RUI: Role and Formation of Tyrosine-Cysteine Protein Cofactors

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This award will investigate the ability of tyrosine and cysteine sidechains of proteins to crosslink through an arylalkylthioether bond (abbreviated as Tyr-Cys). Tyr-Cys crosslinks participate in oxidation catalysis and are formed by two proton-coupled oxidations. Metal-oxygen chemistry and the correct sidechain geometries are necessary for Tyr-Cys crosslink formation. Only four examples have been identified using X-ray crystallography that suggests limitations from the chemistry, sidechain geometries, or detection methods. A search of all protein structures in the protein databank showed many proteins with appropriate sidechain geometries for Tyr-Cys crosslink formation. Based on conditions for dityrosine formation, the chemistry for Tyr-Cys should be facile under biological conditions. Tyr-Cys identification by X-ray crystallography, however, requires careful analysis of high-resolution data. In order in better identify Tyr-Cys crosslinks a proteolysis and HPLC separation method will be employed using mass spectrometry and nuclear magnetic resonance detection. Cysteine dioxygnease contains a Tyr-Cys crosslink and will be examined for method validation. The three other proteins where Tyr-Cys have not been previously observed will be used to test if the combination of these detection methods and a previous protein databank search will expand the number of Tyr-Cys crosslinks simply examining static protein structure. This information, along with reexamining the function of Tyr-Cys in cystiene dioxygenase, will determine if the biological scope of Tyr-Cys containing proteins is significantly broader than currently thought.Broader impacts of this award come from the potential to expand the biological scope of the Tyr-Cys crosslink and parent crosslinked protein derived cofactors. Additionally, this award will use NSF-supported instrumentation and a newly renovated interdisciplinary laboratory. This work will prepare four undergraduate chemistry/biochemistry majors for graduate work in areas related to protein chemistry. This award will also provide indirect training for up to ten additional undergraduate researchers in a shared interdisciplinary research lab. Two high school students who are underrepresented minorities in the sciences will be mentored by the PI and co-PI in conjunction with ACS Project SEED.