Degree

Doctor of Philosophy (PhD)

Department

Chemistry

Document Type

Dissertation

Abstract

NOTCH is a transmembrane protein that transmits signals between cells in direct contact and alters gene expression. The signaling pathway is important because it is involved in cell fate determination. The NOTCH1 extracellular domain has 36 epidermal growth factor-like (EGF) repeats that are responsible for ligand binding. Mutations in the EGF-like repeats cause various neurological and developmental disorders. NOTCH1 signaling initiates when DSL ligands bind to EGF11-12, the minimum Ligand Binding Domain (LBD). The Abruptex region of the NOTCH1 protein (EGF24-29) is a highly glycosylated, flexible region with calcium binding sites that regulates NOTCH1 signaling and binding by interacting with the LBD. In particular, EGF26 is known to be glycosylated catalyzed by the enzyme Pofut1 adding O-fucose and then recognized by Fringe, a Notch regulator that elongates O-fucose by adding N-acetylglucosamine (GlcNAc). Our group has shown that the structural arrangement of EGF27 changes upon the addition of O-linked fucose (O-fuc) and O-linked N-acetylglucosamine (O-GlcNAc). Here, nuclear magnetic resonance spectroscopy (NMR) was used to conduct the structural analysis of unglycosylated EGF26 where 90% of backbone and sidechain resonances were assigned. Nuclear Overhauser experiments were used to determine distance constraints resulting in 90% of residues in the favored region and a high-quality 3D structure was produced. Due to the successful investigation of the structural conformation and dynamics of EGF26, the methods used were applied to SMB-IDDΔ71 of vitronectin in order to understand the dual binding site involved with the plasminogen activator inhibitor-1 and the structural changes throughout vitronectin due to the IDD region.

Date

7-15-2022

Committee Chair

Macnaughtan, Megan

DOI

10.31390/gradschool_dissertations.5899

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Available for download on Thursday, July 03, 2025

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