Proteins play a vital role in all organic life. The structure of a protein is directly related to its function. Hence, how they fold and what they fold into is of great interest. Given the spontaneous manner in which many proteins fold, one would not expect complicated structures like knots to occur in native states. Nevertheless, current research has shown that proteins do indeed contain local knots; some with as many as 6 crossings. In general, the role of knots in proteins and how they are formed is not completely understood. This thesis develops models of protein knotting by using knot theory and tangles. Mathematically, a knot is just a topological embedding of a circle in Euclidean 3-space, R3, or the unit 3-sphere, S3. A tangle is defined as a pair, (B, T), where B is a 3-dimensional ball and T is a set of disjoint arcs properly embedded in B. We begin with 2-string tangles and use the tangle calculus developed by Ernst and Sumners to set up tangle equations. In this model the strings of the 2-tangles represent the protein chain. Solutions to these 2-string tangle equations are then found. Motivated by the hypothesized folding pathway of the knotted protein DehI, a more complicated 3-string tangle model is developed. It is hypothesized that a terminal end of the protein is threaded through two loops. In the proposed model, the threading of a terminal end of the protein through two loops is translated into a Γ;-move on 3-string tangles. A Γ;-move is a special type of 3-string tangle replacement. The 3-braids are utilized as a subset of 3-string tangles to find solutions in a limited case. Additionally, tangle models give insight into how to make specific knot types in proteins. We finish with a general result by proving that any knot of unknotting number 2 can be unknotted by the Γ;-move. With these models we determine which knots are the most biologically possible to occur in proteins.

PSD-95/Dlg/ZO-1 (PDZ) domain - PDZ binding motif (PBM) interactions have been one of the most well studied protein-protein interaction systems through biochemical, biophysical and high-throughput screening (HTS) strategies. This has allowed us to understand the mechanism of individual PDZ-PBM interactions and the re-engineering of PBMs to bind tighter or to gain or lose certain specificity. However, there are several thousand native PDZ domains whose biological ligands remain unknown. Because of the low sequence identity among PDZ domain homologues, promiscuous binding profiles (defined as a PDZ domain that can accommodate a set of PBMs or a PBM that can be recognized by many PDZ domains), and context-dependent interaction mechanism, we have an inadequate understanding of the general molecular mechanisms that determine the PDZ-PBM specificity. Therefore, predicting PDZ specificity has been elusive. In addition, no de novo PBM ligand or artificial non-native PDZ domain have been successfully designed. This reflects the general challenges in understanding the general principles of PDZ-ligand interactions, namely that they are context-dependent, exhibit weak binding affinity, narrow binding energy range, and larger interaction surface than other protein-ligand interactions. Together, PDZ domains make good model systems to investigate the fundamental principles of protein-protein interactions with a wide spectrum of biomedical implications. My studies suggest that understanding PBM specificity with the set of structural positions forming the binding pocket can connect sequence, structure and function of a PDZ domain in a general context. They also suggest that this way of understanding the specificity will shed light on prediction and engineering of specificity rationally. Structural analysis on most of the available PDZ domain structures was established to support the principle (Chapter I). The principle was tested against two different types of PBM; C-terminal PBM (Chapter II) and internal PBM (Chapter III), and shown to support better understanding and design of PDZ domain specificity. We further applied the principle to design de novo PDZ domains, and the preliminary data hints that it is optimistic to engineer PDZ domain specificity (Appendix A and B).

No description available.

No description available.

