This thesis describes some experiments developed to probe the fundamental aspects of the interfacial behaviour of proteins. The contents of this thesis can be broadly divided into two parts. In the first part, we studied how the size of the nanoparticles and other variables such as pH and bulk protein concentration affect the structure of the adsorbed protein layers. We also probed how these factors can influence the binding activity of adsorbed proteins. Study on the adsorption of IgG, Protein A and streptavidin on gold nanoparticles reveals that not all proteins are similarly affected by the size of the adsorbing surface. We found that though the optical properties of all the proteins vary with the size of the nanoparticle, their functionalities are not similarly affected by nanoparticle curvature. Protein A and streptavidin retain their binding capacity to IgG and biotin, respectively, irrespective of the size of the gold nanoparticle that they are attached to. On the other hand, a reduction/ loss in binding of adsorbed IgG to Protein A molecules is observed. The reduction in biological activity further depends on the radius of curvature of the adsorbing surface. The second part of the thesis describes how nanoparticles can used as a probe to study the complex interfacial behaviour of proteins. We have utilized the extreme sensitivity of localized surface plasmon resonance (LSPR) of gold nanoparticles to local refractive index to determine the optical properties of BSA adsorbed on various polymer surfaces. The dielectric properties of the adsorbed protein depend on the nature of the substrate. Further, we have developed a model to determine the refractive index profile of adsorbed protein as a function of the distance from the substrate.

The lumen of the endoplasmic reticulum (ER) provides a specialized environment to assure the folding and oligomerization of secretory proteins to their native conformations. UDP-glucose:glycoprotein glucosyltransferase (UGGT) is a biosensor in the ER that detects the folding state of glycoproteins. UGGT-catalyzed monoglucosylation of incompletely folded glycoproteins leads to their continued retention in the ER through their association with the lectins calnexin and calreticulin for further folding or for degradation. Purified recombinant UGGT from rat liver and glycoprotein substrates from a mutant strain of Saccharomyces cerevisiae were used in an in vitro system to examine the peptide components recognized by UGGT in unfolded glycoproteins and glycopeptides. Mass spectrometry was used to measure and quantitate the levels of glucose incorporation into these substrates that was directly related to their level of recognition by UGGT. To assess the capacity of UGGT for sensing non-native structures in glycoprotein substrates, Exo-1,3-beta-glucanase (beta-Glc) from S. cerevisiae was crystallized and its structure determined. A mutagenesis strategy was used to mutate solvent-exposed residues to yield the beta-Glc F280S point mutant that retained enzymatic activity while still being recognized by UGGT. These data suggest that UGGT recognizes solvent-exposed hydrophobic patches in the primary and tertiary structure of glycoproteins even in near-native conformations.

In this study, the deamidation is involved to modify the structure of barley proteins in terms of prolamin and glutelin in order to improve the functional properties of protein. A wide range of deamidation degrees (0.1% to 45%) were prepared using alkaline method. The results suggested that the optimal deamidation degree of barley prolamin is around 2.4-4.7%, where the solubility, emulsifying and foaming properties of prolamin were significantly improved at both acidic and neutral pHs. The optimal deamidation degree for glutelin is around 2.2 to 5.6%, where deamidated glutelin demonstrated markedly improved solubility at both acidic and neutral pHs. Glutelin performed strong tendency to form aggregates with spherical shape and very large molecular weight. These aggregates are important in stabilizing the emulsions at a broad range of deamidation degree (5.6-43%). These results suggest that barley protein would be an excellent candidate to develop as an emulsifying and foaming ingredient. / Food Science and Technology

Oxidative protein folding within the ER depends on the enzymatic action of numerous chaperones and oxidoreductases. In addition, this process requires the influx of metabolites and energy, including FAD (flavin adenine dinucleotide) and molecular oxygen. Secretory proteins and proteins destined to the secretory pathway need to undergo this process in order to obtain stability and full functionality. Since secretory proteins that fail to fully fold are eliminated by degradation, the process of ER oxidative protein folding is part of a group of ER-associated mechanisms commonly referred to as ER quality control. Interestingly, the proteins that mediate ER quality control can be found in a variety of diverse subdomains of the ER. We have found that the ER-oxidoreductase Ero1 is located on the mitochondria-associated-membrane, the MAM. This specialized subdomain of the ER has been shown to be crucial for a number of processes such as the synthesis of phospholipids as well as calcium-channelling between the ER and mitochondria. The goal of this thesis was to identify possible retention mechanisms and motifs of Ero1 to the MAM.

