This thesis explores roles of 2-oxoglutarate-dependent (2OG) oxygenases as interfaces that modulate steps in the flow of genetic information in cells in response to oxygen availability. Chapter 1 introduces mechanistic, biochemical and physiological aspects of major subfamilies of 2OG oxygenases, and their established regulatory roles in cells. In addition, structural and functional aspects of the ribosome and the translation process are discussed, with a focus on post-translational ribosome modifications. Chapter 2 investigates histone demethylases, which mediate chromatin-dependent regulation of gene expression and provides proof-of-concept for the rational, structure-guided design of small-molecules for selective inhibition of 2OG oxygenases with roles in cancer and inflammatory disease. Chapter 3 suggests regulatory roles for ten-eleven-translocation (TET)- catalysed DNA hydroxylation; calorimetric and thermal analyses reveal a duplex-stabilizing effect of the epigenetic 5-methylcytosine mark that is reversed upon conversion to 5- hydroxymethylcytosine (also termed the ‘sixth’ DNA base), raising the possibility that 2OG oxygenase catalysis might affect transcription via biophysical effects. Chapter 4 investigates fluoride release assays as a technology to enable medicinal chemistry studies on 2OG oxygenases with roles in fat mass regulation and obesity, cancer and inflammation; studies on the ALKBH5 enzyme show that it is a hypoxically upregulated 2OG oxygenase with a substrate preference distinct from previously characterized ALKBH enzymes. Chapter 5 identifies OGFOD1 as a 2OG-dependent ribosomal protein hydroxylase. OGFOD1 catalysis is conserved from yeast to humans. OGFOD1 catalyses formation of trans-3- hydroxy-L-proline in a highly conserved loop of ribosomal protein S23 proximal to the ribosomal decoding centre, possibly to modulate the interactions of eukaryotic ribosomes with tRNA, mRNA and translation factors in an oxygen-dependent manner. OGFOD1 is the functionally most well-conserved protein-modifying 2OG oxygenase; likewise, ribosomal protein S23 hydroxylation is the most well-conserved post-translational ribosome modification in eukaryotes. Some cell lines require OGFOD1 for proliferation, and scaffolds for OGFOD1- selective inhibitors are developed for use as potential antiproliferative agents and probes for cellular function. Chapter 6 shows the development of assays to investigate whether OGFOD1 catalysis affects ribosome assembly and function, including processivity, accuracy of initiation, elongation and termination, in yeast and mammalian cell lines. Chapter 7 concludes that ribosome hydroxylation might present an additional layer of regulatory complexity by which 2OG oxygenases could enable cells to respond to fluctuating oxygen levels.

The first identfied 2-oxoglutarate (2OG) dependent oxygenase was a collagen modifying enzyme in the work by Hutton et al. in 1967. Subsequent work has revealed that 2OG dependent oxygenases are a large family with diverse biological roles. With small molecule substrates, these enzymes catalyse a wide range of oxidative reactions, including those that form part of antibiotic biosynthetic pathways. The currently accepted consensus mechanism for catalysis by 2OG-dependent oxygenases is based on crystallographic data, kinetics and on quantum chemical calculations. The consensus mechanism involves oxidative decarboxylation of 2OG by reaction with an oxygen molecule producing CO₂, succinate and a reactive oxidising species that reacts with the 'prime' substrate. Deacetoxycephalosporin C synthase (DAOCS) is a 2OG-dependent oxygenase involved in cephalosporin biosynthesis. The mechanism of DAOCS is of particular interest because it has recently been proposed to be different from the consensus mechanism. The new mechanism proposal from Valeg ard et al. is primarily based on high-resolution crystallographic data with support from steady-state kinetic experiments and quantum-chemical calculations. The work in discussed in this thesis aimed to test the proposal of Valegård et al. by using a combination of spectroscopic and spectrometric methods analysing enzyme-substrate interactions. Substrate binding was investigated using both protein-observe (Chapter 3) and ligand-observe (Chapter 4.1 and 4.2) methods. Preliminary UV-visible data on enzyme-substrates complex formation was also obtained. The strength of substrate and cosubstrate binding was characterised through dissociation constant measurement. An activity assay (Chapter 2) that allows for direct and simultaneous monitoring of 2OG decarboxylation and penicillin ring expansion was optimised. Both the ligand-observe and protein-observe binding experiments as well as the preliminary UV-visible data indicate that the formation of a ternary complex between DAOCS, 2OG and the penicillin substrate is viable. The activity assay conclusively showed that in the presence of unnatural substrates, such as penicillin G, 2OG oxidation is significantly uncoupled from penicillin oxidation. Uncoupled turnover does not occur in the presence of the natural substrate, penicillin N, which is an aspect that should be considered in the analysis of the steady-state kinetic data. Overall, the results provide evidence that, the consensus mechanism for 2OG-dependent oxygenases is viable for DAOCS, at least in the presence of the natural substrate, penicillin N. It is possible that in the presence of an unnatural substrate, the catalytic process undergoes a more complex mechanism, possibly with the direct involvement of reducing agents in the system.

