Magister Scientiae - MSc / Malaria remains the most important parasitic disease worldwide. It is estimated that over 500 million infections and more that 2.7 million deaths arising from malaria occur each year. Most (90%) of the infections occur in Africa with the most affected groups being children of less than five years of age and women. this dire situation is exacerbated by the emrggence of drug resistant strains of Plasmodium falciparum. The work reported in this thesis focuses on improving the purification of PfHPRT by investigating the characteristics of anion exchange DE-52 chromatography (the first stage of purification), developing an HPLC gel filtration method for examining the quaternary structure of the protein and possible end stage purification, and initialcrystalization trials. a homology model of the open, unligaded PfHPRT is constructed using the atoomic structures of human, T.ccruz and STryphimurium HPRT as templates. / South Africa

This work is focused on the interactome study of 14-3-3ζ protein, a regulatory protein found in all eucaryotic cells. An important 14-3-3 protein feature is the ability to bind a number of structurally and functionally distinct protein ligands. This link is usually implemented through phosphorylated serine and threonine motifs. The first aim of this work is the preparation of sufficient amount of recombinant 14-3-3ζ protein with incorporated photoactivatable analogue of methionine (foto-Met, L-2-amino-5,5- azihexan acid). The four different conditions of recombinant expression in auxotrophic E. coli B834 (DE3) strain were tested to obtain a protein with a maximal rate of photoactivatable methionine analogue incorporation into the sequence 14-3-3 protein. The second aim is to study the methionine 121, 160 and 218 participation in the 14-3-3ζ protein binding groove and finding of potential covalent bond with the phosphorylated peptide 251-266 of Raf-1 kinase (phosphorylation on Ser259). The photo-initiated cross-linking method was used (photolysis), to form a reactive biradical of methionine analogue capable to attack any amino acid residues in close vicinity (till 5Å). Finally, the products of photo-initiated cross-linking were analyzed by cross-linking reactions using MALDI-TOF MS, LC-MS and...

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The chloride/proton antiporter ClC-5 is primarily expressed in the kidney where it aids in re-absorption of proteins from the glomerular filtrate. Functional disruption of ClC-5 causes Dent’s Disease – a renal condition characterized by proteinuria and kidney failure in a third of all cases. The majority of disease-causing mutations translate into premature truncations of the carboxy-terminal (Ct) region of ClC-5 and are predicted to disrupt the protein-protein interactions mediated by this domain. In this thesis, direct ATP binding to the two cystathionine β-synthase (CBS) domains of ClC-5 was demonstrated. ATP binding enhanced the global compactness of the ClC-5 Ct region likely through a clamping motion of the CBS domains around the nucleotide. Along with ATP, the sodium proton exchange regulatory factor 2 (NHERF2) also binds ClC-5; however, the molecular mechanism behind this interaction was unknown as ClC-5 lacked the PDZ binding motif traditionally localized at the Ct end of bait proteins. Here, we also identified a class I PDZ binding motif (657-660; TSII) within the internal sequence of ClC-5. Despite the buried position of this motif in the Ct peptide’s X-ray crystal structure (PDB: 2J9L), the high propensity of this region for dynamic flexibility prompted us to test whether it could mediate NHERF2 interactions. In support of this hypothesis, we demonstrated that the motif is transiently available to interact directly with NHERF2 in vivo and to enable an enhancement in receptor-mediated endocytosis in mammalian cells. Collectively, these results gave further evidence that the intracellular Ct region of ClC-5 serves as a hub to mediate interactions essential for its maturation, stability, and trafficking in renal epithelium, as well as providing further insights into the molecular basis of Dent’s Disease.

