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Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The investigation was aimed to improve the understanding of the binding interactions between DevS and DevR that are implicated in the regulation of the dormancy response in Mycobacterium tuberculosis. These binding interactions could provide good drug targets for the treatment of persistent tuberculosis, the mechanistic understanding of their binding interactions is important for the development of a validated inhibitor screen. A detailed in silico analysis of the amino acid residues that play a role in the binding of receptor DevR to both kinase DevS and the target DNA was undertaken. A reasonable approximation of the DevS structure was produced using homologous protein structures. In silico docking of DevS to DevR merely produced a set of probable candidate structures, since more than one conformation with similar docked energies was observed. The decision on which one is the more correct form can only be estimated by crystallization of this complex. Therefore, the functional expression and purification of the Dev TCS components were pursued. Denaturing HIS™-select nickel affinity gel purification in the form of matrix-assisted refolding led to the production of functional Dev TCS proteins. To understand the binding of DevR to DNA consensus sequences, as well as the nature of these interactions, a model was built of the full length DevR dimer binding to DNA consensus sequences. Based on this model, single mutations were made to DevR in vitro and their effects assessed in order to validate the model built. During Electrophoretic Mobility Shift Assay (EMSA) analysis, it was found that K179I and N183L mutants prevented the binding of DevR to the DNA consensus sequences. If DevR and DevS binding are to be used in a drug development program, it is essential to have the protocols to accurately measure their interaction, in addition to developing a fundamental understanding of how their interactions occur. The binding affinity of DevR to both DevS and the truncated soluble fragment of DevS (DevS201) were explored, using the BIAcore instrument, an SPR-based biosensor. For sufficiently strong binding between a histidine kinase and a response regulator, the KD needs to be in the nM range. The KD was calculated to be 255 nM for DevS201 and 184 nM for DevS. Therefore it can be concluded that DevS201 binds DevR strongly enough to be used in future studies, and that the BIAcore could be used to screen small-molecule inhibitors of DevR-DevS interactions. / AFRIKAANSE OPSOMMING: Die Dev twee komponent sisteem (TKS) bestaan uit ‘n histidine kinase naamlik (DevS) en ‘n reaksie reguleerder DevR. DevS en DevR is betrokke by die regulering van die dormante stadium van Mycobacterium tuberculosis. Hierdie meganisme kan ‘n deurbraak dwelm teiken vir die behandeling van sluimerende tuberkulose wees. Die meganisme van hierdie bindings interaksies is van kritieke belang, tesame met die ontwikkeling van 'n erkende inhibeerder toets. ‘n Gedetaileerde in silico analise van die aminosuur volgordes wat 'n rol speel in die binding van die reseptor DevR aan beide DevS sowel as die teiken DNS is voltooi. ‘n Model van die DevS struktuur is saamgstel met behulp van homoloë proteïen strukture. In silico mering van DevS aan DevR het `n stel van die waarskynlike kandidaat strukture verskaf, aangesien meer as een konformasie met soortgelyke merings energieë waargeneem is. Die mees waarskynlike vorm kan alleenlik geïndentifiseer word na kristallisasie van hierdie kompleks. Die funksionele uitdrukking en suiwering van die Dev TKS proteine is gevolglik uitgevoer. Funksionele Dev TKS proteïene is verkry deur denaturerende HIS-select nikkel affiniteit jel suiwering, in die vorm van matriks-geassisteerde hervouing te gebruik. Ten einde die binding te verstaan tussen DevR en DNS konsensus volgordes, sowel as die aard van hierdie interaksies, is 'n model gebou van die volle lengte DevR dimeer binding aan DNS konsensus volgordes. Hierdie model is gevalideer deur punt mutasies in DevR te skep en die gevolge daarvan te beoordeel met elektroforetiese mobiliteits verskuiwing reaksie analises. Dit is bevind dat K179I en N183L mutante, verhoed die binding van DevR aan die DNS konsensus volgordes. Die gebruik van DevR en DevS bindings in ‘n dwelm ontwikkelingsprogram, benodig die fundamentele begrip van hoe die interaksies plaasvind, sowel as akkurate protokolle om die interaksies te meet. Die BIAcore instrument, ’n SPR-gebaseerde biosensor, is ingespan om die bindings affiniteit van DevR aan beide DevS en die fragment van DevS (DevS201) te ondersoek. Om voldoende sterk binding tussen DevS en die DevR te verseker, moet die KD in die nM omgewing wees. Die KD is bepaal as 255 nM en 184 nM vir DevS201 en DevS, onderskeidelik. Die afleiding kan dus gemaak word dat DevS201 sterk genoeg aan DevR bind om in verdere studies gebruik te kan word, en dat die BIAcore gebruik kan word om klein-molekule inhibeerders van DevR-DevS interaksies te toets.

