Lysine Acylation using Conjugating Enzymes for the Modification of Recombinant Proteins
Embargoed until 2022-03-24
- Doctoral Thesis
Proteins and enzymes perform important functions of life. Further expanding and regulating their function, proteins are dynamically decorated with posttranslational modifications (PTMs). Because of the large number of modifications and pathways involved, it is challenging to isolate a protein with a specific modification from natural sources. Besides native PTMs, artificial modifications can augment the properties and efficacy of proteins for their use in pharmaceutical applications and research. To determine the characteristics of natively existing modifications and to create modified protein products, efficient strategies for their preparation are required. Protein modification in a controlled manner is a difficult task because of the richness of functional groups and chemical environments that the protein structure harbors. While chemical modification of proteins generally offers chemoselectivity for a specific functional group, enzymatic approaches frequently proceed with high site specificity and operate under mild reaction conditions. Such chemoenzymatic methods rely on pathways that operate on proteins, and substrate analogs that can be utilized by these enzymes. Many existing protocols, however, are restricted to modification at the protein termini, rely on non-peptidic metabolites, or require large recognition domains. This dissertation describes the development and application of lysine acylation using conjugating enzymes (LACE), a chemoenzymatic strategy to site-specifically modify folded proteins at internal lysine residues. LACE relies on a minimal genetically encoded tag (four residues) recognized by the E2 small ubiquitin-like modifier-conjugating enzyme Ubc9 (Ube2I), and peptide or protein thioesters. Together, this approach obviates the need for E1 and E3 enzymes, enabling isopeptide formation with just Ubc9 in a programmable manner. Our studies demonstrate that LACE accepts a series of functionalized thioester probes, ranging from synthetic molecules to entire protein domains. The short tag size allowed the modification of diverse substrates, ranging from monomeric to multimeric proteins, and combination of the method with existing strategies for one-pot dual modifications. Importantly, LACE enabled the site-specific installation of native ubiquitin and ISG15 in a programmable manner from entirely recombinant sources in one step. In a complementary approach, this dissertation describes the development of a workflow towards the identification of sequence-specific redox reactivity of cysteine residues. To this end, a solid-supported combinatorial peptide library was prepared, and a high-throughput screen combined with semi-automated peptide sequencing was established for the identification and characterization of library hits. Show more
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SubjectChemistry; Chemical Biology; Biochemistry; Bioconjugation; Protein modification; Site-specific modification; Ubiquitination
Organisational unit03861 - Bode, Jeffrey W. / Bode, Jeffrey W.
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