RUI: Role and Formation of Tyrosine-Cysteine Protein Cofactors

Calvin University, Grand Rapids MI

Investigators

Abstract

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.