Date of Award
Doctor of Philosophy (PhD)
E. James Petersson
Protein modification is an important tool for understanding how a protein moves, folds, and operates in its cellular environment. We aim for a minimalist approach to protein modification in order to learn more about the protein while not perturbing its native state. Our method for ligation of small molecules to proteins has high N-terminal specificity and creates natural amide bond linkages under mild conditions. We utilized E. coli aminoacyl L/F transferase (AaT) in our ligation reaction to recognize a small synthetic substrate analog, which mimics aminoacylated tRNA, and can be chemically synthesized in only three steps. We have studied the mechanism of AaT in order better utilize unnatural substrates and expand the scope of aminoacyl donors and N-terminal acceptors. Using AaT, we can enzymatically attach a wide range of synthetic moieties to the N-termini of peptides and proteins, including natural and unnatural amino acids with reactive azido groups, fluorescent labels, protected disulfides, and photoinducible crosslinkers. This method introduces new opportunities for protein semi-synthesis, which we have applied in two model systems, α-synuclein and calmodulin, and contributes as an effective method to label proteins for biophysical research.
Wagner, Anne, "N-Terminal Protein Modification by Aminoacyl Transferase: Enzymology and Applications." (2013). Publicly Accessible Penn Dissertations. 936.