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Protein and enzyme immobilisation on synthetic material surfaces enables a range of applications from biosensing to industrial biocatalysis. There are several immobilisation techniques, but common methods need multiple preparation steps or are material-dependent, which reduce the effectiveness and success of biosensing/industrial applications. In this thesis, the possibility of using plant-based polyphenol coatings to immobilise a range of proteins and enzymes (avidin, immunoglobulin G, acid phosphatase, chymotrypsin, lactate dehydrogenase, horseradish peroxidase) on polymeric or oxide materials (cellulose, polyester, silica, alumina and stainless steel) was shown for the first time. Polyphenols are in abundance in nature and less costly than dopamine (common immobilisation agent). Polyphenols were more effective than physisorption and polydopamine coatings for certain combinations of materials and proteins. Several parameters that can influence the immobilisation procedure were evaluated showing that there is a dependence on the coating and immobilisation pH as well as the type of coating and material. Polyphenol coatings were also used to functionalise nanoporous anodic aluminium oxide (AAO) membranes in order to measure molecular transport through nanopores. Inspired by the biological nanopores that enable the highly specific and efficient separation of a range of biomolecules, we used AAO membranes with a pore size matching the biological nuclear pore complex for controlling the diffusion of molecules through the pores. AAO membranes also match the requirements for optical waveguide spectroscopy (OWS) that was used to characterise and differentiate processes that occurred inside and above the nanoporous membranes. In a second approach, a nanoporous membrane was placed between two gaskets to be suspended on the flow cell. This work brings a new concept of how the molecular diffusion can be characterised, which is important for controlling the transport of biomolecules. It was possible to monitor in situ biocatalytic reactions as well as nanopore transport control by using a responsive polymer that was able to allow and restrict the transport of molecules.

Meiotic recombination is initiated by DNA double-strand breaks (DSBs) created by the topoisomerase-like protein Spo11. During DSB formation, Spo11 becomes covalently attached to the 5' DSB ends. Removal of Spo11 is essential to repair the DSB by homologous recombination. Spo11 is removed endonucleolytically creating short-lived Spo11-oligonucleotide products. Here I demonstrate that: 1. Spo11-oligonucleotide products are not detected in recombination mutants believed to be defective in meiotic DSB formation. 2. When DSB repair is delayed, Spo11-oligonucleotides persist for longer. 3. Processing of Spo11-DSB ends to create Spo11-oligonucleotides is largely dependent on Mec1 and Tel1 activity. In the process of investigating Spo11-oligonucleotide degradation, it was observed that a mutant defective in both the meiotic recombination checkpoint and in DSB repair failed to accumulate the expected level of DSBs. Work described here leads to the proposal of a DSB feedback mechanism that functions though the Mec1 (ATR) pathway to increase the efficiency of DSB formation. By contrast, Tel1 (ATM) functions to inhibit DSB formation, agreeing with recently published data. However, the data presented also suggests that Tel1 acts alongside the Mec1 pathway to promote DSB formation. It is therefore proposed that such positive and negative regulation creates a homeostatic mechanism to ensure that an optimum frequency of DSBs is formed. In wildtype cells, single –stranded DNA resection relies only on the Exo1 nuclease. In checkpoint defective cells resection length is increased. Results described here demonstrate that in a checkpoint defective strain, resection functions through Exo1, Sgs1/Dna2 and a third currently untested resection mechanism, likely to be Mre11 dependent.

Power harvesting from biological sources has been very popular recently because of the advancements in implantable medical devices. Among all different biofuel cells, utilising enzymes for glucose oxidation plays essential role in developing micro-power sources due to their high bio-catalytic activity. The aim of this study is to develop enzyme electrodes using pyranose-2-oxidase (P2O, wild type and mutants) and investigate the potential use in enzymatic biofuel cell applications as alternative to commercially available glucose oxidase (GOx) for glucose oxidation. Additional work was also carried out with bilirubin oxidase (BOD) for oxygen reduction. The effect of oxygen on enzyme performance, immobilization of the enzymes on carbon surface and biofuel cell performance were mainly investigated. The electrochemical techniques employed in this study were cyclic voltammetry, linear sweep voltammetry and chronoamperometry. Fuel cell test were carried out in glass cells and custom-made stack cells by recording cell potential on different resistances. Polarization curves were obtained by plotting voltage, current and power values. P2O and GOx were first tested in solution in the presence of electron mediator ferrocene carboxylic acid (FcCOOH) to investigate the effect of oxygen on enzyme performances. P2O and its mutants showed similar electrochemical behaviour compared to commercial GOx where P2O-T169G mutant showed better performance, especially when oxygen is saturated in the solution. The immobilization of the mutant P2O-T169G and GOx were then achieved using crosslinking on pyrenyl carbon structures, where either FcCOOH was used in solution or ferrocene (Fc) immobilised with nafion® polymer and carbon nanotubes on electrode surface. BOD was also immobilised on electrode using same method without mediator. Results indicate that enhanced current values was achieved compared to solution studies with good affinity towards glucose for both of the enzymes. Proof of concept biofuel cells were set up using P2O-T169G/GOx and BOD as anodes and cathode, respectively. Initial tests showed that P2O-T169G based enzymatic fuel cell can reach up to a power density of 9.56 μW cm-2 which is ~ 25 % more power output than it was obtained for GOx in aerobic conditions. Finally, a biofuel cell anode using P2O-T169G was combined with air breathing BOD cathode in a stack design enzymatic biofuel cell with an open circuit potential of 0.558 V and maximum power density of 29.8±6.1 μW cm-2 at 0.318 V.