Flavonoids are an important class of secondary metabolites widely distributed in plants. The majority of naturally occurring flavonoids are found in glucosylated form. Glucosyltransferases are enzymes that enable transfer of glucose from an activated donor (UDP-glucose) to the acceptor flavonoid substrates. A flavonol specific glucosyltransferase cloned from Citrus paradisi (Cp3OGT) has strict substrate and regiospecificity. In this study, amino acid residues that could potentially alter the rigidity observed in this enzyme were mutated to position equivalent residues of a putative anthocyanin specific glucosyltransferase from Clitorea ternatea and a GT from Vitis vinifera that can glucosylate both flavonols and anthocyanidins. Using homology modeling followed by site directed mutagenesis to identify candidate regions, three double mutations were made. To test the basis of substrate specificity, biochemical analysis of the three recombinant mutant proteins was carried out. Recombinant protein with mutation S20G+T21S revealed that the enzyme retained activity similar to the wildtype (Cp3OGT) (WT- Km app-104.8 µM; Vmax = 24.6 pmol/min/µg, Mutant- Km app-136.42 µM; Vmax -25pmol/min/µg) but the mutant was more thermostable compared to the WT. The (S290C+S319A) mutant protein retained 40% activity relative to wildtype and has an optimum pH shifted towards the acidic side (pH 6) (Km app-8.27 µM; Vmax-90.9 pmol/min/µg). Mutation of Glutamine87 and Histine154 (H154Y+Q87I) have rendered this recombinant protein inactive with every class of flavonoid tested. Interestingly, the single point mutations H154Y and Q871I had significant activity, slightly greater than that of wildtype enzyme. The two active recombinant proteins will further be analyzed to determine whether the mutations have altered regiospecificity of the original enzyme. Product identification is being conducted using HPLC.

This thesis set out to explore the conformational properties of short designed peptide sequences, in which transitions between structural states may be anticipated. The use of conformationally constrained residues like α-aminoisobutyric acid (Aib) and D-proline (DPro) permits the design of model sequences for structural studies. The principle of imposing conformational constraints by multiple substitutions at backbone atoms in aminoacid residues may also be extended to the higher homologs of α-amino acids, namely β and residues. The experimental results presented in this thesis also examine the potential of using cross-strand interactions between aromatic residues as a probe of structure in designed peptide β-hairpins. Chapter 1 provides a very brief introduction to the necessary background on which the experimental studies in this thesis are based. Chapter 2 describes studies aimed at establishing chain length effects on helix-hairpin conformational distributions in short synthetic sequences, containing centrally positioned Aib-DAla and Aib-Aib segments.The Aib-DAla dipeptide segment has a tendency to form both type-I'/III' and type-I/III β-turns. The occurrence of prime turns facilitates the formation of β-hairpin conformations, while type-I/III turns can nucleate helix formation. The octapeptide Boc-Leu-Phe-Val-Aib-DAla-Leu-Phe-Val-OMe (1) has been previously shown to form a β-hairpin in the crystalline state and in solution. The effects of sequence truncation have been examined using the model peptides Boc-Phe-Val-Aib-Xxx-Leu-Phe-NHMe (2, 6), Boc-Val-Aib-Xxx-Leu-NHMe (3, 7) and Boc-Aib-Xxx-NHMe (4, 8), where Xxx = DAla, Aib. For peptides with central Aib-Aib segments, Boc-Phe-Val-Aib-Aib-Leu-Phe-NHMe (6), Boc-Val-Aib-Aib-Leu-NHMe (7) and Boc-Aib-Aib-NHMe (8) local helical conformations have been established by NMR studies in both hydrogen bonding (CD3OH) and non-hydrogen bonding (CDCl3) solvents. In contrast, the corresponding hexapeptide Boc-Phe-Val-Aib-DAla-Leu-Phe-Val-NHMe (2) favors helical conformations in CDCl3 and β-hairpin conformations in CD3OH. β-Turn conformations (type-I /III) stabilized by intramolecular 4 1 hydrogen bonds are observed for the peptide Boc-Aib-DAla-NHMe (4) and Boc-Aib-Aib-NHMe (8) in crystals. The tetrapeptide Boc-Val-Aib-Aib-Leu-NHMe (7) adopts an incipient 310-helical conformation stabilized by three 4 1 hydrogen bonds. The peptide Boc-Val-Aib-DAla- Leu-NHMe (3) adopts a novel -turn conformation, stabilized by three intramolecular hydrogen bonds (two 4 1 and one 5 1). The Aib-DAla segment adopts a type-I' β-turn conformation. The observation of the NOE Val(1) NH HNCH3 (5), in CD3OH, suggests that the solid state conformation of peptide 3 is maintained in methanol solutions. Peptide hairpins provide an ideal scaffold for exploring cross-strand interactions between residues on facing antiparallel strands. Chapter 3 reports studies directed towards probing, aromatic interactions between facing Phe residues, positioned at the non-hydrogen bonding positions in designed octapeptide β-hairpins. The studies described in this Chapter employ ring current shifted aromatic proton resonances as a means of probing aromatic ring orientations. Crystal structures of eight peptide -hairpins with the sequence