Doctor of Philosophy(PhD) / Soybeans contain around 40% of high quality protein and 20 % of oil. Soy protein has long been used as ingredients for its emulsification and texturizing properties in a variety of foods, soymilk and tofu being the most popular. Soymilk is essentially a water extract of soybeans and there are many variations on the basic soymilk processing steps. Tofu, or bean curd, is made by coagulating soy milk, and then pressing the resulting curds into blocks. This thesis was mainly devoted to thermal denaturation and coagulation of soy proteins and targeted several selected important factors as they relate to the functional properties. The effects of different chemical coagulants as well as proteases on yield and quality of tofu from soybeans were studied. Eight tested chemical coagulants were able to coagulate the soymilk and the results showed that the concentration of soymilk and type of coagulant had a great influence on the properties of the tofu gel. The results also confirmed that the use of a suitable concentration of the quick-acting coagulants is more critical than that of the slow-acting coagulants in tofu making. In general, the extent of soymilk gelation is not determined by a single characteristic but rather results from a combination of factors. The gelation ability of various most common commercially available proteases to coagulate non-defatted soymilk was surveyed and the thermal stabilities of selected protease systems were compared. The difference in the temperature where the enzyme shows its highest activity seemed to be the most significant indicator when choosing a suitable enzyme for a certain industrial application. The three most effective and versatile soymilk coagulants were identified. The presence of small amounts of ficin in the system increased the protein recovery when calcium chloride was used as a coagulant. The most commonly used techniques of analysis of degree of hydrolysis (TNBS, OPA and pH-stat) of soy protein were compared. It was concluded that the pH-stat technique was useful for evaluating the progress of an enzyme-catalyzed protein hydrolysis process on an industrial scale while the OPA method seemed to be the most suitable method to be used for determining DH during the proteolysis of soymilk in laboratory conditions. The roles of soybean proteins, protein fractions and subunits to differences in gelling properties of different soybean varieties were examined. The variability and the interrelationship between soybean seed traits were established and the seed characteristics related to soymilk yield and tofu quality were identified. The results suggested that it is useful to predict the quality of tofu from a combination of characteristics of the soybean seed. It was concluded that large differences exist in soybean seed characteristics and their contributions towards the properties of the final product and implications were made towards the relative importance of individual soybean seed traits to the functional and textural properties of soy products. The SDS gel capillary electrophoresis was applied to characterize soybean storage proteins. The lab-on-a-chip technology was compared with capillary electrophoresis and these two methods were used to quantify the relative amount of 7S and 11S fractions in various soybean cultivars. It was concluded that both lab-on-a-chip instrument and a traditional CGE were adequate for analysis of soy-based products. Both systems were able to reliably quantify the relative amount of protein fractions in samples and thus demonstrate their different genetic origin. The great advantage of the lab-on-a-chip technology is its time-efficiency while the traditional CGE is a preferred instrument for method development. The usefulness of the chemometrical analysis of electrophoretic profiles as a method for objective evaluation, data reduction and interpretation was shown. The possibility of improvement of the protein extraction from soybeans in order to provide a basis for the optimization of soymilk production was studied. The enzyme-assisted extraction using the hydrolytic enzyme treatment to disrupt the soybean cell wall components was expected to improve the protein extraction yield. The results confirmed that the right selection of operational variables led to an increased yield of soymilk as well as its protein concentration. It was also shown that the addition of selected enzyme preparations into the soymilk process design resulted in an increased extraction yield of proteins from seeds into soymilk. The protein quality did not deteriorate during the enzyme-assisted extraction process and a small amount of microbial transglutaminase added together with a coagulant produced tofu with a significantly increased yield while maintaing satisfactory textural properties.

Prion protein (PrP) is best known for its involvement in prion diseases. A normal, dynamic isoform of prion protein (PrP^C) transforms into a pathogenic, compact isoform (PrP^Sc) during prion disease pathogenesis. The PrP^Sc, acting as a template upon which PrP^C molecules are refolded into a likeness of itself, accumulates in the brain neurones and causes disease. It is the only known component of prions, proteinaceous infectious particles. Both prion protein isoforms have the same primary amino acid structure and are encoded by the same prion protein gene (PRNP). PRNP determines susceptibility/disposition to prion diseases and their phenotypes.¶The normal function of PRNP is elusive. The Prnp knock-out mice with disrupted ORF show only very subtle phenotype. A number of hypotheses were proposed on the function of mammalian PRNP. The extracellular, GPI-anchored, glycosylated mammalian PrP^C expressed in a heterogenous set of cells could: transport copper from extracellular to intracellular milieu, buffer copper from synapse, contribute to redox signalling, act neuroprotectively, mediate cell-cell contacts, affect lymphocyte activation, participate in nucleic acid metabolism, be a memory molecule, and be a signal-transduction protein.¶ Experimental evidence demonstrated a redundancy between the PRNP and another, unknown gene. The critical issue therefore is to discover new genes homologous with PRNP, candidates for this redundancy. Using unpublished data, a sequence of zebrafish cDNA sequenced by Prof. Tatjana Simonic’s group (University of Milan, Italy), I discovered a new paralogue of PRNP. By searching manually, and in a targeted fashion, data deposited in public biological databases, I compiled support for the new human gene Shadow of prion protein (SPRN) including the direct evidence, homology-based evidence and ab initio gene prediction. The protein product called Shadoo (shadow in Japanese) is an extracellular, potentially glycosylated and GPI-anchored protein of a mature size of 100-odd amino acids. It is conserved from fish (zebrafish, Fugu, Tetraodon) to mammals (human, mouse, rat), and exhibits similarity of overall protein features with PrP. Most remarkably, the Sho is the first human/mammalian protein apart from PrP that contains the middle hydrophobic region that is essential for both normal and pathogenic properties of PrP. As this region is critical for heterodimerization of PrP, Sho may have potential to interact with PrP and is a likely candidate for the Protein X. Mammalian SPRN could be predominantly expressed in brain (Tatjana Simonic Lab, University of Milan, Italy).¶ Using the same approach to search public databases, I found, in addition, a fish duplicate of SPRN called SPRNB, and defined a new vertebrate SPRN gene family. Further, I also expanded a number of known fish genes from the PRNP gene family. The total number of the new genes that I discovered is 11. With the representatives of two vertebrate gene family datasets in hand, I conducted comparative genomic analysis in order to determine evolutionary trajectories of the SPRN and PRNP genes. This analysis, complemented with phylogenetic studies (Dr. Lars Jermiin, University of Sydney, Australia), demonstrated conservative evolution of the mammalian SPRN gene, and more relaxed evolutionary constraints acting on the mammalian PRNP gene. This evolutionary dialectic challenges widely adopted view on the “highly conserved vertebrate” PRNP and indicates that the SPRN gene may have more prominent function. More conserved Sprn could therefore substitute for the loss of less conserved, dispensable Prnp in the Prnp knock-out mice. Furthermore, the pathogenic potential of PRNP may be a consequence of relaxed evolutionary constraints.¶ Depth of comparative genomic analysis, strategy to understand biological function, depends on the number of species in comparison and their relative evolutionary distance. To understand better evolution and function of mammalian PRNP, I isolated and characterized the PRNP gene from Australian model marsupial tammar wallaby (Macropus eugenii). Marsupials are mammals separated from their eutherian relatives by roughly 180 million years. Comparison of the tammar wallaby and Brazilian opossum PrP with other vertebrate PrPs indicated patterns of evolution of the PrP regions. Whereas the repeat region is conserved within lineages but differs between lineages, the hydrophobic region is invariably conserved in all the PrPs. Conservation of PrP between marsupials and eutherians suggests that marsupial PrP could have the same pathogenic potential as eutherian PrPs. Using the marsupial PRNP gene in comparison with the PRNP genes from eutherian species in which prion diseases occur naturally (human, bovine, ovine) or experimentally (mouse), I defined gene regions that are conserved mammalian-wide and showed the utility of the marsupial genomic sequence for cross-species comparisons. These regions are potential regulatory elements that could govern gene expression and posttranscriptional control of mRNA activity. These findings shed new light on the normal function of mammalian PRNP supporting best the signal-transduction hypothesis. The normal function of PRNP may be triggering of signalling cascades which contribute to cell-cell interactions and may act anti-apoptotically. Yet, in the heterogenous set of cells expressing PrP^C these pathways will contribute to a number of cell-specific phenotypes, such as the synaptic plasticity and activation of lymphoid cells.

