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Bibliography: leaves 94-108. / xi, 108, [88] leaves, [28] 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 aim of this study is to characterize plastid DNA sequence homologies within higher plant nuclear genomes. It is concluded that integrated within the tobacco nuclear genome are multiple copies of large (ie. in excess of 18 kbp), contigous fracts of plastid DNA. The presence of large tracts of plastid DNA in the tobacco nuclear genome contrasts the arrangement of such sequences in the nuclear genomes of other studied plant species. / Thesis (Ph.D.)--University of Adelaide, Dept. of Genetics, 1993

Thesis (Ph. D.)--Rutgers University, 2008. / "Graduate Program in Microbiology and Molecular Genetics." Includes bibliographical references (p. 431-473).

The aim of this work was to study spatial evolution of the chemotaxis proteins of a group of plant-associated soil-dwelling bacteria vernacularly referred to as the B. subtilis group. This was achieved by creating homology models for the chemotaxis proteins if a suitable template was available, and by analysing the selective forces (positive, purifying or neutral) acting upon the chemotaxis proteins. Chemotaxis is the phenomenon in which bacteria direct their movement towards more favourable conditions, and is critical for processes such as obtaining nutrients, escaping toxic compounds, host colonization and bio-film formation. Members of the B. subtilis group exhibit different preferences for certain host plants, and it is therefore feasible that their chemotactic machinery are fine-tuned to respond optimally to the conditions of the various niches that the strains inhabit. Homology models were inferred for the plant growth promoting B. amyloliquefaciens FZB42 proteins CheB, CheC, CheD, CheR, CheW and CheY. The interactions between: CheC-CheD, the P1 and P2 domains of CheA with CheY and CheB, and the P4 and P5 domains of CheA with CheW were also modelled. The hydrophobic interactions contributing to intra- and inter-protein contacts were analysed. The models of the interactions between CheB and the various domains of CheA are of particular interest, because to date no structures have been solved that show an interaction between a histidine kinase (such as CheA) and a multidomain response regulator (such as CheB). Furthermore, evidence that phospho-CheB may inhibit the formation of phospho-CheY by competitively binding to the P2 domain of CheA is also presented. Proteins were analysed to determine if individual amino acid sites are under positive, neutral or purifying selection. The Methyl Accepting Chemotaxis Proteins (MCPs), CheA and CheV were also analyzed, but due to a lack of suitable templates, no homology models were constructed. Site-specific positive and purifying selection were estimated by comparing the ratios of non-synonymous to synonymous substitutions at each site in the sequences for the chemotaxis proteins as well as for the receptors McpA, McpB, and McpC. Homology models were coloured according to intensity of selective forces. It was found that the chemotaxis proteins of member of the B. subtilis group are under strong evolutionary constraints, hence it is unlikely that positive selection in these proteins are responsible for the differences in habitat preference that these organism exhibit.

Three-dimensional (3D) structure of a protein is essential as the guidance of structure-based drug dis-covery. To achieve robust homology modeling with atomic-level accuracy, reliable loop predictions are required. Here, a novel hierarchical protocol of Protein Local Optimization Program (PLOP) is designed to produce sub-2 angstrom predictions on loop regions in homology modeling. Dramatic improvements in both speed and accuracy have been realized with implementation of special-designed clustering and adaptive loop closure algorithm. Four prediction rounds are designed for homology modeling as the high-level protocol of PLOP, which allows latter rounds employ the educated guess of backbone atom positions and hydrogen bonding information inherited from the previous rounds, contributing to additional prediction accuracy. The success of PLOP has been demonstrated with four different data sets, mainly concen-trating on homology modeling of H3 loops of antibodies. GPU-accelerated sampling algorithm and deep learning models are implemented, which are able to produce promising predictions as input templates for PLOP in the context of homology modeling.

The results of deoxyribonucleic acid homology experiments with the type strains of Azospirillum lipoferum, Azospirillum brasilense, and Azospirillum amazonense, and 19 additional strains of A. amazonense, confirmed that A. amazonense is a distinct new species. The description of the genus Azospirillum is emended to accommodate A. amazonense. Ribosomal RNA (rRNA) homology studies indicated 90 to 96% homology between Azospirillum lipoferum and Azospirillum brasilense, and 64 to 70% homology between these species and Azospirillum amazonense. This supports the inclusion of these three species in the genus Azospirillum. In contrast, "Azospirillum seropedicae" had very little homology with the other Azospirillum species (<22% RNA homology) and should not be considered a member of the genus. The taxonomic placement of "A. seropedicae" is uncertain. The nearest relatives of the genus Azospirillum were Aquaspirillum itersonii and Rhodospirillum rubrum (>65% RNA homology); Gluconobacter oxydans and Beijerinckia indica exhibited 30 to 60% RNA homology with Azospirillum species. Deoxyribonucleic acid (DNA) studies indicated that Conglomeromonas largomobilis subsp. largomobilis was related to Azospirillum lipoferum by >45% DNA homology and by 99% RNA homology; moreover, it was found to be a microaerophilic nitrogen fixer. Thus C. largomobilis subsp. largomobilis is a subjective synonym of A. lipoferum. In contrast, DNA homology studies indicated that Conglomeromonas largomobilis subspecies parooensis was unrelated to C. largomobilis or A. lipoferum, or to any other species tested, and its taxonomic position is uncertain. Several strains of azospirilla which form unique star-shaped colonies were identified as A. lipoferum by DNA homology. / Master of Science

