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

S. Chevreux I. Llorens 1 P. Lorenzo Solari S. Roudeau 2 Guillaume Devès 2 A. Carmona 2 Denis Testemale 3 Jean-Louis Hazemann 3 R. Ortega 2
1 SP2M - UMR 9002 - Service de Physique des Matériaux et Microstructures
2 IPCV - Interface Physique et Chimie pour le Vivant
CENBG - Centre d'Etudes Nucléaires de Bordeaux Gradignan
3 MRS - Matériaux, Rayonnements, Structure
NEEL - Institut Néel
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 tomodify 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
keyword : Zinc X-ray Absorption Spectroscopy Superoxide dismutase 1 Native IEF
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Journal articles
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http://hal.in2p3.fr/in2p3-00699389
Contributor : Nathalie Martin <>
Submitted on : Monday, May 21, 2012 - 11:08:39 AM
Last modification on : Thursday, August 22, 2019 - 11:16:01 AM

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S. Chevreux, I. Llorens, P. Lorenzo Solari, S. Roudeau, Guillaume Devès, et al.. 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. Electrophoresis, Wiley-VCH Verlag, 2012, 33, pp.1276-1281. ⟨10.1002/elps.201100596⟩. ⟨in2p3-00699389⟩

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