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

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.