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Subject: search.filters.subject.310109 Proteomics and intermolecular interactions (excl. medical proteomics)

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Now showing 1 - 5 of 5
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    Studies towards thermodynamically stable G-quadruplexes embedded in canonical DNA duplexes : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry at Massey University, Manawatū, New Zealand
    (Massey University, 2023) Chilton, Bruce
    DNA is the polymer responsible for the storage of genetic information and, ultimately, all processes that occur within the cell. Our understanding of DNA structure and function has developed considerably, but some areas are still unclear. In particular, a range of non-canonical DNA secondary structures such as G-quadruplexes (G4s), i-motifs and triplexes, have also been shown to form in genomic DNA sequences and these structures also appear to have a role in genome function. Better understanding of the interactions of non-canonical secondary structures is hindered by their transient nature in the context of larger DNA structures, making it difficult to accurately study them using in vitro analytical techniques (e.g., NMR spectroscopy, X-ray crystallography, etc.). They are typically less thermodynamically stable than canonical DNA duplexes and are formed within the genome only in equilibrium with many secondary structures, typically favouring the canonical duplex. They can often only be formed reliably under specific conditions in vitro (e.g., single-stranded, low pH etc.). This thesis presents several strategies designed to stabilise non-canonical G4 secondary structures, which are of interest because they are often found in the promoter regions of oncogenes. The most commonly used existing G4 stabilisation technique utilises small-molecule ligands which specifically bind to and stabilise G4 structures. This thesis includes an investigation into both a widely used G4-binding ligand and several newly developed ligands, but their potential to block binding sites and disrupt G4 topology makes them less suitable for our intended applications. Hydrophobic modifications can encourage aggregation of DNA strands and therefore increase stability of secondary structures. Hydrophobic phosphate modifications in G4s proved effective at disrupting duplexes and stabilising G4s but was limited by coupling efficiency of the modification and resulting difficulties with purification. Intentional mismatches in the G4-forming sequence were introduced by inverting sequence direction or incorporating α-anomers of nucleotides. This strategy was able to completely disrupt duplex formation while preserving G4 structures, but modification sites have to be carefully considered to avoid significant changes in G4 topology. Internal cross-links were incorporated into DNA using modified nucleotides designed for copper(I)-catalysed azide-alkyne cycloaddition. These cross-links prevent G4 structures from unfolding, but the location of these cross-links must also be carefully considered to prevent disruption of the native G4 topology and blocking protein binding sites. All three of these methods present potential routes for stabilising G4s within larger DNA structures. Furthermore, all three modifications could potentially be expanded to stabilise other non-canonical structures, such as imotifs or triplexes.
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    Untangling the interaction between HP1α and the TERRA G-quadruplex : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand
    (Massey University, 2023) Roach, Ruby
    Over 50 years ago the DNA double-helix structure was solved, revealing the code in which life is written. This set of instructions equates to three billion base-pairs, amounting to two metres in length, fitting into a human nucleus just six microns across. The stable yet dynamic structure that allows for the functional organisation of the genome is chromatin: a complex formed between DNA and proteins. Chromatin is delineated into two microscopically defined compartments: the less dense gene-rich euchromatin and the structurally compact gene-poor heterochromatin. Protective heterochromatin is constitutively maintained for telomere protection, chromosome segregation, DNA repair, and suppression of transposon activity. Essential for the propagation and maintenance of heterochromatin is Heterochromatin Protein 1α (HP1α), which is comprised of a chromodomain and chromoshadow domain separated by a disordered hinge. The histone code model proposes that HP1α is recruited to regions of heterochromatin by its chromodomain-mediated recognition of silent heterochromatin mark trimethylated lysine 9 of histone H3 (H3K9me3); however, this does not account for the specificity of HP1 paralogs, location-specific recruitment, or the contribution of RNA to heterochromatin formation. Here, Telomeric Repeat-containing RNA (TERRA), transcribed from the telomeres and shown to be involved in telomeric maintenance and stability, is investigated in its interaction with HP1α. The interaction with TERRA is proposed as a means for recruitment of HP1α to telomeres for chromosome end protection. Due to its guanine (G)-rich sequence, TERRA folds into a G quadruplex (G4), a topology distinctly different from canonical nucleic acid structures. The interaction between HP1α and TERRA is therefore investigated to establish the means of interaction between HP1α and a G4, and to examine the HP1α specificity towards TERRA G4s. This work showed that binding to TERRA is dependent upon multiple positively charged patches within the disordered hinge of HP1α, and is also affected by mimicking N-terminal phosphorylation, which alters the structure of HP1α. While the hinge of HP1α binds to a myriad of nucleic acid structures, the globular chromoshadow domain provided the specificity for the parallel G4 topology evident in TERRA. Solution structures of HP1α in complex with TERRA show that HP1α undergoes a conformational shift, becoming less flexible. These results show that noncanonical nucleic acid structures such as those formed by TERRA may act as determinants of HP1α function, serving as signposts in the genome for formation of protective heterochromatin. This biophysical study also further implicates non-canonical structures such as G4s as formidable regulators of genomic function.