Fibronectin is an extracellular multidomain glycoprotein that directs and regulates a variety of cell processes such as proliferation, development, haemostasis, embryogenesis, and wound healing. As a major component of blood, fibronectin exists as a soluble disulphide linked dimer, but it can also be incorporated into an insoluble cross-linked fibrillar network to form a major component of the extracellular matrix. Fibronectin is composed of an extended chain of module repeats termed Fn1, Fn2, and Fn3 that bind to a wide range of transmembrane receptors and extracellular matrix components, including collagen. The gelatin binding domain of fibronectin was first isolated as a 45kDa proteolytic fragment and has since been found to be composed of six modules: 6Fn1-1Fn2-2Fn2-7Fn1-8Fn1-9Fn1 (in this notation nFX represents the nth type X module in the native protein). This domain has been reported to bind to both collagen and denatured collagen (gelatin), but with 10-100 times higher affinity to the latter; it can be purified to homogeneity on a gelatin affinity column. In the work presented here, fragments of the gelatin binding domain are expressed in P. pastoris, purified to homogeneity, and investigated at the molecular level. Through a dissection approach, surface plasmon resonance (SPR) is used to characterise the recombinantly produced protein, to accumulate more information about the function of the full domain. NMR is used to assess the folding of the protein fragments at atomic resolution. In particular, the secondary structure of 8Fn1-9Fn1 is mapped using inter-strand NOEs, which suggests that the construct takes the fold of a pair of typical Fn1 modules. Gelatin affinity chromatography is used to confirm that both Fn1 and Fn2 modules contribute to gelatin binding, possibly in two clusters (1Fn2-2Fn2 and 8Fn1-9Fn1). The 7Fn1 module may perform a structural role in linking together these two interaction sites, in the same way as suggested for 6Fn1, which is thought to act in a structural manner to enhance the binding of 1Fn2-2Fn2 to gelatin. Three carbohydrate moieties are found on this domain, one on 2Fn2 and two on 8Fn1. Here, by means of expressing different protein length fragments, and by site directed mutagenesis, the role of each sugar chain is investigated independently. The sugar chain on 2Fn2 does not appear to promote binding to collagen, nor does the first sugar chain on 8Fn1 (N-linked to N497), implying another role for these sugars such as protection from proteolysis. However, the presence of at least a single GlcNAc sugar residue on the second sugar chain site on 8Fn1 (N- linked to N511) is essential for full affinity binding to collagen. Direct binding of the 8Fn1-9Fn1 module pair to collagen is assessed with a short collagen peptide and the binding is monitored by NMR. The peptide appears to bind, predominantly to the final strand of 8Fn1, the first β- strand of 9Fn1, and the linker between the two modules, with μM affinity. A model for bound peptide is proposed. The highly conserved amino acid motif Ile-Gly-Asp (IGD) is found on four of the nine N-terminal Fn1 modules of fibronectin. Tetrapeptides containing the IGD were demonstrated to promote the migration of fibroblast cells into a native collagen matrix. Two of these “bioactive” IGD motifs are found within the gelatin binding domain, one on 7Fn1 and one on 9Fn1. In this study, the motif in the 8Fn1-9Fn1 module pair is shown to be located in a tightly constrained loop within 9Fn1. By site directed mutagenesis, the IGD motifs of 7Fn1 and 9Fn1 are subjected to single amino acid substitutions, and their ability to stimulate cell migration assessed in our assay. By NMR, the fold of the IGD mutant proteins is found to be unaffected by the mutation with respect to the wild type, with the exception of small perturbations around the substitution site. While the wild type module is able to stimulate fibroblast migration, the mutant proteins show reduced or negligible bioactivity. The larger fragments show far more potency in stimulating fibroblast migration, with 8Fn1-9Fn1 (one IGD motif) 104 times more potent than the IGD peptide, and the full gelatin binding domain (two IGD motifs) 106 times more potent than the 8Fn1-9Fn1. Potential mechanisms for this enormous enhancement of the IGD potency in different contexts are discussed.

