Call number: LD2668 .T4 1951 U4 / Master of Science

The three dimensional native structure of multi domain proteins is only achieved when the adjacent domains recognise each other through the domain-domain interface. The domain-domain interface of the Glutathione S-transferase (GST) family has been studied extensively; however, no studies have been conducted on the role of the linker regions in the domain-domain interactions. Glutaredoxin 2 (Grx2) protein, from the GST family was chosen as model to investigate the possible role of linkers in protein stability by mutational analysis. Bioinformatics data revealed a conserved residue within the linker region (Leu78 in Grx2). A Grx2 mutant was created by replacing the conserved residue (Leu78) within the linker region with an alanine. This mutation (Leu to Ala) was performed in order to assess the role of the conserved residue leucine; whilst maintaining Grx2 function. A previous Grx2 mutant (Grx2 Y58W) was utilised because it incorporates tryptophan into domain 1; therefore it was possible to follow tertiary structural changes in this domain. Grx2 Y58W was compared against the mutant created within the linker Grx2 Y58W/L78A. Far-UV CD spectrum indicated that there was an increase of (~30 %) in ellipticity of Grx2 Y58W/L78A protein whereas; tryptophan fluorescence probes indicated no change in tertiary structure. Conformational stability studies showed a decrease of ΔΔG (H2O) = 3.8 kcal.mol-1 due to the impact of the Y58W/L78A mutation. The m-value which is indicative of the co-operativity between the two domains has decreased slightly by ~0.4 kcal.mol-1 M-1. This reduction in the m-value suggested the formation of intermediate however; it was not evident when using ANS as a probe. This study indicates that replacing a leucine with an alanine in the linker region causes a reduction in domain co-operativity. Therefore, the linker region in addition to separating the two domains plays a role in interdomain co-operativity.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. October 2014. / The chloride intracellular channel protein 1 (CLIC1) is a dual-state protein that can exist either as a soluble monomer or in an integral membrane form. Dysfunction in membrane insertion has been implicated in several pathologies including apoptosis, cancer and homeostatic imbalance. The transmembrane domain (TMD) is implicated in membrane penetration and pore formation and is therefore a key target for understanding amphitropism in CLIC1. The mechanism by which the TMD binds, inserts and oligomerises in membranes to form a functional chloride channel is unknown. Here the secondary, tertiary and quaternary structural changes of the CLIC1 TMD and several TMD mutants are reported in an attempt to elucidate the membrane insertion mechanism. A synthetic 30-mer peptide comprising the TMD was examined in 2,2,2-trifluoroethanol (TFE), SDS micelles and POPC liposomes using far-UV CD, fluorescence and UV absorbance spectroscopy. The results suggest a fourstep mechanism whereby the TMD, which is unfolded in buffer, refolds into a helix which partitions onto the membrane, followed by insertion and dimerisation to form a membranecompetent protopore complex. These helices associate via a Lys37-mediated cation-π interaction to form weakly active dimers. The complex is then tethered to the membrane by a cationic motif acting as an electrostatic plug. Thus, electrostatic interactions provide both a strong thermodynamic driving force for helix-helix association as well as structural integrity within the membrane. This represents an important step towards understanding how amphitropism occurs in CLIC1 and offers a unique insight into how CLIC1 and other proteins defy the ‘one-sequence one-fold’ hypothesis.

