Di-Sulfide Bond Localization

Di-Sulfide Bond Localization • Introduction • Disulfide bond is a single covalent bond formed between the sulfur atoms of cysteines. The other sulfur-containing amino acid, methionine, cannot form disulfide bonds. Because it is a covalent bond, disulfide bond is often considered to be the primary structure. However, the function of disulfide bonds are far more than components of primary protein structure, they play a very important role in stabilizing the tertiary and quartenary structures, and are the prerequisite of proteins’ proper biological function.

• Function of disulfide bonds • At tertiary structure level: Disulfide bonds are formed inside the protein subunit at this level. Disulfide bonds play an important role in the folding and stability of some proteins, usually before proteins are secreted to the extracellular medium. The disulfide bond may form the nucleus of a hydrophobic core of the folded protein, i.e., local hydrophobic residues may condense around the disulfide bond and onto each other through hydrophobic interactions. • At quartenary structure level: Disulfide bonds are formed between two protein subunits. This type of disulfide bonds hold two portions of the protein together, biasing the protein towards the folded topology. That is, the disulfide bond destabilizes the unfolded form of the protein by lowering its entropy. The disulfide bond increases the effective local concentration of protein residues and lowers the effective local concentration of water molecules. Since water molecules attack amide-amide hydrogen bonds and break up secondary structure, a disulfide bond stabilizes secondary structure in its vicinity. For example, researchers have identified several pairs of peptides that are unstructured in isolation, but adopt stable secondary and tertiary structure upon forming a disulfide bond between them.

• Given the ability of stabilizing overall structure of proteins, disulfide bridges are cross-linked in many commercialized proteins to increase their resistance to destructive effects of extreme environment used in industrial processes or protect protein-based therapeutics from rapid proteolytic degradation. Manufacturing of these products must take into account oxidative refolding—a formation of native disulfide bonds by specific pairs of cysteines located throughout a sequence of linear protein.

• Creative Proteomics has established a highly sensitive HPLC-MS/MS platform that can analyze disulfide bonds in multiple samples and in both eukaryotic and prokaryotic organisms. In addition, we have optimized our protocol to enable more fast and sensitive site mapping service for disulfide bond analysis.