The Development of New Tools for the Investigation of Protein Function Using Photo-Reactive Unnatural Amino Acids

Simple item page
dc.contributor.advisorCropp, Ashtonen_US
dc.contributor.advisorGerratana, Barbaraen_US
dc.contributor.authorWilkins, Bryan Jasonen_US
dc.contributor.departmentBiochemistryen_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.date.accessioned2010-07-02T05:40:53Z
dc.date.available2010-07-02T05:40:53Z
dc.date.issued2010en_US
dc.description.abstractReported here is the direct synthesis and application of unnatural amino acids for the development of exploratory tools for protein studies. This work takes advantage of an expanded genetic code to extract a more precise chemical understanding of protein function with novel additions to the unnatural amino acid catalogue, as well as the expansion of techniques with previously developed compounds. The photochemical crosslinker, [D11]-p-benzoylphenylalanine (pBpa), is synthesized for isotopic labeling in proteins. When [D11]-pBpa is co-incorporated into protein with [D0]-¬pBpa it is a mass spectral tool for rapid and conclusive identification of crosslinked fragments. Following enzymatic digestion the fingerprint of M, M+ 11 is readily identified allowing for rapid peak identification and the determined site of crosslink formation with single amino acid accuracy. In a means to extract a level of spatiotemporal control over fluorescent labeling of protein, the photo-protected unnatural amino acid, o-nitrobenzyl cysteine (ONBC), is introduced to a small amino acid tag sequence CCPGCC. This tag is required and specifically binds the pro-fluorescent compound 5-bis(1,3,2-dithiasolan-2-yl)fluorescein (FlAsH). This work takes advantage of the inability of FlAsH to bind the cysteine-tag motif in the presence of an ONBC mutation. The photo-protected amino acid is deprotected with light, affording natural cysteine and the successful binding of FlasH to the tetracysteine tag only following ultraviolet irradiation. Finally, fluorinated tyrosine derivatives are synthetically modified to contain photo-protecting groups, which act as a disguise during unnatural amino acid mutagenesis techniques. Fluorinated tyrosines are recognized by endogenous tyrosyl-tRNA synthetases and incorporated globally throughout a protein at tyrosine positions. To circumvent this problem the o-nitrobenzyl photo-protecting group is installed on the tyrosine derivatives 2-fluorotyrosine, 3-fluorotyrosine, and 2,6-difluorotyrosine. The directed evolution of an orthogonal amber-tRNA synthetase, specific for these unnatural amino acids, is performed, providing the translational machinery for site-specific incorporation of these compounds. Following expression of protein with the protected tyrosine derivatives, protein exposed to ultraviolet irradiation subsequently loses the protecting group affording the site-specific incorporation of fluorinated tyrosine. Fluorinated tyrosines are introduced to the critical trysoine residue in the chromophore of super-folder green fluorescent protein to determine how the altered pKa affects its fluorescent properties.en_US
dc.identifier.urihttp://hdl.handle.net/1903/10258
dc.subject.pqcontrolledChemistry, Biochemistryen_US
dc.subject.pquncontrolledAmber Suppressionen_US
dc.subject.pquncontrolledCaged Cysteineen_US
dc.subject.pquncontrolledFluorotyrosineen_US
dc.subject.pquncontrolledp-Benzoylphenylalanineen_US
dc.subject.pquncontrolledPhoto-reactive compoundsen_US
dc.subject.pquncontrolledUnnatural Amino Acidsen_US
dc.titleThe Development of New Tools for the Investigation of Protein Function Using Photo-Reactive Unnatural Amino Acidsen_US
dc.typeDissertationen_US

Files

Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
Wilkins_umd_0117E_11082.pdf
Size:
6.89 MB
Format:
Adobe Portable Document Format
Download

Collections

UMD Theses and Dissertations
Chemistry & Biochemistry Theses and Dissertations