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    In search of novel folds : protein evolution via non-homologous recombination : a dissertation presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Albany, New Zealand
    (Massey University, 2014) Saraswat, Mayank
    The emergence of proteins from short peptides or subdomains, facilitated by the duplication and fusion of the minigenes encoding them, is believed to have played a role in the origin of life. In this study it was hypothesised that new domains or basic elements of protein structure, may result from nonhomologous recombination of the genes coding for smaller subdomains. The hypothesis was tested by randomly recombining two distantly related (βα)8-barrel proteins: Escherichia coli phosphoribosylanthranilate isomerase (PRAI), and β subunit of voltage dependent K+ channels (Kvβ2) from Rattus norvegicus. The aim was to identify new, folded structures, which may or may not be (βα)8-barrels. Incremental truncation (ITCHY), a method for fragmenting and randomly recombining genes, was used to mimic in vivo non-homologous recombination and to create a library of chimeric variants. Clones from the library were selected for right reading frame and solubility (foldability) of the recombined chimeras, using the pSALect selection system. Out of the six clones identified as soluble by pSALect, only one (P25K86) was found to be actually soluble. The protein, P25K86, was found to form oligomers and on treatment with a reducing agent, β-mercaptoethanol the multimeric state disappeared. The protein has three cysteines and one of the cysteines (Cys56) was found to mediate in the bond formation, thus giving a dimeric state. An engineered version of P25K86 that has the Cys56 replaced by serine was expressed as a monomer and additionally it was found to be ! iv! more stable. As the pSALect folding selection system reported false positives, i.e. only one of the six chimeras was actually soluble, it was concluded that the in vivo solubility selection system was leaky. A series of experiments were conducted so as to improve pSALect that led to the creation of pFoldM – a more stringent selection system, discussed in chapter 4. Comparing the newer improved version with the old, two more interesting chimeras were discovered. A total of 240,000 non-homologous recombination events were created in vitro and three soluble chimeras (evolutionary solutions) were found. Data from circular dichroism spectroscopy (CD) combined with heteronuclear single quantum coherence (HSQC) spectra suggest that the proteins, P24K89 and P25K86, are present in a molten globule state. ITCHY, as a means of mimicking the subdomain assembly model, was applied in vitro. The discovery of two interesting chimeras (P25K86 and P24K89) using highthroughput engineering experiments widens the possibilities of exploring the protein structure space, and perhaps offers close encounters with these never born proteins that may be trapped in an ensemble of fluctuating (structured and unstructured) states.
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    Eukaryotic signature proteins : guides to pathogenic eukaryotic parasites : a thesis presented in partial fulfilment of the requirements of the degree of PhD in Genetics at Massey University, Palmerston North, New Zealand
    (Massey University, 2012) Han, Jian
    Eukaryotic Signature Proteins (ESPs) are proteins that delineate the eukaryotes from the archaea and bacteria. They have no homologues in any prokaryotic genome, but their homologues are present in all main branches of eukaryotes. ESPs are thus likely to have descended from ancient proteins that have existed since the first eukaryotic cell. This project looks at ESPs of some eukaryotic parasites and human (Homo sapiens) as their host organism and focuses on Giardia lamblia, a fresh water pathogenic basal eukaryote. The ESP datasets from Giardia and two other parasites, Trichomonas vaginalis and Plasmodium falciparum, as well as the host human were calculated in light of available genomic data and the datasets contained a range of proteins associated with membrane, cytoskeleton, nucleus and protein synthesis. ESPs have great potential in phylogenetic studies since these proteins are present in all eukaryotes and are expected to have a slow and constant rate of evolution. Phylogenetic analyses were performed on the 18 eukaryotic organisms including some basal eukaryotes, and also for mammals, using orthologues of the all ESPs from these organisms. Strategies such as concatenating sequences and constructing consensus networks were tested to evaluate their potential with large numbers of ESP alignments. The results were promising, and ESPs hold great potential for their use in future phylogenetic analyses of eukaryotes. RNA interference is hypothesised to be an ancient mechanism for gene regulation and like the ESPs, it is typically found in all main branches of eukaryotes. High throughput sequencing data from Giardia and Trichomonas small RNAs (15-29mers) were re-analysed showing two length peaks for Giardia RNAs: a “larger peak” and an “ultra small peak”, the former of which is likely to be the product of the enzyme Dicer, which processes miRNA. The “ultra small peak” but not the “larger peak” was also found in Trichomonas. The two peaks possibly represent two different mechanisms of RNA interference (RNAi) in these parasites, but analysis of potential target sites from the Dicer-processed RNAs has not yet shown any indication that ESPs are regulated any differently from other parasite proteins. Sugar metabolic pathways including glycolysis and citric acid cycle were searched for ESPs, this was done to determine the relationship between the conservation of eukaryotic metabolic pathways and conservation of individual proteins. However no ESPs were identified from these pathways because Giardia has enzymes that show more similarity to those from prokaryotes than eukaryotes. These enzymes are significantly different from that of the host‟s, and these alternative enzymes offer potential as novel drug targets. In addition, ESPs that are present from host but lost in some parasites were analysed, and these ESPs are involved in many understudied pathways. It is these differences which can provide a guide in determining which pathways we should examine when designing drug targets. Overall, numerous proteomic similarities and differences in ESPs were identified between host and parasite. These proteins show potential for future evolutionary studies, and will guide future directions in ancestral eukaryotic regulation and metabolism.