X-ray crystallography is extensively deployed to determine the structure of proteins, both unbound and bound to different molecules. Crystallography has the power to visually reveal the binding of small molecules, assisting in their development in structure-based lead design. Currently, however, the methods used to detect binding, and the subjectivity of inexperienced modellers, are a weak-point in the field. Existing methods for ligand identification are fundamentally flawed when identifying partially-occupied states in crystallographic datasets; the ambiguity of conventional electron density maps, which present a superposition of multiple states, prevents robust ligand identification. In this thesis, I present novel methods to clearly identify bound ligands and other changed states in the case where multiple crystallographic datasets are available, such as in crystallographic fragment screening experiments. By applying statistical methods to signal identification, more crystallographic binders are detected than by state-of-the-art conventional approaches. Standard modelling practice is further challenged regarding the modelling of multiple chemical states in crystallography. The pervading modelling approach is to model only the bound state of the protein; I show that modelling an ensemble of bound and unbound states leads to better models. I conclude with a discussion of possible future applications of multi-datasets methods in X-ray crystallography, including the robust identification of conformational heterogeneity in protein structures.

Carboxylesterases are glycosylated general detoxification enzymes belonging to the serine esterase superfamily and play a critical role in the hydrolysis of numerous ester- and amide- containing molecules, including active metabolites, drugs and prodrugs. Three functionally active carboxyleterases have been identified in man (CES1-3), which all show differential tissue expression and critically overlapping, yet specific substrate selectivities. Elucidating the basis of their exact substrate preference would help facilitate the design of clinical prodrugs which are activated by carboxylesterases. Because of their widespread applications, carboxylesterases have attracted much attention in recent years, with CES1 being the most extensively studied human carboxylesterase to date. The work presented here addresses the structure-function relationship of the three human carboxylesterases using a combination of X-ray crystallography, kinetic analysis and biophysical techniques. Recombinant proteins were successfully produced using a mammalian expression system in high yield (5.0 – 84.0 mg/ L cell culture). Analytic ultracentrifugation and size-exclusion chromatography coupled to multi-angle laser light scattering were used to investigate the proteins in solution. These results showed CES1 exists primarily in a trimeric arrangement, whilst CES2 and CES3 are monomeric. Interestingly, atypical mechanisms of substrate inhibition, positive cooperativity and biphasic kinetics were observed for both CES1 and CES2. Three structures of CES1 were solved: wild type, an aglycosylated form and a catalytically inactive form, to 1.48, 1.86 and 2.01 Å respectively. The novel structure of CES2 was solved to 2.04 Å, which revealed that the enzyme forms a strand exchange dimer in contrast to the trimeric CES1. To summarise, this thesis documents a platform that has been generated for the production, characterisation and crystallization of human carboxylesterases. This will aid future structural work for protein ligand binding studies.

