Doctor of Philosophy (PhD)
Cysteine proteases (CPs) are enzymes with a nucleophilic thiol in their active sites. Inhibitors of cysteine proteases (ICPs) occur naturally in bacterial pathogens and some protozoa. In parasites, ICPs are often virulence factors, contributing to the formation and survival of amastigotes within host cells. These amastigotes have higher CP activity, therefore making both ICPs and CPs potential drug targets. Despite great genetic variability, ICPs contain highly conserved structural features, including a series of defined loops that play a significant role in binding CPs. Papain, a CP from Carica papaya, complexes with ICP from Leishmania mexicana. Although the individual 3-D structures of ICP and papain have been determined, as of this work, the structure of the papain-ICP complex has only been predicted, not solved. This research details the development of a technique for determining quaternary structure of the papain-ICP complex using paramagnetic relaxation enhancement NMR (PRE-NMR). A paramagnetic tag (MTSL) was added to various cysteine-mutants of ICP to measure distances to reductively 13C-methylated papain. The modification of ICP with MTSL was quantified using EPR, and the effects of labeling on the binding kinetics of papain and ICP were determined using SPR. 13C-methyl peak perturbations due to PRE were observed when papain was bound to spin-labeled E102C-ICP and K27C-ICP. Intermolecular distances were predicted using modeling software and a working model of the complex was created. Data from additional mutants will help to further determine complex structure and perfect the model.The penultimate chapter of this dissertation includes work towards the development of a method for studying protein-protein interactions using atomic force microscopy. Papain-ICP was used as a model system, with the intention to apply this method to the study of another system: filamentous actin (f-actin) and the actin-binding domain of abelson tyrosine-protein kinase (ABL2-FABD). The creation of nanopores on an AFM sensor chip surface was successful. ICP monomers bound selectively into the pores. Attempts to form the papain-ICP complex on the chip surface were unsuccessful, and future work is needed to perfect this method. The final chapter of this dissertation is a literature review outlining previous work in this area.
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Thomasson, Margaret Sara, "Investigating the Structure of the Papain-Inhibitor Complex using SPR and NMR" (2016). LSU Doctoral Dissertations. 493.