Proteomics is a rapidly growing discipline dealing with structure, molecular interactions, conformational dynamics, modifications and the functions of proteins. Mass spectrometry (MS) and (nuclear magnetic resonance) (NMR) have been used comprehensively to study protein interactions. Acyl carrier protein (ACP) interacts with more than 30 partner proteins during either fatty acid or polyketide biosynthesis. In order to be fully activated ACP gains a 4'-phosphopantetheinyl (4'-PP) group from coenzyme A using acyl carrier protein synthase (AcpS) via posttranslational modification. Protein-protein interactions of the ACP from the actinorhodin (act) polyketide synthase (PKS) complex and AcpS were investigated using oxidative footprinting with hydroxyl radicals generated from the Fenton reaction. Chemical modification of acidic residues was also used to investigate the interaction between these proteins using l-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) this acted as a zero length cross linker to induce modification with Me-Glycine. MS was used to identify the modified residues and peptides and the extent of modification. Several residues were found to be protected in the complex between the two proteins and these may participate in the interaction interface between ACP and ACPS. Isotopically labelled ACP was expressed and purified and multidimensional NMR experiments were recorded to investigate this interaction interface identified using oxidative footprinting techniques. Chemical shift perturbations for ACP residues were calculated, and these revealed that many residues were affected by oxidation of ACP. Oxidation of methionine to methionine sulfoxide was confirmed. Metabolomics is a discipline which deals with metabolites in a biological system. It provides a wealth information for disease diagnosis, drug discovery, toxicology and genetic modification. Attempts have been made in this thesis to utilize metabolomics in biometrics. Mice were used as a model to attempt to determine individuals' age by their scent. In this part of the project chemometric methods were used to discriminate mice using a gas chromatography-mass spectrometry dataset of volatile organic compounds obtained from their urine. Principal component regression (PCR), partial least squares regression (PLSR) and support vector regression (SVR) were used to determine mouse age. Mice could be discriminated by their age using SVR without overfitting the data.

Over the past 40 years there has been substantial interest in continuous glucose monitors (CGMs) for diabetes management. Much of the work has focused on the use of enzymes, lectins (carbohydrate binding proteins) or boronic acids as the sensing element, but instability and biocompatibility has hindered development. This has presented an 0pp0l1unity for supramolecular chemists to make synthetic lectins for glucose that respond in real time to glucose in the blood. However, carbohydrate recognition in water presents a challenge due to the hydromimetic nature of the substrate. Moreover, subtle structural differences between monosaccharides makes selectivity problematic. Despite these difficulties, our group has met with some success in designing synthetic lectins over the past decade. Recently we reported a simple monocyclic receptor An-L 34 for glucose which is accessible in only 5 steps with 23% overall yield. The receptor binds glucose with excellent selectivity over physiological blood glucose levels, displaying increases in fluorescence emission suggesting its potential as a CGM. We report the development of a series of receptors based on the new architecture. New synthetic procedures have provided access to asymmetrical systems that have highlighted the driving forces behind binding. 2nd and 3rd generation dendritic solubilising groups have been synthesised leading to receptors that are less prone to aggregation and with enhanced affinity for glucose and improved optical properties for sensing. The improvements are attributed to the interaction of the carboxylate groups with the sugar from the larger solubilising groups. Using this approach a new receptor with 100 fold selectivity for positively charged monosaccharide glucosamine has been demonstrated. The new generation systems have been screened in human blood plasma, with the 2nd generation systems showing enhancements in response to glucose. Furthermore work has started towards immobilisation of receptors for fibre optic sensing.

The de novo design of active biomolecules, where both structure and function are constructed from first principles, requires the precise positioning of chemical functionality within stable and thoroughly characterised scaffolds. The first hexameric coiled coil, CC-Hex, is a homo-hexameric peptide assembly with a contiguous central channel of ~6 A in diameter, which presents clear opportunities for its use as a scaffold for the installation· of catalytic function. In Chapter 3, the reactivity of cysteine (Cys) residues installed within the lumen of CC-Hex with small-molecule alkylating agents is investigated. Comparison of the rates of reaction with iodoacetamide and unfolding of the assembly shows that the former proceeds largely via the assembled channel, rather than via the unfolded state. Furthermore, iodoacetate does not react, suggesting selectivity for uncharged reagents by the hydrophobic lumen of the assembly. Further mutations to the CC-Hex sequence, however, result in largely unfolded peptides. To address this issue, a computational design approach to higher-order coiled coils - the a-helical barrels - is described in Chapter 4, with the aim of delivering highly stable and mutable assemblies. This treats the two interfaces on either side of each helix of the barrel as heterodimer-like interfaces. This allows one million sequences to be generated and scored for the ability to form a-helical barrels. This process is used to generate 22 sequences for synthesis and experimental validation. All of these are a-helical, and, as determined by analytical ultracentrifugation, 8 match the computationally predicted oligomeric state. X-ray crystal structures reveal the first de novo pentameric and bluntended heptameric (CC-Hept) barrels, as well as further hexameric architectures. An iterative approach is described to install a Cys-His-Glu catalytic triad into CC-Hept in Chapter 5. All of the peptide designs are a-helical and heptameric in solution, and X-ray crystal structures are obtained for each of the iterations. For each mutant, hydrolase activity is assessed using p-nitrophenyl acetate (PNPA) as the substrate. Enhanced hydrolysis rates over baseline are observed for the Cys-His dyad peptide and particularly for the Cys-His-Glu triad peptide. Moreover, an increase in catalytic efficiency is observed when homocysteine is installed as the nucleophile, consistent with the increased conformational flexibility of the side-chain thiol. In Chapter 6, metal binding to the a-helical barrels is described, with up to 18 HgII ions binding to multiple Cys residues installed within a lengthened hexameric coiled coil. This once more highlights the mutability of the a-helical barrels and presents their use as monomers for metallated peptide nanotubes and as scaffolds for bioremediation.