Boc-Leu-Phe-Val-Xxx-Yyy-Leu-Phe-Val-OMe revealed that the Phe(2) and Phe(7) aromatic rings are in close spatial proximity, with a centroid-centroid distance (Rcen) of 4.4Å to 5.4Å between the two phenyl rings. Proton NMR spectra in chloroform and methanol solutions reveal a significant upfield shift of the Phe(7) C , ′ H2 protons (6.65 ppm to 7.04 ppm). Specific assignments of the aromatic protons have been carried out in the peptide Boc-Leu-Phe-Val-DPro-LPro-Leu-Phe-Val-OMe (6). The anticipated ring current shifts have been estimated from the aromatic ring geometries observed in crystals for all eight peptides. Only one of the C , ′ H proton lies in the shielding zone, with rapid ring flipping, resulting in averaging between the two extreme chemical shifts. An approximate estimate of the population of conformations which resemble crystal state orientations may be obtained. Key nuclear Overhauser effects (NOEs) between facing Phe sidechains provide support for close similarity between the solid state and solution conformations. Temperature dependence of aromatic ring proton chemical shifts and line widths for peptide 6 (Boc-Leu-Phe-Val-DPro-LPro-Leu-Phe-Val-OMe) and the control peptide Boc-Leu-Val-Val-DPro-Gly-Leu-Phe-Val-OMe establish an enhanced barrier to ring flipping, when the two Phe rings are in proximity. Modeling studies suggest that small, conformational adjustments about the C -C ( 1), and C -C ( 2) bonds of the Phe residues may be required in order to permit unhindered, uncorrelated flipping of both the Phe rings. The maintenance of specific aromatic ring orientations in organic solvents provides evidence for significant stabilizing interactions. Earlier studies from this laboratory established that a centrally positioned DPro-LPro-DAla segment could induce hairpin formation in nonapeptide sequences, facilitated by a three residue loop segment. The DAla residue at position 6 in the nonapeptide Boc-Leu-Phe-Val-DPro-LPro-DAla-Leu-Phe-Val-OMe has been shown to adopt a left handed helical (αL) conformation. The studies described in Chapter 4, examine the effects of aminoacid replacements at positions 5 and 6. NMR studies on eight nonapeptides, with the general sequence Boc-Leu-Phe-Val-DPro-Xxx-Yyy-Leu-Phe-Val-OMe are described. In the case of peptides with a central DPro-LPro-Yyy sequence, two kinds of hairpin conformations are formed in solution. These are; i) β-hairpin structures with a central three residue loop, resulting in registered antiparallel tripeptide strands, and ii) a slipped hairpin structure, nucleated by a central DPro-LPro type-II β-turn, with residue 6 being incorporated into the C-terminal strand. The three residue loop β-hairpins are favored for DAla(6) and Aib(6), while the LAla(6) peptide favors a “slipped” hairpin structure. Replacement of the Pro(5) residue by LAla results in a reduced population of three residue hairpins in the nonapeptide with the DPro-LAla-DAla segment. Replacement of Pro(5) by Aib, abolished hairpin formation. Aromatic proton chemical shifts provide a convenient diagnostic for the presence of three residue loop hairpin conformations in these nonapeptides. A great deal of current interest has focused on the conformations of peptides incorporating β and γ aminoacid residues. Earlier studies from this laboratory have focused on the conformational properties of the β,β -disubstituted γ residue gabapentin (1-aminomethylcyclohexane acetic acid). Subsequent work with the related β aminoacid β3,3Ac6c (1-aminocyclohexaneacetic acid) revealed that intramolecularly hydrogen bonded conformations are infrequently observed in short peptides. The studies described in Chapter 5, examine the conformational properties for model peptides containing the isomeric β-aminoacid, β2,2Ac6c (1-aminomethylcyclohexane-1-carboxylic acid). The effect of gem dialkyl substituents on the backbone conformations of amino acid residues in peptides, has been investigated using four model peptides, Boc-Xxx-2,2Ac6c-NHMe [Xxx = Leu (1), Phe(2)] and Boc-Xxx- 3,3Ac6c-NHMe [Xxx = Leu (3), Phe(4)]. Tetrasubstituted carbon atoms restrict the ranges of stereochemically allowed a C11 helical turn, which is a backbone expanded analog of the type III -turn in sequences. The crystal structure of the peptide Boc-Phe- 3,3Ac6c-NHMe (4) establishes a the asymmetric unit adopt backbone torsion angles of opposite signs. In one of the molecules, the Phe residue adopts an unfavourable backbone conformation, with the energetic penalty being offset by favourable aromatic interactions between proximal molecules in the crystal. NMR studies provide evidence for the maintenance of folded structures in solution, in these hybrid sequences. The result presented in this thesis suggests that it should be possible to construct designed synthetic peptides, which can undergo transitions between two distinct and energetically favourable conformational states. The ability to design peptide sequences that can undergo switching between helical and β-hairpin states, or between hairpin structures with variations in connecting loop length may prove valuable in providing further insights into the factors influencing conformational dynamics.