| Malaria is a disease afflicting people in tropical regions globally. Plasmodium falciparum causes the most lethal form of malaria, and intense biochemical research is focused on understanding and disrupting its biology. P. falciparum contains a unique organelle called the apicoplast which is essential for parasite viability. Fatty acid biosynthesis is an important metabolic pathway in the apicoplast, and acyl carrier protein (ACP) plays a central role by covalently binding nascent acyl chains. While fatty acid biosynthesis is critical for liver stage parasites, the role of ACP during the erythrocytic stages of P. falciparum had not been characterized. I investigated the redox status of ACP during erythrocytic stages and found ACP to be reduced but not acylated. Using X-ray crystallography I solved the tertiary structure of ACP, which is a four helix bundle with a conserved phosphopantetheine group at the start of helix 2. ACP is genetically encoded in the nucleus, and ACP protein is trafficked to the apicoplast. An N-terminal transit peptide is responsible for apicoplast localization, but the specific mode of transit peptide recognition for trafficking is unknown. I have biophysically characterized the transit peptide of ACP by circular dichroism and nuclear magnetic resonance to discover that its conformation in solution is primarily disordered, yet a short helix is formed transiently. I disrupted this helix by proline mutagenesis and assayed these mutant transit peptides by determining whether they can localize green fluorescent protein to the apicoplast in transgenic parasites. Proline mutants successfully traffic to the apicoplast, suggesting that a structural motif is not required for apicoplast transit peptide recognition and that transit peptides are likely recognized in the unfolded state. To facilitate colocalization within living parasites, I composed an internal ribosome entry site (IRES) expression cassette using IRES sequences from cricket paralysis virus or Rhopalosiphum padi virus, but found neither of them to be active in P. falciparum. To examine rare codon usage in P. falciparum genes heterologously expressed in E. coli, I identified rare codon clusters that cause translation pauses using a sliding window average of the geometric mean of codon usage frequencies. |
