In nature, highly crosslinked networks of proteins and other biopolymers are common materials. In many cases, dityrosine crosslinks form spontaneously after tyrosine sidechains are enzymatically oxidized into reactive intermediates within a restricted region. The overall objective of the proposed research is to develop a novel site-specific protein immobilization chemistry similar to natural dityrosine crosslinking mechanisms. Crosslinks between strategicallly placed phenolic groups (e.g., tyrosine) will be catalyzed in the presence of a mild oxidant by a metal complex between a synthetic metal ligand and a metal binding peptide genetically appended to the protein. Specifically, this objective will be pursued by: i.) solid-state synthesis of combinatorial libraries of peptidic metal binding ligands, ii.) rapid on-bead library screening with labelled tyrosine-containing model peptides to discover a ternary metal complex that catalyzes dityrosine formation, iii.) optimization of catalytically active leads through refined searching within the positive parameter space, and iv.) testing of active complexes with model proteins to demonstrate the utility of self-immobilizing proteins. The proposed chemistry may have major advantages over existing protein modification methodologies. First, the proposed method does not rely on diffusible reagents that react with all accessible members of a particular class of nucleophilic functional group. Rather, protein modification will be localized to specific sites determined by the pre-formation of a ternary metal complex. Second, the protein modification site is determined genetically, eliminating the need for post-translational modification and allowing specific protein immobilization from complex mixtures. An important health related application of the proposed technology will be more efficient immobilization of proteins into arrays on solid supports. The widely expected future impact of protein arrays on clinical diagnosis and other areas of human health care may be realized more quickly with new and effective protein modification technology.
| Endrizzi, Betsy J; Huang, Gang; Kiser, Patrick F et al. (2006) Specific covalent immobilization of proteins through dityrosine cross-links. Langmuir 22:11305-10 |
| Stayner, R Scott; Min, Dong-Joon; Kiser, Patrick F et al. (2005) Site-specific cross-linking of proteins through tyrosine hexahistidine tags. Bioconjug Chem 16:1617-23 |
