Goldring, James Philip Dean.Kwangu, Mwenya Chitembusha.2025-08-052025-08-0520242024https://hdl.handle.net/10413/23874Doctoral Degree. University of KwaZulu-Natal, Pietermaritzburg.Plasmodium falciparum (Pf) is responsible for most malaria deaths in humans, however, but most Pf infections do not lead to death of the infected individual. Elimination strategies have not worked well due to the emergence of drug resistance from parasites, creating a need for further research on novel drug targets against malaria. Copper is a cofactor for enzymes like the cytochrome c oxidase in various organisms. If not tightly regulated, its redox activity may lead to a free radical generation that may be destructive to the cellular structure. In infected red blood cells, excess or absence of copper leads to the growth inhibition of malaria parasites. This is because copper is an essential mineral to the parasite. Therefore, acquisition, distribution both in the cytosol and mitochondria, and efflux of this metal must be understood. The current study aimed to identify and determine the copper-binding abilities of PfGrx1 and PfSAHH, proteins that could have a role in the cytosolic distribution of copper and secretory pathway, thereby maintaining optimum copper levels in P. falciparum. The in-silico analysis revealed copper binding CXXC and GXGXXGXG motifs in the amino acid sequences for PfGrx1 and PfSAHH proteins, respectively. These binding motifs which are present in the two proteins, have been found to bind copper in other organisms. PfGrx1 and PfSAHH were cloned, expressed as His-tag proteins, purified, and resolved on SDS-PAGE gel, then detected as ~12 and ~53 kDa proteins respectively, using mouse anti-His-tag antibodies on a western blot. Just like proteins in other systems, the purified rPfGrx1 and rPfSAHH bound copper in-vitro and in-vivo using ascorbic acid oxidation, and Bicinchoninic acid (BCA) copper release assays. When the growth of E. coli cells with or without the plasmid expressing the recombinant proteins was monitored in the presence or absence of 8 mM copper, E. coli cells with expressed recombinant proteins were able to grow suggesting that copper was bound to the recombinant proteins in-vivo, thus enabling growth of the bacteria. Protein-to-protein interaction modeling studies showed a possibility of PfGrx1 and PfSAHH interacting with each other and other copper proteins, suggesting a possible transfer of copper from protein to protein, and potential copper pathways in P. falciparum. The binding of copper was supported by docking studies that established copper binding sites for PfCtr1, PfGrx1, PfSAHH, PfCox17, PfCox11, PfCox19, and PfSco1. Characterization of Plasmodial copper-binding proteins can provide an understanding of Plasmodia copper homeostasis with potential novel drug target determination using bioinformatic tools. More interaction studies may provide novel and likely offer the basis for understanding of mechanisms for drug deliveries to the parasite, though further studies are needed to elucidate the existing channels.enMalaria.Parasites.Thesis