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    Unraveling the dynamics of protein-protein interactions in the Gcn2 signal transduction pathway : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology and Genetics, Massey University, Albany, New Zealand
    (Massey University, 2016) Ramesh, Rashmi
    Eukaryotic cells regulate protein synthesis (translation) for a rapid response to various types of stress, and this involves several protein-protein interactions (PPIs) and protein phosphorylation. Phosphorylation of eukaryotic initiation factor-2 α (eIF2α) is a common regulatory mechanism to adjust protein synthesis in response to various stimuli. Gcn2 (General Control Non-derepressible) is an eIF2α kinase that is conserved from yeast to mammals, that is activated in response to amino acid starvation. Gcn2 activation leads to a reduction in global protein synthesis and simultaneous augmented translation of GCN4, a transcriptional activator of genes that are necessary to overcome stress. This cascade of events that allows cells in stress adaptation constitutes the General Amino Acid control (GAAC) pathway in yeast. Gcn2 activity is controlled by a large array of proteins that directly or indirectly regulate Gcn2. Gcn2 has to bind another protein called Gcn1, in order to be activated in response to amino acid starvation. Yih1 (Yeast IMPACT homolog 1) in yeast and its counterpart IMPACT (IMPrinted and AnCienT) in mammals are homologous proteins that indirectly regulate Gcn2. Yih1/IMPACT inhibit Gcn2 by competing for Gcn1 binding. Yih1 associates with Actin, and studies so far have suggested that Yih1 only inhibits Gcn2 when it dissociates from Actin. The focus of this thesis work was to shed more light on those interactions relevant for Gcn2 regulation. Firstly, we have identified that the Yih1 mediated interactions occur at distinct cellular locations within the cell, supporting the idea that spatially restricted cellular interactions controlled Gcn2 function. Using in vitro studies we have identified the regions on eEF1A that are involved in Gcn2 and Yih1 binding. The distinct binding sites for both proteins on eEF1A led to further investigations on how the dynamics of these interactions involving eEF1A might affect Gcn2 function. Together with unpublished observations by E Sattlegger and B Castilho, a function for the Yih1 ancient domain in interacting with eEF1A has been identified. Finally, the mechanisms by which Actin might control Gcn2 function were studied. In this regard, we have identified the Yih1-Actin interaction as one of the key PPIs involved in the crosstalk between the cytoskeleton and Gcn2 regulation. Together, the findings presented in this thesis, support the hypothesis that Gcn2 activity is spatiotemporally controlled by dynamic PPIs that occur at specific time at particular locations.
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    Rheo-NMR studies of macromolecules : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Physics at Massey University, Palmerston North, New Zealand
    (Massey University, 2008) Kakubayashi, Motoko
    In this thesis, the effects of simple shear flow on macromolecular structure and interactions are investigated in detail via a combination of Nuclear Magnetic Resonance (NMR) spectroscopy and rheology, namely Rheo-NMR. A specially designed NMR couette shear cell and benchtop shear cell, developed in-house, demonstrated that the direct measurement of the above phenomena is possible. First, to determine whether the shear cells were creating simple shear flow, results were reproduced from literature studies of liquid crystal systems which report shear effects on: Cetyl Trimethyl Ammonium Bromide (CTAB) in deuterium oxide, and Poly(gamma-benzyl-L-glutamate) (PBLG) in m-cresol. Next, the possible conformational changes to protein structure brought about by shear were investigated by applying shear to Bovine -lactogobulin ( -Lg). As the protein was sheared, a small, irreversible conformational change was observed by means of one-dimensional and two-dimensional 1H NMR with reasonable reproducibility. However, no observable change was detected by means of light scattering. A large conformational change was observed after shearing a destabilized -Lg sample containing 10% Trifluoroethanol (TFE) (v/v). From an NMR point of view, the sheared state was similar to the structure of -Lg containing large amounts of -helices and, interestingly, similar to the structure of -Lg containing -sheet amyloid fibrils. Gel electrophoresis tests suggested that the changes were caused by hydrophobic interactions. Unfortunately, this proved to be difficult to reproduce. The effect of shear on an inter-macromolecular interaction was investigated by applying shear during an enzyme reaction of pectin methylesterase (PME) on pectin. Experimental method and analysis developments are described in detail. It was observed that under the conditions studied, shear does not interfere with the de-esterification of pectin with two types of PME, which have different action mechanisms at average shear rates up to 1570 s-1.