The Mechanism of Binding Between the Plasmodium falciparum H-protein and Lipoate Ligase 1

Plasmodium falciparum;lipoate;Biochemistry

Rodriguez, Ryan E.

Johns Hopkins University

关键词: Plasmodium falciparum;    lipoate;    Biochemistry;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/39477/RODRIGUEZ-THESIS-2016.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Malaria is a devastating tropical disease, accounting for 2 million infections and 440,000 deaths in 2015. The most effective treatment for malaria is chemotherapy to kill the blood stage parasite. Recent spread of drug resistance against the most effective anti-malarial known, raises concerns about the future efficacy of existing blood stage chemotherapy.It is therefore necessary to identify and study unique biochemical pathways in the parasite that hold the potential for chemical intervention. Mitochondrial lipoylation in Plasmodium falciparum is one such pathway. Recent evidence suggests that mitochondrial lipoylation in the blood stage uses a novel redox mechanism. This pathway appears to be a suitable drug target for blood stage malaria and further characterization of the mechanisms involved are needed.In this study two of the key proteins involved in this pathway, lipoate ligase 1 (LipL1) and the H-protein, were expressed in E. coli and purified using FPLC. The purified proteins along with the cofactors necessary to drive the protein-protein interaction were screened for crystal formation in an attempt to collect x-ray diffraction data that could be used for structural determination. Crystals were grown and subjected to x-ray radiation, but produced poor quality diffraction data and no structural determinations could be made.The dissociation constant and binding kinetics of the protein-protein interaction under reducing and non-reducing conditions were studied using isothermal titration calorimetry (ITC). Data collected from ITC experiments yielded an average dissociation constant of 0.097µM under non-reducing conditions and 4.8µM under reducing conditions. Surface Plasmon Resonance (SPR), which uses a fraction of the protein sample compared to ITC, was also used to study the binding between LipL1 and the H-protein. Data collected from SPR experiments yielded a dissociation constant of 0.39µM under non-reducing conditions. The above dissociation constants suggest that lipoylation of the H-protein takes place due to its increased affinity for the LipL1-lipoyl-AMP complex, which dominates under non-reducing conditions. Under very reducing conditions, the LipL1-dihydrolipoyl-AMP complex dominates and as can be inferred from the dissociation constants, binding and subsequent lipoylation of the H-protein is diminished allowing for binding or lipoylation of other substrates.