<p> 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-NH₂). 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>i</i>-1 carbonyl and the <i>i</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.</p><p> 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>i</i>+2, <i>i</i>+3, <i>i</i>+4, and <i>i</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</i> / <i>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.</p><p> 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 <i>tau.</i> Hyperphosphorylation of <i>tau</i> is associated with numerous neurodegenerative diseases, most notably Alzheimer's disease (AD). Within <i>tau,</i> 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 Arg₄₀₆ 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 <i>tau.</i> 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 <i>tau,</i> 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 <i>tau_395-411</i> 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 <i>tau.</i> This increase in cis-Pro amide bond population may be directly correlated to the onset of AD.</p>

<p> Several <i>Saccharomyces cerevisiae</i> mutant tRNA-^Q2 species (glutamine isoacceptor, CUG anticodon) were synthesized and assayed for aminoacylation activity with <i>Saccharomyces cerevisiae </i> glutaminyl-tRNA synthetase. The derived steady state parameters were compared to similar datasets from the literature. The mutants behaved analogously to similar mutant species based on tRNA from <i>Escherichia coli</i>, but with slightly relaxed specificity as revealed by comparison of <i>k</i>_cat/<i>K</i>_M values relative to wild type <i>in vitro</i> transcribed tRNA. Additionally the eukaryotic N-terminal domain appendage, as found in <i>Sce</i> glutaminyl-tRNA synthetase, is considered in light of the discovery of non-canonical aminoacyl-tRNA synthetase functions, including its role in the assembly of the multiple aminoacyl-tRNA synthetase complex.</p>

Thesis (Ph. D.)--University of California, San Francisco, 2005. / Source: Dissertation Abstracts International, Volume: 66-12, Section: B, page: 6424. Adviser: Erin O'Shea.

Thesis (Ph. D.)--University of California, San Francisco, 2005. / Source: Dissertation Abstracts International, Volume: 66-12, Section: B, page: 6597. Adviser: Kevan M. Shokat.

Thesis (Ph. D.)--University of Wisconsin - Madison, 2006. / (UMI)AAI3234626. Adviser: Michael M. Cox. Source: Dissertation Abstracts International, Volume: 67-09, Section: B, page: 5050.

Thesis (Ph. D.)--University of California, San Francisco, 2007. / Source: Dissertation Abstracts International, Volume: 68-04, Section: B, page: 2350. Adviser: David O. Morgan.

Thesis (Ph. D.)--University of California, San Francisco, 2006. / Source: Dissertation Abstracts International, Volume: 68-01, Section: B, page: 0094. Adviser: Elizabeth H. Blackburn.

Thesis (Ph. D.)--University of California, San Francisco, 2005. / Source: Dissertation Abstracts International, Volume: 66-12, Section: B, page: 6439. Adviser: Hiten Madhani.

Thesis (Ph. D.)--University of Illinois at Urbana-Champaign, 2008. / Source: Dissertation Abstracts International, Volume: 69-05, Section: B, page: 2986. Adviser: Zaida Luthey-Schulten. Includes bibliographical references (leaves 174-187). Available on microfilm from Pro Quest Information and Learning.

Thesis (Ph. D.)--Indiana University, Dept. of Biology, 2009. / Title from PDF t.p. (viewed Oct. 8, 2009). Source: Dissertation Abstracts International, Volume: 70-02, Section: B, page: 0830. Adviser: David L. Daleke.