This thesis describes improvements to our protein loop structure prediction algorithm and use of this algorithm to inform a computational investigation of anti-HIV antibodies. First, in Section I, we outline improvements to the Protein Local Optimization Program ("Plop") that allow us to reliably restore long loops containing secondary structure elements, and predict nativelike conformations for loops whose surroundings deviate from the native crystal structure context. Shifting to focus exclusively on antibody hypervariable loop prediction, we also benchmark our results in the community-wide Second Antibody Modeling Assessment. Plop can now be reliably deployed as a tool for understanding important biological systems. In Section II, we start from a system of interest - broadly neutralizing antibodies against HIV-1 - with the long-term goal of computationally identifying more potent VRC01-class anti-HIV antibodies. We show proof of concept results for predicting relative binding affinity upon mutation using free energy perturbation (FEP) simulations for the VRC01 antibody binding to glycoprotein gp120. Using the protocols developed in Section I, we provide case studies for using Plop to understand key FEP results and guide future experiments.

A lessor understood co-chaperone, the Hsp70/Hsp90 organising protein (Hop), has been found to play an important role in modulating the activity and co-interaction of two essential chaperones; Hsp90 and Hsp70. The best understood aspects of Hop so far indicate that residues in the concave surfaces of the three tetratricopeptide repeat (TPR) domains in the protein bind selectively to the C-terminal motifs of Hsp70 and Hsp90. Recent research suggests that P. falciparum Hop (PfHop), PfHsp90 and PfHsp70 do interact and form complex in the P. falciparum trophozooite and are overexpressed in this infective stage. However, there has been almost no computational research on malarial Hop protein in complex with other malarial Hsps.The current work has focussed on several aspects of the in-silico characterisation of PfHop, including an in-depth multiple sequence alignment and phylogenetic analysis of the protein; which showed that Hop is very well conserved across a wide range of available phyla (four Kingdoms, 60 species). Homology modelling was employed to predict several protein structures for these interactions in P. falciparum, as well as predict structures of the relevant TPR domains of Human Hop (HsHop) in complex with its own Hsp90 and Hsp70 C-terminal peptide partners for comparison. Protein complex interaction analyses indicate that concave TPR sites bound to the C-terminal motifs of partner proteins are very similar in both species, due to the excellent conservation of the TPR domain’s “double carboxylate binding clamp”. Motif analysis was combined with phylogenetic trees and structure mapping in novel ways to attain more information on the evolutionary conservation of important structural and functional sites on Hop. Alternative sites of interaction between Hop TPR2 and Hsp90’s M and C domains are distinctly less well conserved between the two species, but still important to complex formation, making this a likely interaction site for selective drug targeting. Binding and interaction energies for all modelled complexes have been calculated; indicating that all HsHop TPR domains have higher affinities for their respective C-terminal partners than do their P. falciparum counterparts. An alternate motif corresponding to the C-terminal motif of PfHsp70-x (exported to the infected erythrocyte cytosol) in complex with both human and malarial TPR1 and TPR2B domains was analysed, and these studies suggest that the human TPR domains have a higher affinity for this motif than do the respective PfHop TPR domains. This may indicate potential for a cross species protein interaction to take place, as PfHop is not transported to the human erythrocyte cytosol.

The papain-like cysteine protease family (C1 proteases) is highly important because of its involvement in research and industrial applications and its role in various human diseases. Protein inhibitors are an important aspect of C1 protease biology and are relevant to its clinical, industrial and research importance. To study the interaction between the proteases and the inhibitors it is very useful to have accurate structural models of the protease-inhibitor complexes. To this end, a high-throughput pipeline for modelling complexes of papain-like cysteine proteases and protein inhibitors was implemented and tested (Tastan Bishop & Kroon, 2011). The pipeline utilizes a novel technique for obtaining modelling templates by using superpositioning to combine coordinates from separate experimental structures. To test the pipeline, models of complexes with known structures (test set) were modelled using many different templates and the resultant models evaluated to compare the quality of the different templates. It was found that use of the new technique to obtain templates did not introduce significant errors, while allowing closer homologs to be used for modelling - leading to more accurate models. The test set models were also used to evaluate certain steps of the modelling protocol. The effect of Rosetta energy minimization on model accuracy and the use of Rosetta energy and DOPE Z-score values to identify accurate models were investigated. Several complexes were then modelled using the best available templates according to criteria informed by the previous results. A website was built that allows a user to download any of the metrics or models produced in the study. This website is accessible at http://rubi.ru.ac.za/cpmdb

Thesis (MSc (Biochemistry))--University of Stellenbosch, 2005. / This study describes: 1. The cloning of baboon cytochrome P450 side-chain cleavage (CYP11A1) cDNA by in vitro site-directed mutagenesis. 2. The identification and sequencing of three baboon CYP11A1 mutants: CYP11A1a, CYP11A1b and CYP11A1c. 3. The expression and characterisation of baboon and human CYP11A1 cDNA, CYP11A1a, CYP11A1b and CYP11A1c in nonsteroidogenic COS-1 cells. The Km and V-values for the metabolism of 25-hydroxycholesterol were determined. 4. The construction of the first homology model of CYP11A1, using both mammalian (CYP2C5) and bacterial (CYP102) cytochromes P450 crystal structures as templates. 5. A structure/function study into the role of the amino acid residues Ile98, Lys103 and Thr291 in substrate binding and enzymatic activity. 6. The proposal of a topological model of the CYP11A1 active pocket as determined by substrate docking studies.