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| - Selectivity of Methylation of Metal-Bound Cysteinates and ItsConsequences
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| - Alkylation of metal-bound cysteinate residues forms an integral step in both the activation of theDNA-damage sensing Ada protein from E. coli and the reaction mechanisms of several zinc-dependent enzymes.The roles of metal ions and the protein structure in regulating the reactivity of bound cysteinate residues is notwell-understood. Variants of a consensus zinc finger peptide were used to determine the effects of alkylationof cysteine residues on both metal binding and stability of the peptide structure. The ability of thioethers to actas ligands was probed through the direct synthesis of peptides with methionine or S-methylcysteine replacingthe second histidine within the zinc finger framework. This position can be substituted with cysteine with nosignificant loss of structure or stability. Two-dimensional 1H NMR studies and water exchange experimentsof the peptide with S-methylcysteine in this position showed that methylation affected the structure of thepeptide−zinc complex in the last turn of the helix, adjacent to the site of methylation, without disrupting therest of the structure. Titrations with cobalt revealed that the peptides with methionine or S-methylcysteine donot bind metal ions as tightly as do peptides with histidine or cysteine in this position. Similar to peptideslacking a fourth ligand, these thioether containing peptides form two-to-one peptide-to-cobalt complexes atlow metal concentrations. Alkylation of the cobalt complex of the peptide with cysteine as the fourth ligandwith dimethyl sulfate in aqueous solution yielded a product with absorption spectral features essentially identicalwith those of the S-methylcysteine derivative. Methylation of either of the other two cysteine residues withinthis peptide resulted in the loss of detectable metal binding. The carboxyl terminal cysteine was alkylated ata rate approximately 5-fold higher than the other cysteine residues, potentially due to the relative accessibilityof this cysteine sulfur compared with the others which are shielded by peptide amide to sulfur hydrogen bonds.Other studies suggest that all of the cysteine residues in this peptide are less prone to alkylation in the cobaltcomplex than they are in the unfolded, metal-free form under similar solution conditions. These results indicatethat thioether residues have a significantly lower affinity for cobalt(II) and zinc(II) than cysteine or histidine.Thus, significant modulation of metal-bound cysteinate reactivity can be achieved through the position of thecysteinate within the three-dimensional structure of a metal−peptide complex.
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