Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)


Biotechnology Science and Engineering

Committee Chair

Robert L. McFeeters

Committee Member

Hana McFeeters

Committee Member

Luis R. Cruz-Vera

Committee Member

William Setzer

Committee Member

Bernhard Vogler


Radiolabeling--Methods., Phenylalanine., Tyrosine.


Structural insight into biological macromolecules furthers understanding of function and provides a variety of information about cellular processes. Though NMR spectroscopy can be utilized to provide such insight, limitations, including size, are significant barriers. Site specific isotope labeling can be used to provide information on a variety of systems including large, slow tumbling systems and membrane proteins that are not amenable to study by traditional means. In effect, site specific isotope labeling increases the size barrier and thereby number of macromolecular systems able to be studied by NMR spectroscopy. Methyl labeling has been established as a leading method for site specific studies. However, more options are needed to extend utility and supplement systems where methyls are sparse or inadequately located. Aromatic amino acids are well positioned to be additional site specific probes, complementing existing methyl labeling. Aromatics are often found at important interaction interfaces and play significant roles in terms of structure and interactions. They have the added properties of having distinct 13C side chain chemical shifts, multiple magnetically equivalent 1H positions in the side chains, and reduced effective correlation times due to ring flipping. Thus, they are excellent probes for studying large systems. Site specific isotope labeled (SSIL) phenylalanine and tyrosine with α, γ and ε carbons 13C labeled are produced by recombinant and inexpensive means. Reduced number of peaks per residue and elimination of C-C scalar coupling in SSIL provides numerous advantages in improving the NMR spectra of large protein systems. Optimized production conditions resulted in increased yield efficiency. Also presented, is a strategy for the use of SSIL phenylalanine and tyrosine for study of a novel antibiotic target enzyme Pth1 with its natural substrate, peptidyl-tRNA.



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