Photoaffinity labeling is a quintessential technique in studying and analyzing the interaction between a ligand and receptor. Diazirines are one of the important photo-labile moieties used in photoaffinity labeling due to their superior photo labeling characteristics. Herein, we report the investigations we conducted with diazirine photolabels on (a) photochemical aspects leading to enhancement of their ambient light stability and (b) equipping them with fluorous tags to enable fluorous enrichment of labeled proteins. Furthermore, we report a pilot study to develop BACE-1 inhibitors, which have potential to be developed into photoaffinity probes. 3-Trifluoromethyl-3-phenyldiazirine offers good selectivity and protection against pseudolabeling but due to its photo lability, it undergoes decomposition even under ambient light. Thus the laboratory handling, including synthesis, of 3-trifluoromethyl-3-phenyldiazirine is cumbersome and restricted under constant darkness. Herein, we have designed, synthesized and evaluated two photolabels with enhanced stability to ambient light conditions in addition to the good selectivity and protection against pseudolabeling as offered by 3-trifluoromethyl-3-phenyldiazirine. It was also found that the aqueous solubility, a vital physical property for a photolabel, was also improved in the modified ambient light stable photolabels. Fluorous tags have found wide use in synthetic applications; herein we explore the possibility of its application in photoaffinity studies. We designed, synthesized and conducted photoactivation studies on two fluorous diazirine photolabels. The photoactivation studies unraveled an unanticipated photoreaction when the fluorous tag was directly connected to the diazirine ring, yielding a fluorous alkene. The more practical photolabel of the two was chosen as the target specific photoaffinity labeling moiety for fluorous proteomics. Upon conducting photolabeling experiments under various conditions, we found that the strong hydrophobic character of the fluorous tag renders the photoaffinity label insoluble in aqueous solutions and significantly alters the binding mode and affinity of the photoaffinity label to its target receptor. A library of 1,3-disubstituted 2-propanols was combinatorially prepared and tested as small molecule inhibitors of β-secretase (BACE-1). The initial screening of the 1,3-disubstituted 2-propanol library revealed a few low micromolar inhibitors for BACE-1. The compound that showed the best activity was chosen for further SAR studies, which resulted in a potent BACE-1 inhibitor with nanomolar inhibition. Investigation on the selectivity of these compounds for BACE-1 inhibition over cathepsin D revealed that these compound series possess very high selectivity. Furthermore, the physicochemical properties study showed that these compounds possessed the calculated parameters advantageous to cross the blood-brain barrier (BBB).

The chloride/proton antiporter ClC-5 is primarily expressed in the kidney where it aids in re-absorption of proteins from the glomerular filtrate. Functional disruption of ClC-5 causes Dent’s Disease – a renal condition characterized by proteinuria and kidney failure in a third of all cases. The majority of disease-causing mutations translate into premature truncations of the carboxy-terminal (Ct) region of ClC-5 and are predicted to disrupt the protein-protein interactions mediated by this domain. In this thesis, direct ATP binding to the two cystathionine β-synthase (CBS) domains of ClC-5 was demonstrated. ATP binding enhanced the global compactness of the ClC-5 Ct region likely through a clamping motion of the CBS domains around the nucleotide. Along with ATP, the sodium proton exchange regulatory factor 2 (NHERF2) also binds ClC-5; however, the molecular mechanism behind this interaction was unknown as ClC-5 lacked the PDZ binding motif traditionally localized at the Ct end of bait proteins. Here, we also identified a class I PDZ binding motif (657-660; TSII) within the internal sequence of ClC-5. Despite the buried position of this motif in the Ct peptide’s X-ray crystal structure (PDB: 2J9L), the high propensity of this region for dynamic flexibility prompted us to test whether it could mediate NHERF2 interactions. In support of this hypothesis, we demonstrated that the motif is transiently available to interact directly with NHERF2 in vivo and to enable an enhancement in receptor-mediated endocytosis in mammalian cells. Collectively, these results gave further evidence that the intracellular Ct region of ClC-5 serves as a hub to mediate interactions essential for its maturation, stability, and trafficking in renal epithelium, as well as providing further insights into the molecular basis of Dent’s Disease.

Soluble guanylyl cyclase (sGC) is the receptor for nitric oxide and a highly sought-after therapeutic target for the management of cardiovascular diseases. New compounds that stimulate sGC show clinical promise, but where these stimulator compounds bind and how they function remains unknown. Here, using a photolyzable diazirine derivative of a novel stimulator compound, IWP-051, and MS analysis, we localized drug binding to the 1 heme domain of sGC proteins from the hawkmoth Manduca sexta and from human. Covalent attachments to the stimulator were also identified in bacterial homologs of the sGC heme domain, referred to as H-NOX domains, including those from Nostoc sp. PCC 7120, Shewanella oneidensis, Shewanella woodyi, and Clostridium botulinum, indicating that the binding site is highly conserved. The identification of photoaffinity-labeled peptides was aided by a signature MS fragmentation pattern of general applicability for unequivocal identification of covalently attached compounds. Using NMR, we also examined stimulator binding to sGC from M. sexta and bacterial H-NOX homologs. These data indicated that stimulators bind to a conserved cleft between two subdomains in the sGC heme domain. L12W/T48W substitutions within the binding pocket resulted in a 9-fold decrease in drug response, suggesting that the bulkier tryptophan residues directly block stimulator binding. The localization of stimulator binding to the sGC heme domain reported here resolves the longstanding question of where stimulators bind and provides a path forward for drug discovery.

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