2015 Beckman Symposium   

Susan Hromada

Presentation Date:
August-6-2015

Presenter:
Susan Hromada

Title:
Investigation of Tyrosine-Cysteine crosslinks in a model protein

Institution:
Calvin College

Department:
Chemistry and Biochemistry

Description:
Proteins, made up of amino acid building blocks, undergo various modifications after translation. One of these post-translational modifications is bond formation between Tyrosine and Cysteine amino acids. The covalent bond formed between these two amino acids has been recognized to have active functions inside its parent protein. In galactose oxidase, the Tyr-Cys crosslink functions as a co-oxidant in redox chemistry. In cysteine-dioxygenase, Tyr-Cys is proposed to contribute rigid hydrogen bond donation. The Tyr-Cys crosslink also has implications as an antioxidant in the protein, as seen in cytochrome c oxidase where His-Tyr, an analogous crosslink, reduces reactivity of the heme site. In light of this bond's significance, an exploration into the mechanistic details of Tyr-Cys crosslink formation has been examined using an orphan protein from Bacteroides fragilus, BF4112, as a chemical model. BF4112 has a tyrosine and cysteine side chain geometrically predisposed for Tyr-Cys crosslink formation adjacent to a His2Glu coordinated metal binding site. This project has investigated the previously reported copper-mediated formation of the Tyr-Cys bond as well as iron-mediated and non-metal conditions. New robust and quantitative ways are needed to study this crosslink as current detection methods of gel electrophoresis or proteolytic mass spectrometry coupled with X-ray crystallography are unable to provide precise quantitative measurements of amount of crosslink in solution and absorbance assays are only viable in small proteins with little tryptophan interference. In BF4112, a protein with only one tryptophan, an absorbance change assay can be used to study crosslink formation under different conditions. Cu(I)O2 and Fe(II)O2 have been shown to form Tyr-Cys crosslink. Cu(II)peroxide has shown small crosslink formation but primarily other oxidation products. Metal free pathways using HOCl and OSCN- oxidants have shown no crosslink formation. Fluorescence spectrophotometry and two-dimensional NMR have been studied in this project as new detection methods for study of the Tyr-Cys crosslink.


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