The self-assembly of structured particles into monodisperse clusters is a challenge on the nano-, micro- and even macro-scale. While biological systems are able to self-assemble with comparative ease, many aspects of this self-assembly are not fully understood. In this thesis, we look at the strategies and rules that can be applied to encourage the formation of monodisperse clusters. Though much of the inspiration is biological in nature, the simulations use a simple minimal patchy particle model and are thus applicable to a wide range of systems. The topics that this thesis addresses include: Encapsulation: We show how clusters can be used to encapsulate objects and demonstrate that such `templates' can be used to control the assembly mechanisms and enhance the formation of more complex objects. Hierarchical self-assembly: We investigate the use of hierarchical mechanisms in enhancing the formation of clusters. We find that, while we are able to extend the ranges where we see successful assembly by using a hierarchical assembly pathway, it does not straightforwardly provide a route to enhance the complexity of structures that can be formed. Pore formation: We use our simple model to investigate a particular biological example, namely the self-assembly and formation of heptameric alpha-haemolysin pores, and show that pore insertion is key to rationalising experimental results on this system. Phase re-entrance: We look at the computation of equilibrium phase diagrams for self-assembling systems, particularly focusing on the possible presence of an unusual liquid-vapour phase re-entrance that has been suggested by dynamical simulations, using a variety of techniques.

F₁-ATPase, the sector of ATP synthase where the synthesis of cellular ATP occurs, is a rotary molecular motor in its own right. Driven by ATP hydrolysis, direct observation of the rotation of the central axis within single molecules of F₁ is possible. Operating at close to 100% efficiency, F₁ from thermophilic Bacillus has been shown to produce ~40pN˙nm of torque during rotation. This thesis details the groundwork required for the direct measurement of the torque produced by F₁ using a rotary angle clamp, an optical trapping system specifically designed for application to rotary molecular motors. Proof-of-concept experiments will be presented thereby demonstrating the ability to directly manipulate single F₁ molecules from Escherichia coli and yeast mitochondria (Saccharomyces cerevisiae), along with activation of F₁ out of its inhibited state by the application of external torque. Despite in-depth knowledge of the rotary mechanism of F₁ from thermophilic Bacillus, the rotation of F₁ from Escherichia coli is relatively poorly understood. A detailed mechanical characterization of E.coli F₁ will be presented here, with particular attention to the ground states within the catalytic cycle, notably the ATP-binding state, the catalytic state and the inhibited state. The fundamental mechanism of E.coli F₁ appears to depart little from that of F₁ from thermophilic Bacillus, although, at room temperature, chemical processes occur faster within the E.coli enzyme, in line with considerations regarding the physiological conditions of the different species. Also presented here is the verification of the rotary nature of yeast mitochondrial F₁. The torque produced by F₁ from thermophilic Bacillus, E.coli and yeast mitochondria is the same, within experimental error, despite their diverse evolutionary and environmental origins.

Alpha-hemolysin is a pore-forming toxin secreted by pathogenic Staphylococcus aureus. Its spontaneous oligomerization and assembly into a trans-bilayer beta-barrel pore is a model for the assembly of many other pore-forming toxins. It is studied here in vitro as a means to probe general membrane protein oligomerization and lipid bilayer insertion. This thesis details the results of experiments to develop and implement a novel in vitro lipid bilayer system, Droplet-on-Hydrogel Bilayers (DHBs) for the single-molecule imaging of alpha-hemolysin assembly. Chapter 2 describes the development of DHBs and their electrical characterization. Experiments show the detection of membrane channels in SDS-PAGE gels post-electrophoresis and DHBs use as a platform for nanopore stochastic sensing. Chapter 3 describes the engineering and characterization of fluorescently-labelled monomeric alpha-hemolysin for use in protein assembly imaging experiments described in Chapter 6. Chapter 4 describes the characterization of DHB lipid fluidity and suitability for single-molecule studies of membrane protein diffusion. In addition, a novel single-particle tracking algorithm is described. Chapter 5 describes experiments demonstrating simultaneous electrical and fluorescence measurements of alpha-hemolysin pores embedded within DHBs. The first multiple-pore stochastic sensing in a single-lipid bilayer is also described. Chapter 6 describes experiments studying the assembly of alpha-hemolysin monomers in DHBs. Results show that alpha-hemolysin assembles rapidly into its oligomeric state, with no detection of long-lived intermediate states.

