Honors College Thesis
 

Characterizing the Interactome of Nitrotyrosine-Calmodulin using Genetic Code Expansion

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https://ir.library.oregonstate.edu/concern/honors_college_theses/z029p9762

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  • A specific tyrosine post-translational modification, called 3-nitro-tyrosine (nitroTyr), has been known to be present on an essential calcium signaling protein called calmodulin (CaM) during oxidative stress. While protein-bound 3-nitro-tyrosine has long been considered a biomarker of OS, it is also hypothesized to be a mechanism for protein signaling. However, purification of nitroTyr-CaM binding proteins has not been possible because the tyrosine residues on CaM could not be nitrated at the amino acid resolution. In this study, nitroTyr has been site-specifically incorporated into traditional tyrosine residues 99 and 138 using techniques of genetic code expansion. Columns of wild-type CaM, 99 nitroTyr-CaM, and 138 nitroTyr-CaM, were made for isolation of protein binding partners. In a protein extraction experiment using bovine heart tissue, myoglobin was found to bind preferentially to nitroTyr-CaM. Surprisingly, in vitro binding studies between CaM ± nitroTyr and recombinant myoglobin shows the opposite trend – WT CaM bound preferentially to myoglobin instead. In summary, this project demonstrated that pulldown of WT and nitroTyr-CaM binding partners was possible – however, teasing out meaningful differences in binding partners remains a challenge. Key Words: 3-nitrotyrosine, affinity chromatography, calmodulin, protein purification, endothelial nitric oxide synthase eNOS, genetic code expansion, myoglobin, oxidative stress, post-translational modification, protein binding, unnatural amino acid.
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