A dissertation submitted to the Faculty of Health Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science in Medicine. 2017 / Mycobacterium tuberculosis assembles a complex cell wall consisting of mycolic acids, arabinogalactan and peptidoglycan layers. The peptidoglycan is important for structural maintenance and osmotic protection of the cell. Beta-lactam antibiotics, such as penicillin, perturb biogenesis of cross-linked peptidoglycan by inhibition of penicillin-binding proteins and cause cell death. As a result, penicillin-binding proteins have been extensively used in antimicrobial development. However, penicillin insensitive enzymes involved in peptidoglycan biogenesis such as amidases, transglycosylases and endopeptidases remain to be exploited for anti-TB drug development, a field that urgently requires new drugs in light of the rapid emergence of drug resistant strains. In this study, we functionally characterize a novel class of LytM domain containing peptidoglycan endopeptidases (also known as M23 peptidases) in mycobacteria. Bio-informatics tools were used to identify LytM domain-containing homologues in Mycobacterium smegmatis, designated MepB1-MepB4. These were deleted using standard allelic exchange mutagenesis and recombination techniques and the resulting mutants were assessed for cell wall related defects. We found that mycobacterial LytM endopeptidases have important roles in bacterial growth as demonstrated by delayed cell growth kinetics in a ΔmepB1 deletion mutant. We noted no growth defects in ΔmepB2 and ΔmepB3 single deletion mutants but observed defective cell division in a ΔmepB2 ΔmepB3 double deletion mutant. In this double mutant, spatial localization of new cell wall biosynthesis revealed the inability to degrade the septal bridge joining two daughter cells, pointing to a critical role for these enzymes in cell separation. MepB1 is sequestered from the peptidoglycan by cytosolic localization and its absence causes a septal and polar buldging phenotype. To further investigate the biological roles of these putative peptidoglycan endopeptidases, protein interaction studies were conducted using the bacterial two-hybrid mycobacterial protein fragment complementation assay. This analysis identified FtsX, a key cell division protein, as an interacting partner for both MepB2 and MepB3, thus identifying these proteins as novel components of the mycobacterial divisome. Collectively, these observations provide the first insight into a new group of potential drug targets for tuberculosis disease and notably enhance the overall understanding of peptidoglycan turnover, which is of general revelevance in bacterial pathogens. / MT2017

Ph.D, Faculty of Science, University of the Witwatersrand, 2011 / Chloride intracellular channel proteins (CLICs) are able to exist in a soluble or membrane-bound state. The mechanism by which the transition between the two states takes place is yet to be elucidated. It is proposed that structural rearrangements of the N-terminal domain take place when CLICs encounter the lower pH environment of the membrane surface (pH 5.5). This prompts the CLICs to form a soluble membrane-ready state prior to pore formation and membrane transversion. Since the insertion of CLIC1 into membranes occurs at low pH, perhaps protonation and electrostatic effects of key conserved residues at the domain interface situated within the transmembrane region bring about the structural changes necessary for this transition. Structural and sequence alignments revealed that a conserved salt-bridge interaction between conserved residues on helices 1 and 3 of the N-terminal domain is present at the domain interface of CLICs. Therefore, this interaction was proposed to play an important role in maintaining the structural integrity and conformational stability of the N-terminal domain. This hypothesis was tested by mutating conserved CLIC1 residues Arg29 and salt-bridge partner Glu81 to methionine, thus removing the salt-bridge interaction. The conformational stabilities of each mutant at pH 7 (cytosol) and pH 5.5 (membrane surface) in the absence of membranes was then measured and compared to that of the wild type protein. The mutations did not impact upon the structural integrity of the protein. However, removal of the salt-bridge and hydrogen bonding interactions caused a loss in the cooperativity of unfolding from the native to unfolded state that resulted in the formation of an intermediate species. The intermediate species are less stable than the intermediate species of wild type CLIC1 at pH 5.5. Nevertheless, the properties (secondary and tertiary structure, ANS binding and cooperative unfolding (N ↔ U)) of the intermediate species are the same for all mutants and wild type protein. It can be concluded that the salt-bridge and more importantly hydrogen bonding interactions between helices 1 and 3 stabilise the Nterminal domain of CLIC1. It can be hypothesised that in the absence of membranes under acidic conditions, such as those at the surface of the membrane, protonation of acidic amino acid residues at the domain interface cause destabilisation of the Nterminal domain. This causes a reduction in the activation energy barrier for the conversion of soluble CLIC1 to its membrane-insertion conformation.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. July, 2017 / Eukaryotic protein synthesis occurs in three phases: initiation, elongation and termination. The elongation phase is mediated by elongation factors. Elongation factors are divided into elongation factor 1 (eEF1) and elongation factor 2 (eEF2). Elongation factor 1 complex are proteins that mediates the extension of growing polypeptide chains by adding one amino acid residue at a time. The eEF-1 complex comprises of four subunits, eEF1α, eEF1β, eEF1γ and eEF1δ. The β-subunit of elongation factor 1 complex (eEF1) plays a central role in the elongation step of eukaryotic protein biosynthesis, which essentially involves interaction with the α-subunits (eEF1α) and γ-subunits (eEF1γ). To biophysically characterise heEF1β, three E. coli expression vector systems was constructed for recombinant expression of the full length (FL-heEF1β), amino terminus (NT-heEF1β) and the carboxyl terminus (CT-heEF1β) regions of the protein. NT-heEF1β was created from the FL-heEF1β by site-directed mutagenesis using mutagenic forward and reverse primers. The results suggest that heEF1β is predominantly alpha-helical and possesses an accessible hydrophobic cavity in the CT-heEF1β. Both FL-heEF1β and NT-heEF1β forms dimers of size 62 kDa and 30 kDa, respectively, but the CT-heEF1β is monomeric. FL-heEF1β interacts with the N-terminus GST-like domain of heEF1γ (NT-heEF1γ) to form a 195 kDa complex, or a 230 kDa complex in the presence of oxidised glutathione. On the other hand, NT-heEF1β forms a 170 kDa complex with NT-heEF1γ and a high molecular weight aggregate of size greater than 670 kDa. This study affirms that the interaction between heEF1β and heEF1γ subunits occurs at the N-terminus regions of both proteins, also the N-terminus region of heEF1β is responsible for its dimerisation and the C-terminus region of heEF1β controls the formation of an ordered eEF1β-γ oligomer, a structure that may be essential in the elongation step of eukaryotic protein biosynthesis. / MT 2018

