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Coupling of native IEF and extended X ‐ray absorption fine structure to characterize zinc‐binding sites from p I isoforms of SOD 1 and A 4 V pathogenic mutant
Author(s) -
Chevreux Sylviane,
Llorens Isabelle,
Solari Pier Lorenzo,
Roudeau Stéphane,
Devès Guillaume,
Carmona Asuncion,
Testemale Denis,
Hazemann JeanLouis,
Ortega Richard
Publication year - 2012
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201100596
Subject(s) - extended x ray absorption fine structure , metalloprotein , chemistry , mutant , isoelectric focusing , gene isoform , zinc , crystallography , absorption spectroscopy , biochemistry , enzyme , organic chemistry , physics , quantum mechanics , gene
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 p I isoforms. We applied this methodology to SOD 1, wild‐type, and A la4 V al mutant ( A 4 V ), a mutation found in amyotrophic lateral sclerosis ( ALS ) because decreased Z n affinity to SOD 1 mutants is suggested to be involved in the pathogenesis of this neurodegenerative disease. We observed similar coordination structures for Z n in wild‐type and mutant proteins, in all measured p I isoforms, demonstrating the feasibility of EXAFS on electrophoresis gels and suggesting that the Z n‐binding site is not structurally modified in A 4 V SOD 1 mutant.