Magister Pharmaceuticae - MPharm / In this study, new solid supramolecular derivatised forms of bis(adamantine-1-aminium) carbonate (ADTCO3) were prepared. ADTCO3 is a derivative of amantadine used for Parkinson’s disease and has antiviral properties against influenza-A, dengue fever and pharmacological activity towards Parkinson’s disease. The new forms prepared were polymorphic and co-crystal forms of ADTCO3. Polymorphism is a phenomenon where the ability of a substance to exist in two or more crystalline forms occurs when crystallised under different conditions and co-crystallization is the process of formation of multicomponent crystals of a drug substance. New solid forms often display different mechanical, physicochemical and thermal properties that can remarkably influence the bioavailability, hygroscopicity and stability of active pharmaceutical ingredients (APIs). For the formation of polymorphs of ADTCO3, techniques such as dry grinding, solvent-drop grinding, co-precipitation, sublimation and vapour diffusion were applied. For the development of co-crystals and/or complex formation, ADTCO3 was treated in combination with ten selected co-formers viz; benzoic acid, 4-hydroxybenzoic acid, cinnamic acid, 4-hydroxycinnamic acid, succinic acid, tartaric acid, salicylic acid, L-glutamic acid, citric acid monohydrate and L-glutaric acid using similar techniques as applied in the polymorphism study. The first four co-formers were selected for their potential biological activity and the latter six were selected for their generally regarded as safe (GRAS) status. All products were isolated and characterized using different analytical techniques to assess the thermal behaviour of the products by hot stage microscopy (HSM), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). FTIR spectroscopy and proton-nuclear magnetic resonance (1HNMR) were used to identify and determine the purity of the parent compounds and the modified forms. X-ray powder diffraction was used to determine the formation of a new phase and single crystal X-ray diffraction was applied at the initial stages to identify ADTCO3 by its unit cell parameters. Furthermore, the Cambridge Structural Database (CSD) and other resources were used to generate information on the molecular structures of all elucidated parent compounds, their polymorphs and reported co-crystals. Four different polymorphic forms of ADTCO3 were identified (viz. ADTCO3 Forms I to IV) and sixteen co-crystals (viz. ADTCO3BA1 to ADTCO3BA5, ADTCO3HBA, ADTCO3CIN, ADTCO3HCIN, ADTCO3SUC, ADTCO3LTTA, ADTCO3SA, ADTCO3CA, ADTCO3GLA, ADTCO3GA) were synthesised. Of the sixteen co-crystals 5 were identified as ADTCO3BA “salt” co-crystal polymorphic forms and 2 as ADTCO3SUC co-crystal polymorphic forms. Two solvated “salt” co-crystal forms were also identified, namely; ADTCO3GLA and ADTCO3LTTA. ADTCO3GLA had a mass loss of 10.3% (n = 2.4) and ADTCO3LTTA had a mass loss of 5.25% (n = 0.86). Finally, the rest of the co-crystals ADTCO3HBA, ADTCO3CIN, ADTCO3HCIN, ADTCO3SA, ADTCO3CA and ADTCO3GA all crystallised as “salt” co-crystals.

No description available.

Histone H3 lysine 4 (H3K4) methylation is an evolutionarily conserved mark commonly associated with transcription activation in eukaryotes. In mammals, this post-translational modification is deposited by the KMT2 family of H3K4 methyltransferases. Biochemical studies have shown that the enzymatic activity of the KMT2 enzymes is regulated by a core complex of four evolutionarily conserved proteins: WDR5, RbBP5, ASH2L and DPY30, collectively known as WRAD, which are all important for global H3K4 methylation. However, how these proteins interact and regulate the activity of the KMT2 enzymes is not well investigated. During my PhD, I have used structural and biochemical approaches to determine the interactions underlying formation of the core complex and regulation of KMT2 enzymatic activity. My research have shown that 1) WDR5 uses two peptide-binding clefts on opposite sides of its β-propeller domain to bridge the KMT2 enzymes to the regulatory subunit RbBP5, 2) the WDR5 peptidyl-arginine-binding cleft exhibits plasticity to accommodate the binding of all KMT2 enzymes and 3) RbBP5 S350 phosphorylation stimulates formation of the RbBP5-ASH2L complex and H3K4 methylation by the mammalian KMT2 enzymes. Collectively, these studies have provided the structural basis for understanding the important interactions governing KMT2 complex assembly and activity.

