Characterizing the Interactome of Nitrotyrosine-Calmodulin using Genetic Code Expansion Public

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

Descriptions

Attribute NameValues
Creator
Abstract or Summary
  • 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.
License
Resource Type
Date Available
Date Issued
Degree Level
Degree Name
Degree Field
Degree Grantor
Commencement Year
Advisor
Academic Affiliation
Non-Academic Affiliation
Rights Statement
Publisher
Peer Reviewed
Language
Replaces
Additional Information
  • description.provenance : Submitted by Garth Kong (kongg@oregonstate.edu) on 2017-07-26T21:27:48ZNo. of bitstreams: 2license_rdf: 1379 bytes, checksum: da3654ba11642cda39be2b66af335aae (MD5)KongGarthL2017.pdf.pdf: 2227569 bytes, checksum: 82aa3ae81010a74e60745ed71dc1056c (MD5)
  • description.provenance : Made available in DSpace on 2017-08-01T23:34:19Z (GMT). No. of bitstreams: 2license_rdf: 1379 bytes, checksum: da3654ba11642cda39be2b66af335aae (MD5)KongGarthL2017.pdf.pdf: 2227569 bytes, checksum: 82aa3ae81010a74e60745ed71dc1056c (MD5)
  • description.provenance : Approved for entry into archive by Steven Van Tuyl(steve.vantuyl@oregonstate.edu) on 2017-08-01T23:34:19Z (GMT) No. of bitstreams: 2license_rdf: 1379 bytes, checksum: da3654ba11642cda39be2b66af335aae (MD5)KongGarthL2017.pdf.pdf: 2227569 bytes, checksum: 82aa3ae81010a74e60745ed71dc1056c (MD5)

Relationships

Parents:

This work has no parents.

Last modified

Downloadable Content

Download PDF

Items