Dityrosine cross-linking

Dityrosine cross-links are widely observed in nature in structural proteins such as elastin and silk. Natural oxidative cross-linking between tyrosine residues is catalyzed by a diverse group of metalloenzymes. Dityrosine formation is also catalyzed in vitro by metal-peptide complexes such as Gly-Gly-His-Ni(II). The aim of this dissertation is to exploit the unique redox chemistry of tyrosine using a chemical and electrochemical stimulus to specifically immobilize proteins in an oriented and covalent manner. Tyrosine also adheres to various substrates possibly through DOPA conversion.;A novel method was also developed for modifying gold electrode surfaces through electrochemically triggered adsorption of acrylamide copolymers containing a low percentage of tyrosineamide side chains. The amount of copolymer adsorbed, as monitored by SPR, was proportional to the percent of tyrosineamide side chains in the polymer over the range 0–3 mol %. The modified gold surfaces were hydrophilic and resisted nonspecific adsorption of GFP. Incorporation of NTA side chains into the tyrosineamide copolymers allowed specific immobilization of His6-tagged GFP. The Ni(II)-dependent GFP binding was measured by SPR and verified by fluorescence microscopy. The method may find utility as a means to electrically address the immobilization of unique ligands in biosensors or other diagnostic devices based on arrayed ligands.;On the basis of these observations a system was developed to specifically and covalently surface immobilize proteins through dityrosine cross-links. Methacrylate monomers of the catalytic peptide Gly-Gly-His-Tyr-OH (GGHY) and the Ni(II)-chelating group nitrilotriacetic acid (NTA) were copolymerized with acrylamide into microbeads. Green fluorescent protein (GFP), as a model protein, was genetically tagged with a tyrosine-modified His6 peptide on its carboxy terminus. GFP-YGH6, specifically associated with the NTA-Ni(II) groups, and was covalently coupled to the bead surface through dityrosine bond formation catalyzed by the colocalized GGHY-Ni(II) complex. After extensive washing with EDTA to disrupt metal coordination bonds, we observed that up to 75% of the initially bound GFP-YGH6 remained covalently bound to the bead while retaining its structure and activity. Dityrosine cross-linking was confirmed by quenching the reaction with free tyrosine. This oxidative property of tyrosine was also used to crosslink water-soluble polymers into hydrogels upon addition of an oxidant.