The molecular and crystal structures of dibenzobarrelene and a number of its diester derivatives, and three dibenzosemibullvalenes have been determined by the use of X-ray crystallography. The objectives of the study were to investigate whether or not the di-π-methane photorearrangement could be carried out in the solid state, and how such solid state results would differ from results obtained in solution. In addition, we were interested in investigating the extent to which intermolecular steric effects would modulate or change the course of the photorearrangement in the solid state as compared to its solution pathway with the view of developing a structure-reactivity correlation for the reaction in the solid state. All the dibenzobarrelenes underwent the di-π-methane photorearrangement in the solid state to give the corresponding dibenzosemibullvalene photoproduct(s). In the symmetrical 11,12-diester derivatives of dibenzobarrelene, only one di-π-methane photoproduct could be identified for each of the reactants. The ethyl/ethyl and isopropyl/iso-propyl diester derivatives displayed polymorphism. An absolute asymmetric synthesis was performed on one of the dimorphs of the iso-propyl/iso-propyl derivative which crystallized in a chiral space group, and a quantitative enantiomeric excess yield was obtained. The molecular structures of all the compounds studied showed different degrees of conjugation of the ester carbonyl groups to the central vinyl bond. The unsymmetrical 11,12-diester derivatives yielded regioisomeric dibenzosemibullvalene photoproducts. Generally, the reactions in the solid state were found to be more regioselective than the same reactions in solution. The observed differences of the relative quantities of regioisomeric photoproducts in the solid state are interpreted on the basis of intermolecular steric effects. In appropriate systems, intermolecular steric effects may be used to predict successfully not only the major regioisomeric product of a di-π-methane photorearrangement in the solid state, but also the major enantiomeric product. One regioisomeric photoproduct is obtained for each 9,11 and 10,11-diester derivative. The photoproducts obtained from these unsymmetrical 9,11 and 10,11-diester derivatives of dibenzobarrelene show that electronic effects may be the dominant factor which governs the photochemical reaction pathway of this class of compounds in the solid state. Overall, intermolecular steric hindrance and electronic factors affect the solid state photochemical pathway of each compound to different extents. There appears to be an interplay of electronic and steric factors in determining the reaction pathway which leads to the major product in the solid state. The dominance of one factor (steric versus electronic) over the other in the determination of the most favorable photochemical pathway is dependent upon the conformations of the ester groups and their relative extents of conjugation to the central vinyl bond, and the relative intermolecular steric environments of the ester groups or substituents involved in the first step (vinyl-benzo bridging) of the photochemical reaction. / Science, Faculty of / Chemistry, Department of / Graduate

Flavonoids are a specialized group of compounds produced by plants that give them greater adaptability to their environment and ultimately enhance their ability to survive. In plants, one function of flavonoids is to attract pollinators by their various flavor and scent profiles. They also protect the photosynthetic machinery from photo-oxidation. In humans, flavonoids have been shown to act as antioxidants, exhibit antimicrobial activity, and have shown potential as cancer treatments. In nature, flavonoids are most often found coupled with a sugar group (glucose, rhamnose, and others) which imparts stability and increases bioactivity. The process of adding a sugar (glycosylation) is catalyzed by a class of enzymes called glycosyltransferases (GT). One such enzyme found in grapefruit only glucosylates the flavonol class of flavonoids at the 3-OH position and is of interest due to its unique substrate and regio-specificity. Called Cp3GT (Citrus paradisi flavonol 3-O glucosyltransferase), this enzyme is similar in structure to other plant GT’s yet differs in the flavonoids it can glucosylate and where the glucose can be added. To date, the literature has not reported a structural mechanism for a flavonol specific 3-O glucosyltransferase’s unique catalytic activity. High-resolution structural imagery of enzymes, elucidated using X-ray crystallography, can be used to direct custom enzyme development to produce bioavailable natural products. Furthermore, structural research on enzymes with high specificity strengthens enzyme-ligand docking simulations, which are commonly used to test the binding affinity of potential pharmaceuticals. This research hypothesizes Cp3GT has structural features that confer its unique substrate and regiospecificity that are not revealed by homology modeling. This hypothesis will be tested using x-ray crystallography of purified Cp3GT protein bound to its preferred flavonol substrates. The gene for Cp3GT was transformed into Pichia pastoris and was recombinantly expressed using methanol induction. Cp3GT was purified to 80% purity using cobalt metal affinity chromatography. Cp3GT was subjected to additional purification measures using anion exchange chromatography with the goal of increasing purity to ≥95% for crystallization experiments. Purity analysis was conducted using SDS-PAGE (Coomassie/silver stain, western blot) and UV-Vis spectrophotometry. While initial results are promising, additional purification steps may be needed to achieve the purity necessary for crystallization.

