Multifunctionalization of Proteins: Strategies for Combining Semi-Synthetic and Bioorthogonal Techniques

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Doctor of Philosophy (PhD)
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alpha synuclein
amber codon suppression
aminoacyl transferase
click chemistry
unnatural amino acids
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Proteins play important roles in biological processes including muscle movement, initiating immune responses, and memory formation. Full understanding of protein function requires determining its structure, assignment of function, and elucidation of interactions with other proteins or metabolites. Protein modification is useful for installing chemical entities that allow researchers to specifically probe function, attach fluorophores for localization, covalently trap protein interactions, or perform affinity enrichment from a complex milieu. Installation of functional groups otherwise not accessible from canonical protein expression requires a toolbox of protein modification strategies. We have developed strategies for using a combination of techniques to functionalize proteins, including E. coli aminoacyl transferase (AaT) mediated N-terminal protein modification, Amber codon suppression for incorporation of unnatural amino acids (Uaas), auxotrophic strain incorporation of Uaas, and click chemistry reactions. A chemoenzymatic approach is used with AaT mediated N-terminal transfer of an azide functionality and subsequent click- chemistry to fluorescently label protein N-termini. We also use AaT to install a unique N-terminal protein semi-synthesis ligation site. Additionally, Amber codon suppression is used to site-specifically incorporate Uaa side chains with click-chemistry handles, intrinsic fluorophores, and photocrosslinkers into proteins. Click-chemistry is used with Amber codon suppression to control protein function in a highly selective manner. We also use auxotrophic strain incorporation to globally replace a natural amino acid with an isostere Uaa to introduce azide and alkyne functional groups. Protein multifunctionalization is achieved by combining these modification methods in a single expressed protein. Effectively achieving this combination has required careful consideration of both molecular biology and chemical reactivity. We demonstrate the combination of these techniques to label alpha-synuclein, a protein which is involved in neurodegenerative diseases. Moreover, the protocols for multiple labeling that we have developed should be generally practicable for a variety of proteins.

E. James Petersson
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