The biochemistry of cytochrome P450 is an interesting topic in biology and chemistry due to the importance of their functions to human health, but also has relevance to biotechnology. As such, understanding the catalytic mechanism of the P450s is crucial to assigning their applications. In this regard, it is often required to develop biomimetic models or synthetic analogues of their active sites for chemical investigation. In this thesis, I have investigated the mechanistic details of a novel cytochrome P450 Peroxygenase, OleTJE whose reaction with long chain fatty acids gives a mixture of alpha- and beta-hydroxy fatty acids and terminal alkenes. Our QM/MM calculation on its reaction mechanism reveals regioselective formation of these products and suggests how it can be bioengineered to alter the product distribution towards terminal alkene. We also studied the reactions of cytochrome P450 in drug metabolism and reveal the factors that determine the regioselectivity of substrate hydroxylation over desaturation in P450 isozymes. Spin-selective products formation was observed; the energy gaps between O-H and C-O bonds formed, and the pi-conjugation energy determines the extent of desaturation in addition to perturbations by environmental influences in the binding pocket as revealed by QM/MM study. The reaction of [(L52)FeIII(OOH)]2+ biomimetic model with aromatic compounds such as benzene and anisole as studied with DFT shows that hydroxylation occurs by direct C-O bond formation rather than an initial low-energy homolytic O-O bond cleavage which is slightly higher in energy. Moreover, the homolytic cleavage activates the oxidant toward reaction unlike in the heme where heterolytic cleavage occurs to form an active oxidant. This was followed by determining why phenol is a dominant product over ketone which is the primary product in reaction with aryl compounds. We also investigated the chemical and reactivity differences between nonheme iron(IV)-tosylimido and iron(IV)-oxo oxidants as biomimetic models of reactive intermediates in certain enzyme reactions. The iron(IV)-tosylimido complex has larger electron affinity and will react better with sulfides in an electrophilic addition than the iron(IV)-oxo which react faster in hydrogen atom transfer. These studies have employed computational analysis in most cases, and used to provide support for experimentally-obtained data where they exist; and have revealed fascinating bio(chemistry) of heme and nonheme iron-containing enzymes and oxidants with various substrates.

End binding protein 1 (EB1) is a key element in the complex network of protein-protein interactions at microtubule growing ends which has a fundamental role in microtubule polymerisation. EB1 regulates the microtubule dynamic behaviour, through protein recruitment, and has been associated with several disease states, such as cancer and neuronal diseases. Diverse EB1 binding partners are recognised through a conserved SxIP motif within an intrinsically disordered region enriched with basic, serine and proline residues. Crystal structure of EB1 in complex with a peptide containing the SxIP motif demonstrated that the isoleucine-proline dipeptide is bound into a well‐defined cavity of EB1 that may be suitable for small molecule targeting. The research described herein reports the use of a multidisciplinary approach for the discovery of the first small molecule scaffold to target the EB1 recruiting domain. This approach included virtual screening (structure and ligand based design) and multiparameter compound selection. Solution NMR structures of the C-terminal domain of EB1 in the free form and in complex with the small molecule are also reported. A key finding from these structures is that the hydrophobic binding pocket reported to be essential for recruiting SxIP proteins is not pre-formed but highly dynamic in solution. This brings new insights to the protein recruitment mechanism regulated by EB1 and for the identification of new small molecule inhibitors for the EB1-SxIP protein interactions. The interaction of short length peptides containing the SxIP motif with EB1 was characterised through the use of solution NMR and ITC methods. The contributions for the binding of the SxIP motif and neighbouring residues to EB1 were quantified in terms of binding energy. A structural model shows that the binding pocket of EB1 is largely extended when in complex. This research describes not only the first chemical scaffold that targets EB1, it details important structural features of the interaction of this protein with SxIP containing peptides. This structural information provides fundamental understanding of this interaction that can be exploited in the future to discover higher affinity ligands.