Investigation of the Ligand-binding Mechanism of Methionine Sulfoxide Reductase A of E.coli

Abstract

The rise of free oxygen in the atmosphere over 2.5 billion years ago made it possible for large land-based plants and animals to thrive. But oxygen, and the energy it provides, comes at great cost. Aerobic metabolism generates highly reactive intermediates and by-products in the form of hydroxyl radicals, superoxide anions and hydrogen peroxide. Within the cell, these reactive oxygen species attack biological macromolecules, producing covalent modifications that can affect both function and structure. One amino acid residue in proteins that is particularly sensitive to oxidation is the sulfur-containing side chain of methionine. Fortunately, methionine oxidation can be reversed by the actions of peptide methionine sulfoxide reductase (MsrA), which reduces methionine sulfoxide back to methionine and restores function to damaged proteins. We have used multiple spectroscopic techniques to investigate the mechanism by which MsrA recognizes and binds to a wide range of oxidized substrates in need of repair. Substrates studied include proteins, peptides and the non-steroidal anti-inflammatory drug Sulindac. Competition experiments with the fluorescent reporter ANS suggest the existence of weak, but specific, hydrophobic interactions between MsrA and unstructured and/or hydrophobic ligands.