Graduation date: 1981

Protein synthesis (mRNA) is tightly regulated under numerous conditions in cardiomyocytes. It can be activated by hormones such as insulin and also by other agents such as phenylephrine (PE) that activates hypertrophy in the heart. Cardiac hypertrophy involves an increase in the muscle mass of the heart, principally in the left ventricular muscle, and the increase is due to enlarged cell size, not increased cell number. A pivotal element of cardiac hypertrophy is an elevation in the rates of protein synthesis, which drives the increase in cell size causing hypertrophy. Unfortunately, we currently lack the understanding of the basic mechanisms that drives hyperactivated protein synthesis. Cardiac hypertrophy is clinically important because it is a major risk factor for heart failure. It initially serves as an adaptive response to increase cardiac output in response to higher demand, but ultimately leads to deterioration of contractility of the heart if hypertrophy is sustained. The main goal of this research project is to understand how hypertrophic agents, such as phenylephrine (PE), activate protein synthesis using adult rat ventricular cardiomyocytes as a model. Specifically, this study focuses on how the translational initiation is controlled by upstream signalling pathways.

Research Doctorate - Doctor of Philosophy (PhD) / This study described in this work examines the potential for derivatives of the potent PP1 (IC50 9.0 µM) and PP2A (IC50 3.0µM) inhibitor, norcantharidin, the demethylated cantharidin analogue, and their protein phosphatase inhibition, namely PP1 and PP2A and their cytotoxicity across a range of human cancer cell lines. A variety of derivatives were examined, paying particular attention to modifications to the anhydride moiety. These included a series of ring opened and ring closed cantharimides, a series of α-hydroxylactams, a series of lactone analogues and derivatives, and a series of heteroatom substituted analogues. Of the analogues developed, the ring opened and ring closed cantharimides displayed moderate to excellent activity, in cases, an improvement over the lead compound norcantharidin was observed. The ring closed dodecyl-linked bis analogue (63) was the most potent analogue displaying µM potent cytoxicities against all the cell lines examined. Of the ring opened analogues, the morpholino analogues proved most active.

Milk protein content of cows in subtropical Queensland declines from the beginning of winter to the end summer and is a repeatable consistent observation across years. The genesis of such an event is hypothesised to lie in the nutritional and environmental changes that occur over this period with a change in forage type from the temperate species to the tropical species, a change in cereal grain supplementation and a reduction in intake associated with heat stress causing a change in the nutritional supply to the animal. A series of experiments examined the effect of high levels of ammonia absorption on milk production and milk components in dairy cows. The experiments used extensive, grazing trials with supplements of urea to increase ammonia absorption, and intensive, infusion of ammonia into cows indoors. Feeding urea in the supplement of cows grazing ryegrass increased milk protein significantly by 0.05 percentage units but had no effect on milk volume. Infusing ammonia intra-venously reduced milk volume (~ 20%) and milk protein content (<0.1 percentage units). The primary finding of these experiments was that infusion of high ammonia levels into dairy cows will depress milk protein production and content. However, the levels of ammonia absorption experienced under usual strip grazing conditions in Queensland over winter are not high enough to affect milk protein production and content. The effect of altering substrate supply and pattern associated with the shift in feed supply from winter to summer was examined by infusing different amino acid patterns and acetate into cows by a close arterial infusion method. Infusate was delivered to the external iliac artery, allowing the mammary gland first pass use of the nutrients. Supplying amino acids by infusion increased milk protein yield 18.7 vs 19.7 g/h (5.3 %) for infused half of the udder. Milk protein yield was increased more (19.2 vs 20.2 g/h for infused half of the udder) by infusion of a milk amino acid profile compared to a microbial amino acid profile. Infusing acetate has no effect on milk protein yield but decreased milk yield for infused half of the udder (560 vs 510 g/h). It may be concluded, that under the practical levels of crude protein and ammonia supply which arise from different agronomic practices (N fertiliser) and grazing management (stage of growth, strip grazing), that any practical increase in ammonia supply per se to the liver does not explain the depression in milk protein percentage seen in Queensland dairy herds.

xxi, 126 leaves : ill., tables ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1978

117 leaves : ill. ; 29 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1975