Background: A human ribosomal protein L9 (RPL9) encodes a protein that is a component of the 60S subunit. RPL9 is located on chromosome 4p14 and is approximately 5.5 kb in length and contains 8 exons. The message for human RPL9 is 712 nucleotides long. Some of the functions of RPL9 documented so far include the crucial involvement of the gene product in cell proliferation and protein biosynthesis. Lipoic acid synthetase (LIAS) is a 1.73 kb gene also located at chromosome 4p14. Alternative splicing occurs at these locus and two transcript variants encoding distinct isoforms have been identified but in this study the results represents both isoforms together. The protein encoded by LIAS gene belongs to the biotin and lipoic acid synthetases family and localizes in the mitochondrion. Function of lipoic acid synthetase is not yet well documented. Some studies have attempted to characterise its function by looking at the biological pathways at which LIAS gene product plays a crucial role, for example the biosynthesis of alpha-lipoic acid. Alpha lipoic acid is a natural antioxidant and it is also naturally-occurring enzyme co-factor found in a number of multi-enzyme complexes regulating oxidative metabolism. Motivation for study: RPL9 and LIAS were previously found to be mutated in CHO (Chinese Hamster Ovary) cell lines and these mutant lines had gained resistance to apoptosis. Aim: The main objective of this study was to evaluate the expression pattern of RPL9 and LIAS in lung cancer and to characterise their role in apoptosis and also to determine if the expression pattern of this genes varies between normal and diseased state of the tissue. Methods: In Situ hybridization, quantitative Real Time PCR, TUNEL and Bio-informatics have been employed in order to attain the objectives of this study. Results: In Situ hybridization showed that RPL9 localises in the cytoplasm and it is up-regulated in lung cancer relative to normal lung. LIAS localises in the cytoplasm and it is also up-regulated in lung cancer. The expression of RPL9 was relatively higher than that of LIAS determined by the intensity of localisation. Quantitative real time PCR confirmed the up-regulation of RPL9 and LIAS in lung cancer. RPL9 and LIAS were found to be up-regulated 8 and 4 fold respectively in lung A549 lung adenocarcinoma relative to MRC5 normal lung fibroblast cell lines. TUNEL showed the highest DNA fragmentation in adenocarcinoma, followed by squamous cell lung carcinoma then large cell lung carcinoma which is the same pattern observed in RPL9 and LIAS mRNA localisation by In Situ hybridization. To further characterise the role of RPL9 and LIAS in human, Bio-informatics tools were used and the results revealed that RPL9 is highly conserved through evolution, up-to 100 % identical to chimpanzee and 98 % to mouse. LIAS was found to be 91 % identical to rat and 90 % identical to mouse. It has been documented that the rate of conservation of a gene in evolution is believed to be correlated with its biological importance and its number of protein–protein interactions. Conclusion: All these discoveries coupled with resistance to apoptosis of CHO cell line in which RPL9 and LIAS were found to be mutated following promoter-trap mutagenesis, strongly suggests that RPL9 might be playing a role in cell cycle and apoptosis. RPL9 has been highly conserved through evolution. Manipulation of this gene can lead to greater biological discoveries in cancer research and the elevated expression of RPL9 can be used as a molecular marker for early detection of cancer.