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    Establishing a screening procedure for finding amino acids important for protein-protein interaction : a thesis presented in fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Albany, New Zealand
    (Massey University, 2020) Ghuge, Aditi Ashok
    According to recent findings, the Gcn2 kinase is a global regulator of biological processes in the cell. In Saccharomyces cerevisiae (yeast), it has been shown that Gcn2 is activated by its interaction with Gcn1 in the presence of uncharged tRNAs, inducing the starvation response. The binding of Gcn2 to the RWDBD domain of Gcn1 is necessary to activate Gcn2 on medium supplemented with 3AT (3-Amino-1,2,4-Triazole) - which induces starvation for histidine. In addition, an R2295A mutation in the RWDBD domain prevents the activation of Gcn2 by disrupting the Gcn1-Gcn2 interaction. We predict that the Gcn1-Gcn2 interaction is also mediated by several other amino acids in the Gcn2 binding region of Gcn1. Therefore, to be able to test this hypothesis, a screening method was established in this research that will allow the determination of all possible amino acids in Gcn1 required for Gcn2 binding. In this thesis we aimed to establish and optimize each module of the screening procedure. One of the modules is competitive growth. This approach will take advantage of the fact that overexpressed RWDBDs in yeast can bind to Gcn2. As a result, the RWDBD disrupts Gcn1- Gcn2 interaction, and prevents Gcn2 activation, thereby preventing yeast from overcoming starvation induced by 3AT. Therefore, yeast will not be able to grow. In contrast to that, yeast strains expressing a mutated RWDBD domain incapable of disrupting the Gcn1-Gcn2 interaction will allow yeast cells to grow on starvation medium. We found that, a competitive growth assay on medium with 0.5 mM 3AT, with a duration of 120 hours, will allow for the enrichment of yeast strains expressing mutated RWDBD domains which have lost the ability to bind to Gcn2. TRP1 was fused in frame to the C-terminal end of the RWDBD domain which will help to eliminate truncated RWDBD domains from the library of mutated RWDBD domains. The idea was that the presence of a nonsense mutation in the RWDBD domain will lead to the premature termination of translation. As a result, TPR1 is not translated, and the strain remains auxotrophic for the amino acid tryptophan, whereas RWDBD domains without a nonsense mutation allow the TRP1 translation, conferring prototrophy for the amino acid tryptophan. For easy insertion of mutations, the Gcn2 binding region within the RWDBD was flanked with unique new restriction sites, recognized by AvrII and PmeI. With the help of these new restriction sites, it is now possible to replace the Gcn2 binding region with randomly mutagenized versions. To retrieve the sequences of mutated RWDBD domains easily and efficiently, a cell culture PCR procedure was optimized. In conclusion, in this thesis the major parameters of the screening procedure were optimised.
