<p dir="ltr">Post-translational modifications (PTMs) play a crucial role in regulating protein function and location. Protein AMPylation, the addition of adenosine monophosphate (AMP), significantly influences protein trafficking, stability, and pathogenic virulence. The Fic Domain family of proteins targets hydroxyl-containing amino acid residues (Ser, Thr, or Tyr), catalyzing the addition of various phosphate-containing moieties such as nucleoside monophosphates (NMPs), phosphocholine, and phosphate. Using gene mining techniques, Dr. Seema Mattoo’s group has identified a clade of Fic domain containing proteins typified by the enzyme originating from <i>Bordetella bronchiseptica</i> (BbFic) which prefers utilizing guanosine triphosphate (GTP) as a substrate over other nucleotides. To understand the physiological role of GMPylation, identifying the proteins modified by BbFic is a first critical step and can be accomplished via mass spectrometry-based proteomics. For a low stoichiometry PTM like GMPylation, however, there is a need to develop chemical tools that enable the targeted enrichment of modified protein. Identifying key interactions between substrate proteins and the BbFic nucleotide binding site will enable development of highly specific molecular tags for Fic substrates.</p><p dir="ltr">The goal of this research project, therefore, is to design chemical probes to tag Fic enzyme substrates, thereby facilitating the identification of GMPylated proteins in chemical proteomics workflows. A set of ATP and GTP analogues carrying either alkyne or azide handles were proposed as possible probes. While 8-azido guanosine showed a high docking score in our in-silico study, literature reports highlight its chemical instability upon exposure to air and light. An alternative probe, the 8-ethynyl guanosine, also showed a high docking score and docks in the same position and orientation as guanosine (the natural ligand) but necessitates synthetically challenging via cross-coupling reactions.</p><p dir="ltr">We considered multiple GMP analogues as potential molecular tags with the assistance of molecular docking with the BbFic enzyme. With predicted binding affinities in hand, we prioritized candidate GTP analogs for synthesis to probe the BbFic-mediated protein GMPylation process. While N6 propargyl guanosine serves as a lead probe for AMPylation, computational analysis reveals challenges with O6 due to its altered hydrogen bond donor/acceptor presentation. The distinctive chemical properties of guanosine, compared to adenosine, require a thorough evaluation of protective group strategies, as not all synthetic methodologies used for ATP analogue synthesis are applicable to GTP analogues. Isolating the triphosphate analogue proved challenging, although purification of the monophosphorylated counterpart is feasible. The Protide analogue benefits from phosphate charge masking, which facilitates purification. While much work remains until the physiological role of GMPylation can be determined, important progress has been made in the design and synthesis of chemical tools for studying this newly discovered PTM.</p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/26359639 |
| Date | 25 July 2024 |
| Creators | Sara Sedky Elshaboury (19200796) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/thesis/_b_Development_of_Chemical_Probes_to_Study_Protein_Guanosine_Monophosphorylation_b_/26359639 |
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