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.

Protein charge ladders reveal that the net charge of ALS‐linked superoxide dismutase can be different in sign and magnitude from predicted values

“…In the current study, we only examined the electrostatic effects of formally non-isoelectric ALSlinked amino acid substitutions. When considering the strong correlation between the structural environment of an ionizable amino acid residue and its pK a (a point that has been recognized for over 50 years 54 ) it is reasonable to hypothesize that a destabilizing isoelectric ALS-linked mutation such as A4V-which alters the structural environment of at least 20 residues in the apo-SOD1 dimer 55,56 and raises pI values during native isoelectric focusing 28 by up to DpI 5 10.25-might lower the net charge of SOD1 by a degree that will increase its propensity to self-assemble into toxic oligomers.…”

“…In cases where accurate measurements have been made, the measured values (denoted Z CE in this article) can differ in magnitude by up to seven-fold from predicted values that are calculated from the standard pK a of side-chains (denoted Z seq ), presumably because of anomalous pK a . 24,25 The isoelectric points of a few ALS-variants have been measured, [26][27][28][29][30] however, pI is not an expression of-and does not scale with-net charge at pH 6 ¼ pI. 24 Experimentally measuring the net charge of ALS-variant SOD1 instead of predicting it is especially important because SOD1 is an electrostatically peculiar metalloenzyme.…”

Reduced net charge and heterogeneity of pI isoforms in familial amyotrophic lateral sclerosis mutants of copper/zinc superoxide dismutase

“…In our study, copper and zinc ratios in active hWT and fALS mutant p I isoforms were determined by PIXE analysis using native IEF protocols as previously described (Fig. ).…”

“…Cells were collected and washed twice in cold 20 mM Tris‐HCl pH 7.4 and once in ultrapure water. Protein extraction was performed under non‐denaturating conditions, without demetallation/remetallation steps . Soluble protein extracts were prepared at 4°C by glass‐bead lysis of the cell pellet resuspended in an equal volume of 20 mM Tris‐HCl pH 7.4, 1 mM PMSF and 1 mM benzamidine.…”

“…For each protein extract, native IEF were performed simultaneously with typically eight IPG strips from the same batch. Three IPG strips were used for the identification of active isoforms by nitroblue tetrazolium (NBT) assay according to published methods . Mean p I value was determined according to a linear pH gradient.…”