Degree

Doctor of Philosophy (PhD)

Department

Chemistry

Document Type

Dissertation

Abstract

NOTCH1 has been associated with a variety of human ailments, such as cancer and heart disease. NOTCH1 is a transmembrane protein that contains 36 epidermal growth factor-like (EGF) repeats, which facilitate cell signaling by binding proteins on neighboring cells. Each repeat contains about 40 amino acids and has to be folded in a specific manner to function properly. The amino acid sequence, carbohydrate pattern, and calcium-binding sites of EGF-like repeats vary, but the pattern along the 36-repeat chain is conserved. The Abruptex region (EGF24-29) of NOTCH1 is a highly glycosylated (having undergone sugar addition) and flexible region, which gained its name by a series of mutations in the NOTCH protein in Drosophila melanogaster that result in hyperactivation of NOTCH1’s activity. Even though the Abruptex region is important for NOTCH1 signaling; currently, there are no 3-dimensional (3D) structures for most of this region. Certain EGF repeats are greatly modified with the addition of O-linked glycans, and many have calcium binding sites, which gives each EGF repeat a unique function. EGF27 has been identified as a functionally important repeat; specifically, modification of EGF27with fucose and N-acetylglucosamine (O-GlcNAc) plays a role in NOTCH1 function and regulation. 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). In order to investigate the role of EGF27’s glycosylation in relation to NOTCH1’s signaling, nuclear magnetic resonance (NMR) spectroscopy has been employed to determine the 3-dimensional (3D) structure of EGF27, Fucα1-O-EGF27, and GlcNAc-β1,3- Fucα1-O-EGF27. For EGF27, the nuclear resonances of the backbone and sidechain 1H, 13C, and 15N nuclei have been assigned to the 15N-1HN (100%) 57 of 57 Cα (100%), 51 of 51 Cβ(100%), and 50 of 57 CO (87.72%) resonances. Nuclear Overhauser effect experiments have been used to determine distance constraints. Using these data, the first 3D structure was determined for unmodified EGF27, backbone and sidechain assignment have been identified for Fucα1-O-EGF27, and backbone resonances were assigned for GlcNAc-β1,3- Fucα1-O-EGF27.

Committee Chair

Macnaughtan, Megan A.

Available for download on Saturday, November 02, 2024

Share

COinS