Membrane functionalization is a promising strategy for augmenting cell performance in regenerative medicine. To this end, the design, construction, characterisation and cell affinity of protein-polymer surfactant nanoconstructs are presented. Nanoconstructs of eGFP were synthesised that exhibited near-native structure and function, as well as effective and persistent membrane affinity. Human mesenchymal stem cells were labelled for up to ten days in culture, without affecting cell viability or differentiation capacity. This "cell priming" technology has been used to address the issue of hypoxia-related central necrosis during in-vitro tissue engineering. Specifically, nanoconstructs of myoglobin, with enhanced oxygen-binding affinity, were synthesised and used to prime mesenchymal stem cells prior to hyaline cartilage engineering. The myoglobin-primed cells produced tissue constructs with a 62 % increase in type II : type I collagen ratio and, significantly, a reduction in cell necrosis from 42 ± 24 % to 7 ± 6 %.

Fungal natural products are secondary metabolites produced by complex biosynthetic pathways, many of which include the activities of multidomain polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) megasynthases. This thesis reports investigations of the intrinsic programming of PKS-NRPS hybrid enzymes and of the reconstruction of polyketide biosynthetic pathways by heterologous expression in Aspergillus oryzae. Rational domain swaps were performed between megasynthases involved in the synthesis of structurally-related 2-pyridones. Swaps between tenellin and desmethylbassianin synthases (TENS and DMBS) from closely related isolates of Beauveria bassiana confirmed that the C-methyltransferase domain controls methylation pattern and the ketoreductase domain contributes to chain-length programming; the hybrid enzymes produced were highly active. Hybrid enzymes from more distant swaps had reduced activity but showed potential for the production of chimaeric compounds. Exchanges between even more distantly-related enzymes resulted in complete loss of enzyme activity. Attempts to reconstruct the militarinone A biosynthetic pathway in A. oryzae met more success when MILS was co-expressed with enzymes from the DMB pathway than with its cognate enzymes. In contrast, heterologous expression of five genes from Aspergillus terreus resulted in full reconstruction of the lovastatin biosynthetic pathway. However, efficient lovastatin production was found to require additional co-expression of the thioesterase responsible for releasing the lovastatin nonaketide from the nonaketide synthase. Curiously, co-expression of the subset of enzymes required for synthesis of lovastatin intermediates was unproductive. The genome of Phoma sp. C2932 was sequenced and analysed to identify the squalestatin biosynthetic gene cluster. The putative squalestatin hexaketide synthase (SQHKS) expressed in isolation in A. oryzae did not produce novel compounds. SQHKS employs an unusual starter unit, the production of which is likely to be governed by other genes identified in the cluster. Co-expression of SQHKS with phenylalanine ammonia lyase and an AMP-dependent Co-A ligase, in the presence of cinnamic acid, yielded novel compounds, albeit in insufficient yield to confirm a structure related to squalestatin hexaketide. The results of lovastatin-production experiments suggest that co-expression of additional genes from the squalestatin pathway may improve product yields.

Spirotetronates are a subclass of polyketide natural products that exhibit an array of potentially exploitable bioactivities. They are characterised by the presence of a tetronic acid ring spiro-linked to cyclohexene. A general route to their biosynthesis has been elucidated through numerous in vitro and in vivo studies. These studies have shown a conserved cassette of enzymes to be responsible for both the incorporation of the eponymous tetronic acid ring, and subsequent chemical modification of this ring. In addition to this, a highly unusual acetylation/deactylation event has been shown to be instrumental in priming spirotetronate intermediates for the [4+2] cyclo-addition that leads to the formation of the spiro-centre. Until recently it remained unclear as to whether or not [4+2] cyclo-addition in the biosynthesis of spirotetronates proceeded via an enzymaticallycatalysed transformation. Herein I will present the crystal structures of the first tetronic acid forming condensing enzyme abyA1, the first acetyl lyase abyA5 and the first reported naturally occurring Diels-Alderase abyU. This work provides a significant insight into the molecular basis of spirotetronate biosynthesis and lays a foundation upon which enzymes from spirotetronate pathways could be used in synthetic biology tbwards the production of non-natural tetronate containing polyketides.

The 'marker of self' protein C047 is an unusual component of the band 3 macrocomplex because it is already expressed on the erythroblast prior to band 3 and protein 4.2 expression, upon which C047 is dependent for its membrane stability. We hypothesised that either an alternative C047 isoform is initially expressed before switching to the erythroid C047 isoform 2, or that C047 isoform 2 is expressed throughout terminal differentiation and is dependent upon an alternative interaction to maintain its membrane stability before incorporation into the band 3 macrocomplex. The work presented in this thesis showed that C047 isoform 2 and C047 isoforms 3 and 4, were expressed during terminal differentiation. However, unlike isoform 2, C047 isoforms 3 and 4 were not detected at the erythroblast membrane, indicating that C047 does not undergo isoform switching.

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