Metal catalyzed tyrosine-tyrosine crosslinking: Progress toward the site-specific, oriented immobilization of proteins to surfaces

The ability to covalently couple proteins to artificial surfaces in an oriented manner has many applications. Such applications include the immobilization of proteins to biosensor chip surfaces for the diagnosis of diseases, ligand binding interaction studies, and protein microarrays for proteomics applications. Current techniques available for immobilization of proteins on artificial surfaces have significant limitations. For example, nonspecifically adsorbing proteins to surfaces can result in improper orientation or denaturation of the protein. Covalently coupling proteins to surfaces by functional groups prevalent on the protein surface such as terminal carboxyl groups, primary amines, or sulfhydryl groups is also commonly practiced; however, this method can also result in the random orientation of the bound protein.; Metal coordination chemistry has also been used to immobilize proteins to surfaces by hexahistidine tags that can be genetically engineered into proteins, usually at either the amino or carboxy terminus. This method has the advantage that metal-chelating groups such as Ni+2-nitrilotriacetic acid (Ni+2-NTA) have strong affinity for the imidazole group on histidine (H) but not other amino acids in proteins. Therefore, the orientation of the protein after immobilization can be controlled by placement of a H tag in the protein. However, because the interaction between H residues and the metal-chelating groups are reversible, proteins tend to wash away from the surface after initial binding in microchip applications.; This dissertation focuses on the progress toward the development of a method for the oriented immobilization of proteins and peptides to artificial surfaces using metal coordination chemistry to initially attach the hexahistidine tagged proteins to the surface in an oriented manner and then convert the reversible linkage into a permanent covalent bond. The chemical basis for this approach is the addition of a tyrosine (Y) residue to the H tag of a recombinant protein or peptide and a Y residue placed in proximity to a surface-bound, metal-chelating molecule. Oxidation chemistry, preferably using the chelated metal as a catalyst, was then used to attempt to catalyze the formation of covalent Y-Y crosslinks.