Dissertation/Thesis Abstract

Structural effects of phosphorylation and β-O-GlcNAcylation on α-helices and structural effects of phosphorylation and R406W on tau395-411
by Elbaum, Michael Birch, M.S., University of Delaware, 2014, 326; 1567802
Abstract (Summary)

The dynamic interplay between phosphorylation and beta-O-GlcNAcylation (OGlcNAc) of serine and threonine plays critical roles in numerous intracellular processes. Changes in phosphorylation and OGlcNAcylation are linked to Alzheimer's disease, diabetes, and cancer. We have conducted a systematic study on a model alpha-helix to determine the structural effects of phosphorylation and OGlcNAcylation of serine and threonine residues, on the N-terminus, C-terminus, and internal positions (Ac-XKAAXAKAAXAKAAGY-NH2, Ac-YGAKAAAAKAAAAKAX-NH 2). We found that both phosphorylation and OGlcNAcylation on the N-terminus increase alpha-helix stability, with phosphorylation exhibiting a greater increase in alpha-helix stability than OGlcNAcylation. These stabilizing effects were found to be greater for threonine than serine, and for the dianionic phosphate over the monoanionic phosphate. In contrast, both phosphorylation and OGlcNAcylation reduced helix stability on internal and C-terminal positions relative to serine or threonine. These effects are not simply electrostatic interactions; we observe a unique cyclization of serine and threonine residues due to an intra-residue phosphate-amide hydrogen bond. Furthermore this interaction is greater for threonine than serine and for the dianionic phosphate over the monoanionic phosphate. On the N-terminus NMR data are consistent with an alpha-helix capping mechanism in which the phosphate hydrogen bonds to its own amide, organizing and nucleating the first turn of the alpha-helix through an induced n to pi star interaction between the i-1 carbonyl and the i (phosphorylated) carbonyl. On internal and C-terminal positions, alpha-helix destabilization is due to this phosphate amide intra-residue hydrogen bond disrupting the backbone hydrogen bonding network. Interestingly, the overall effects of phosphorylation and OGlcNAcylation on an alpha-helix are analogous to the effects observed for proline, with the effects of phosphothreonine greater than the effects of proline on the alpha-helix at all positions.

To further understand the effects of serine GlcNAcylation on alpha-helices, we sought to explore possible side-chain interactions with neighboring residues, such as hydrophobic, CH/pi, or boronic acid/diol conjugates. A series of Baldwin model alpha-helices (Ac-AKAAAAKAAAAKAAGY-NH2) were designed to explore i+2, i+3, i+4, and i+7 interactions utilizing 4-iodo-phenylalanine (4-iodo-Phe) or 4-B(OH)2-phenylalanine (4-B(OH)2-Phe) as interacting residues. Although no interaction was found to exist between GlcNAc and either 4-iodo-Phe or 4-B(OH)2-Phe, an alpha-helix stabilizing interaction was found involving lysine and boronic acid in a relative i / i+4 relationship. The interaction between lysine and boronic acid exhibited an increase in alpha-helix stability as the concentration of KF was increased, yet showed no increase in alpha-helix stability when NaCl was used rather than KF. This observation is consistent with fluoride ions playing a crucial role in the alpha-helix stabilizing interaction, as well as a mechanism involving more than a simple electrostatic model.

Phosphorylation is known to affect protein structure when there is no defined secondary structure such as an alpha-helix. Many examples exist where phosphorylation sites have been identified in natively disordered proteins, such as the microtubule binding protein tau. Hyperphosphorylation of tau is associated with numerous neurodegenerative diseases, most notably Alzheimer's disease (AD). Within tau, phosphorylation of Ser404 has shown to occur within patients with AD. Furthermore, elevated rates of AD onset have been observed due to a mutation of Arg 406 to Trp406. Given the roles of both Ser404 phosphorylation and R406W mutation in the onset of AD and other neurodegenerative disorders, I sought to determine the local structural effects of both phosphorylation and R406W mutation within tau. Due to the lack of organized secondary structure, one way to study regions of disordered proteins is to study small peptide fragments. Tetra-peptides of the sequence TSPX, representing residues 403-406 of tau, were synthesized, containing either or both phosphorylated Thr403 and Ser404 residues, containing either the native arginine or tryptophan mutation. We have found that phosphorylation of Ser404 and R406W mutation both independently and dependently lead to a higher population of cis amide bonds. To validate the data obtained from tetra-peptides, larger tau395-411 peptides with synthesized with both unmodified and phosphorylated Ser404 and either Arg406 or Trp406. The data herein are consistent with both phosphorylation of Ser404 and R406W mutation leading to a higher population of cis amide bonds within tau. This increase in cis-Pro amide bond population may be directly correlated to the onset of AD.

Indexing (document details)
Advisor: Zondlo, Neal J.
Commitee:
School: University of Delaware
Department: Department of Chemistry and Biochemistry
School Location: United States -- Delaware
Source: MAI 53/06M(E), Masters Abstracts International
Source Type: DISSERTATION
Subjects: Molecular chemistry, Biochemistry
Keywords:
Publication Number: 1567802
ISBN: 978-1-321-29059-2
Copyright © 2019 ProQuest LLC. All rights reserved. Terms and Conditions Privacy Policy Cookie Policy
ProQuest