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    Molecular dynamics simulation of inter-molecular interactions : a thesis submitted to Massey University in Albany, Auckland in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Computational Biochemistry
    (Massey University, 2019) Shadfar, Shamim Zahra
    Many aspects of the operation of chemical and biological systems are based on intermolecular interactions. In this work, binding modes and interactions between molecules at a range of scales have been studied, using molecular dynamics (MD) simulations. The first chapter provides an introduction of each of the different chemical and biological systems that are studied in this work. It also introduces MD and its role in the context of this research. The second chapter corresponds to the study of host-guest interactions for cyclodextrin- bullvalene complexes. Bullvalene is a shapeshifter molecule, which interconverts between different isomers at room temperature. The goal of this chapter is to capture one favourable isomer of bullvalene (guest molecule) by binding it to cyclodextrin as a host molecule. This chapter consists of two smaller chapters (2i and 2ii). The former details the development and validation of a “host-guest binding potential energy profiling” (HGBPEP) method, which is a rotational interaction energy screening method designed for prediction of the most favourable orientation and position of bullvalene isomers with respect to cyclodextrin. The latter investigates the interaction of bullvalene isomers and cyclodextrin molecules, and finally binding free energy values of the complexes are calculated. The third chapter describes KstR, a transcriptional repressor in Mycobacteria. KstR is required for Mycobacterium tuberculosis (Mtb) pathogenesis as well as regulating the initial steps in cholesterol degradation by controlling the expression of the enzymes that carry out the early stages of cholesterol catabolism. Therefore, this protein is of great interest for development of new tuberculosis treatments. In this chapter, the stability and conformational changes of KstR in its different states – apo, DNA-bound and ligand-bound –have been studied. The main goal is to investigate the binding mechanism of KstR to DNA, as well as the effect of DNA and ligand binding on the structure and dynamics of KstR more generally, using MD simulations. In the fourth chapter, KstR2, another Mtb transcriptional repressor, is studied. KstR2 represses a 14-gene regulon involved in the later steps of cholesterol degradation. It is structurally similar to KstR, but has been proposed to act through a novel scissor-like mechanism. This chapter investigates two key questions regarding the mechanism of action of KstR2: first, the effect of mutating the key switch residue ARG170 to ALA, and second, the effect of ligand binding on its structure and motion. The focus of the fifth and final chapter is phosphatidylinositide 3-kinases (PI3Ks), which are proteins that take part in signalling pathways regulating factors like cell growth, survival and proliferation, which in turn are involved in cancer. The interaction between PI3Kα and another protein, RAS, is very important in the formation, growth and maintenance of RAS- driven tumours. A model of PI3Kα (class IA PI3K) has therefore been built, as well as of RAS associated with a model cell membrane, and MD simulations used to investigate the process by which the two proteins interact with one another and with the lipid bilayer. Altogether, this thesis uses MD simulations to provide insight into intermolecular interactions at a range of scales, with a particular focus on proteins involved in tuberculosis and in cancer.
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    Identification of large ribosomal proteins required for the full activation of the protein kinase Gcn2 in Saccharomyces cerevisiae : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biological Sciences at Massey University, Albany, New Zealand. EMBARGOED indefinitely.
    (Massey University, 2019) Anderson, Reuben Andrew
    Protein synthesis is a fundamental biological process that all organisms require for maintaining life, growth and development. The maintenance of amino acid levels, the building blocks of proteins, is essential for maintaining protein synthesis under all biological conditions. Hence, amino acid shortage can be deleterious to the cell. Therefore, cells harbour mechanisms to cope and overcome amino acid starvation. When eukaryotes are subjected to amino acid starvation, the resulting accumulation of uncharged tRNAs activates the protein kinase Gcn2, leading to phosphorylation of eIF2α and activation of the amino acid starvation response. Uncharged tRNAs are the signal of starvation, directly detected by Gcn2. Gcn2 must bind to the effector protein Gcn1 and both must contact ribosomes for Gcn2 activation. The current working model for how the starvation signal is delivered to Gcn2 postulates that these uncharged tRNAs bind in the A-site of the ribosome in a codon specific manner, which are subsequently transferred to Gcn2. Gcn1 is directly involved in this process but its exact involvement is unknown. To test the working model, it is paramount to investigate where Gcn1 and Gcn2 bind on the ribosome. Ribosomes consist of a large and small subunit, each containing multiple ribosomal proteins placed in unique locations. Identification of ribosomal proteins contacting Gcn1 or Gcn2 will allow for deduction of where Gcn1 and Gcn2 bind on the ribosome. This study aimed to determine Gcn1 and Gcn2 contact points on the large ribosomal subunit, usinga genetic approach. The hypothesis was that if an interaction of Gcn1 or Gcn2 with a particular large ribosomal protein (Rpl) is important for Gcn2 activation, then its overexpression would impair Gcn2 function. Overexpression of several large ribosomal proteins impaired cell growth on a medium triggering amino acid starvation, suggesting Gcn2 activation was impaired. Groups of two or more of these Rpls were found in several regions which contain ribosomal proteins shown or suggested to interact with Gcn1 or Gcn2 previously. This included a region containing the P-stalk proteins (part of the large ribosomal complex) known to contact Gcn2. A region close to the small ribosomal protein Rps10, known to contact Gcn1, was also identified. Another region with Rpls which contacts a protein eEF3, which is suggested to share similar ribosomal contacts as Gcn1, was identified.