Protein modification by ubiquitination regulates protein abundance, function and localisation. Specificity of ubiquitination is largely determined by ubiquitinprotein ligases (E3s). The Nedd4–family proteins are a group of E3s containing a conserved domain structure of a C2 domain, multiple WW domains and a carboxyl terminal HECT domain, which is responsible for E3 activity. The prototypical member of this family, Nedd4, is known to down-regulate the epithelial Na+ channel (ENaC) by ubiquitination. This process requires interactions between ENaC and specific WW domains of Nedd4. Mutation or deletion of WW domain binding sites in ENaC leads to Liddle's syndrome, an autosomal dominant form of hypertension. At the beginning of this study there was evidence to suggest that Nedd4–2, a Nedd4–family protein closely related to Nedd4, could also regulate ENaC. The focus of this study was to characterise the ability of Nedd4–2 to regulate ENaC and other potential substrates. Two major splice variants of Nedd4–2 were identified, which were both found to down–regulate ENaC in Xenopus oocytes. In vitro binding studies and whole cell functional analysis showed that interactions between ENaC and Nedd4– 2 occur via two of the four Nedd4–2 WW domains. The E3 activity of Nedd4–2 was further examined, revealing that it can use the same ubiquitin–conjugating enzymes as Nedd4 and exhibits strongest activity in the presence of UbcH5b. An in vitro ubiquitination assay and whole cell functional analysis provided evidence that Nedd4–2 down-regulates ENaC via ubiquitination. The possibility that Nedd4 and Nedd4–2 could down-regulate a number of voltage–gated Na+ channels (Navs) by a similar mechanism to regulation of ENaC was investigated. Not only were Nedd4 and Nedd4–2 found to interact with seven Navs, but these channels and ENaC have conserved WW domain binding specificity. Ubiquitination studies indicated that these channels can be ubiquitinated by Nedd4 and Nedd4–2. Co–expression of Nedd4 or Nedd4–2 with neuronal Navs in Xenopus oocytes reduced channel activity to varying degrees. These data indicate that Nedd4 and Nedd4–2 are likely to be key regulators of neuronal Nav channels in vivo. / Thesis (Ph.D.)--Department of Medicine, 2004.

1v. (various pagings) : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (D.Sc.)--University of Adelaide, Dept. of Physical and Inorganic Chemistry, 1974

Bibliography: leaves 133-157. / xiii, 157, [22] leaves, [31] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The principle aim of the work described in this thesis was to use protein engineering to increase the thermostability of barley (1->3,1->4)-B-glucanases / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 2000

Erlangen, Nürnberg, Universiẗat, Diss., 2007. / Enth. 1 Sonderabdr. aus: International journal of radiation biology ; Vol. 83. 2007. - Beitr. teilw. dt., teilw. engl.