In prokaryotes, protein glycosylation can be a determinant of pathogenicity as it plays a role in host adherence, invasion, and colonization. Impairment of glycosylation in some organisms, for example N-linked glycosylation in Campylobacter jejuni, leads to decreased pathogenicity; thus, opening new avenues for the development of antivirulence agents. A member of the protein glycosylation (pgl) gene locus in C. jejuni, PglC, is predicted single-pass transmembrane (TM) protein, that catalyzes the phosphoglycosyl transferase (PGT) reaction in the first membrane-committed step of the N-linked glycosylation pathway. The small size of PglC (201 aa) compared to homologous PGTs suggests it may represent the minimal catalytic unit for the monotopic PGT superfamily. Herein, the structure of C. concisus PglC including its putative TM domain has been solved to 2.74 Å resolution to reveal a novel protein fold with a unique alpha-helix-associated beta-hairpin (AHABh) motif and largely solvent-exposed structure. There is noted a parsimony of fold in the form of short-range motifs underpinning the structural basis for critical functions of PglC: membrane association and active-site geometry. Biochemical and bioinformatics studies support structural evidence suggesting the crystallographically-observed, kinked TM helix is re-entrant on the cytoplasmic face of the membrane rather than membrane spanning. Thus, PglC represents a first-in-class structure of a novel membrane interaction mode for monotopic membrane proteins. Additionally, the AHABh-motif and active-site helical geometry establishes co-facial positioning of the catalytic-dyad. Molecular docking of PglC substrates, undecaprenyl phosphate (UndP) and UDP-N,N-diacetylbacillosamine (UDP-diNAcBac), within the active-site reveals co-incident binding sites, consistent with the proposed ping-pong enzymatic mechanism. Loading of PglC into membrane-bilayer nanodiscs (ND) allows for the investigation of PglC structure and function within a native-like membrane environment by small-angle x-ray scattering (SAXS). Observation of PglC in ND via SAXS confirms the application of the method for studying small, integral, monotopic membrane proteins in a membrane environment. Moreover, development of a mathematical approach by which resident-protein: ND stoichiometry can be deduced from measured scattering intensity enables independent confirmation of loading stoichiometry. Overall, the membrane-interaction modality observed for PglC is the first structurally characterized example of a new membrane association mode for monotopic proteins with the membrane. These studies provide insight into the structural determinants of the chemical mechanism and substrate-binding for C. concisus PglC and for the extensive homologous monotopic PGT superfamily, thus allow homology modeling and enabling future inhibitor design. / 2019-06-12T00:00:00Z

No description available.

CD16 is a physiologically essential Fc and #947; receptor III as either a single- pass transmembrane protein (CD16A) or as a glycosylated phosphatidylinositol (GPI) anchored protein (CD16B) on the surface of immune cells that have been implicated in many autoimmune and immune complex-mediated diseases. Its functions include binding and clearing antibody (IgG) coated foreign pathogens, receptor-mediated phagocytosis, and triggering antibody dependent cellular cytotoxicity. It is well established that these functions depend on protein-protein interaction between CD16 and the Fc domain of IgG. However, the molecular details of CD16-IgG interactions are less well defined, but are essential to developing therapeutic compounds to treat many autoimmune and IC diseases. Stable mammalian cell lines expressing wild-type CD16 isoforms and site-specific mutants, including extracellular soluble fragments of CD16 have been established. Soluble forms of wild type CD16A and these CD16 mutants were expressed in a bacterial pathway in order to amass sufficient quantities for x-ray crystallographic studies. The soluble portions of wild-type CD16A and several site-specific CD16A and CD16B mutants were constructed by PCR amplification and ligation with a pET vector. The proteins were expressed in a prokaryotic pathway, BL21 AI, for 8-10 hours and lysed to obtain inclusion bodies. A hand-held sonicator was used to wash the inclusion bodies, while a Urea solution separated and dissolved the proteins. The target proteins were then refolded by rapid dilution, concentrated with a stir cell, and purified. Wild type sCD16A and four site specific mutants were constructed with good sequencing, while wild type sCD16A, sCD16A F176V, and sCD16A G147D were expressed and refolded to optimal levels. X-ray crystallographic data has been collected from sCD16A F176V as a result of these studies and crystals are currently being grown from wild type sCD16A and sCD16A G147D.