Thesis (M.S.)--Chinese University of Hong Kong. / Includes bibliographical references.

by Wing-Hoi Tam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 95-99). / Abstracts in English and Chinese. / Title --- p.1 / Acknowledgement --- p.2 / Contents --- p.3 / Abbreviations --- p.5 / List of figures --- p.7 / List of tables --- p.9 / Primers and their sequences used in the projcet --- p.10 / Abstract --- p.12 / Introduction / Small heat shock proteins --- p.16 / Human heart sequencing EST project --- p.17 / Small heat shock protein B3 (HSPB3) --- p.18 / The alpha crystallins --- p.18 / Point mutation of a conserved arginine in the alpha-crystallin domain perturbs the overall alpha crystallins structure and function --- p.19 / HSP27 Confers thermotolerance in cells --- p.19 / Chaperone properties of sHSP --- p.20 / In Viro-chaperone function of sHSPs --- p.21 / Does the alpha-crystallin domain necessary for chaperone activity? --- p.21 / Expression HSPB3 in heart diseases patients and in porcine model --- p.22 / Small heat shock proteins in development and differentiation --- p.23 / HSPB3 role in mammalian embryogenesis --- p.24 / Structure of the small heat shock proteins --- p.25 / Crystal structure of a small heat shock protein --- p.25 / Subunit contacts --- p.26 / Muscle is the only tissue that simultaneously expresses all five sHSPs --- p.26 / "Two independent sHSP systems in muscle cells, to serve and to protect" --- p.27 / Methods and Materials / Small scale preparation of plasmid DNA --- p.29 / Large scale preparation of plasmid DNA --- p.29 / Amplification of mouse and rat HSPB3 coding region --- p.30 / Northern Blotting and Hybridisation --- p.31 / Purification of DNA fragments by GENECLEAN --- p.31 / Preparation of competent Escherichia coli for transformation --- p.32 / Transformation of DNA plasmid into competent Escherichia coli --- p.32 / 5´ة Rapid Amplification of cDNA Ends (5´ةRACE) --- p.33 / 3´ة Rapid Amplification of cDNA Ends (3´ةRACE) --- p.35 / Upstream and Downstream Genomic sequence --- p.36 / Isolation of genomic DNA from human tissue --- p.37 / Multiple tissue cDNA panel (MTC) --- p.37 / The yeast two hybrid screenings and confirmation --- p.43 / Isolation of total RNA from human tissues --- p.43 / Automated Sequencing --- p.44 / Alignment of DNA and amino acid sequence --- p.44 / Radiation hybrid mapping --- p.45 / Results / Determination of the foll length of human HSPB3 cDNA by 5' and 3' RACE --- p.46 / Identification of human HSPB3 gene in mouse and rat heart tissues --- p.49 / "Sequence homology between human, mouse and rat HSPB3" --- p.51 / Chromosomal mapping of the HSPB3 gene --- p.60 / Expression of human HSPB3 is not confined to the heart and skeletal muscle --- p.62 / Mouse HSPB3 expression is confined to the heart tissue --- p.65 / HSPB3 genomic PCR of downstream sequence --- p.67 / HSPB3 upstream genomic sequence --- p.67 / No intronic sequences between the open reading frame --- p.68 / The yeast two hybrid screening --- p.71 / HSPB3 can not form dimers --- p.82 / Expression of Prepro-CDD-ANF in human adult and fetal --- p.83 / Discussions / Previously reported sequence HSPL27 is a chimera --- p.87 / HSPB3 monomers do not dimerises --- p.88 / No intronic sequence in the HSPB3 gene --- p.89 / HSPB3 and CDD-ANF may play an important role during stress to the heart --- p.91 / Change of expression pattern of Prepro-CDD-ANF during development --- p.93 / Reference --- p.95

Chan, Man Hon. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 141-144). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.