authors.lastname: Ortega
mesh_terms: D000690:Amyotrophic Lateral Sclerosis / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods*; D009154:Mutation; D020033:Protein Isoforms; D013052:Spectrometry, X-Ray Emission; D013482:Superoxide Dismutase / Q000097:blood / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000072105:Superoxide Dismutase-1; D056928:X-Ray Absorption Spectroscopy / Q000379:methods*; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
chemical_list: D020033:Protein Isoforms; C000606290:SOD1 protein, human; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D015032:Zinc