Carbon-based materials are poised to be an important class of 21st century materials, for bio-medical, bio-electronic, and bio-sensing applications. Diamond and polymers are two examples of carbon-based materials of high interest to the bio-materials community. Diamond, in its conductive form, can be used as an electrochemical bio-sensor, whilst its nanoparticle form is considered a non-inflammatory platform to deliver drugs or to grow neuronal cells. Polymers, especially when chemically modified, have been used extensively in biological environments, from anti-microbial use to drug delivery. The large-scale use of either material for biological use is limited by two factors: ease of chemical modification and the paucity of knowledge of their surface chemistry in aqueous media. This thesis addresses aspects of both these issues. The first study reported is an in situ study of the adsorption dynamics of an exemplar globular protein (bovine serum albumin, BSA) on nanodiamond using the relatively novel quartz crystal microbalance with dissipation (QCM-D) technique. For the first time, QCM-D enabled the detailed study of protein dynamics (i.e. kinetics, viscoelastic properties, overlayer structure, etc.) onto nanodiamond thin films having various surface chemistry and roughness. The dynamics of protein adsorption is found to be sensitive to surface chemistry at all stages of adsorption, but it is only sensitive to surface roughness during initial adsorption phases. Our understanding of the nanodiamond-biology interface is enhanced by this study, and it suggests that QCM-D is useful for the study of the surface chemistry of nanoparticle forms of inorganic materials. A second study concerns a novel surface functionalization scheme, based on carbene and azo-coupling chemistry, which has been recently introduced as a practical, facile method for modifying the surfaces of polymers. Using modern surface characterization techniques, it is demonstrated that a chemical linker can be attached to polystyrene surfaces using carbene-based chemistry, and that further chemical functionality can be added to this chemical linker via an azo-coupling reaction. In situ studies of protein dynamics at these interfaces were conducted using QCM-D, thus enabling a link between specific protein behaviour and the polymer surface chemical termination chemistry to be made. A third area of study of investigates the use of diamond electrodes as a bio-sensor for dopamine under physiological conditions. For these conditions, ascorbic acid interferes with the dopamine oxidation signal, in ways that render the two signals irresolvable. Various modifications are used in attempts to reduce this interference, including: small and large cathodic treatments, grafting of electro-active polymers, addition of carbon nanotubes, and hydrogen plasma treatment. Those modifications leading to the hydrogen-termination of diamond are shown to work the best. Notably, hydrogen plasma treatment effects the complete electrochemical separation of dopamine and ascorbic acid at a diamond electrode. This is the first time this has been accomplished without adding non-diamond materials to the diamond electrode surface.

Molecular biophysics is a rapidly evolving field aimed at the physics-based investigation of the biomolecular processes that enable life. In this thesis, we explore two such processes: the thermodynamics of DNA bending, and the mechanism of the RecQ DNA helicase. A computational approach using a coarse-grained model of DNA is employed for the former; an experimental approach relying heavily on single-molecule fluorescence for the latter. There is much interest in understanding the physics of DNA bending, due to both its biological role in genome regulation and its relevance to nanotechnology. Small DNA bending fluctuations are well described by existing models; however, there is less consensus on what happens at larger bending fluctuations. A coarse-grained simulation is used to fully characterize the thermodynamics and mechanics of duplex DNA bending. We then use this newfound insight to harmonize experimental results between four distinct experimental systems: a 'molecular vise', DNA cyclization, DNA minicircles and a 'strained duplex'. We find that a specific structural defect present at large bending fluctuations, a 'kink', is responsible for the deviation from existing theory at lengths below about 80 base pairs. The RecQ DNA helicase is also of much biological and clinical interest, owing to its essential role in genome integrity via replication, recombination and repair. In humans, heritable defects in the RecQ helicases manifest clinically as premature aging and a greatly elevated cancer risk, in disorders such as Werner and Bloom syndromes. Unfortunately, the mechanism by which the RecQ helicase processes DNA remains poorly understood. Although several models have been proposed to describe the mechanics of helicases based on biochemical and structural data, ensemble experiments have been unable to address some of the more nuanced questions of helicase function. We prepare novel substrates to probe the mechanism of the RecQ helicase via single-molecule fluorescence, exploring DNA binding, translocation and unwinding. Using this insight, we propose a model for RecQ helicase activity.

<p>The realm of molecular physical chemistry ranges from the structure of matter and the fundamental atomic and molecular interactions to the macroscopic properties and processes arising from the average microscopic behaviour.</p><p>Herein, the conventional electrodic problem is recast into the simpler molecular problem of finding the electrochemical, real chemical, and chemical potentials of the species involved in redox half-reactions. This molecular approach is followed to define the three types of absolute chemical potentials of species in solution and to estimate their standard values. This is achieved by applying the scaling laws of statistical mechanics to the collective behaviour of atoms and molecules, whose motion, interactions, and properties are described by first principles quantum chemistry. For atomic and molecular species, calculation of these quantities is within the computational implementations of wave function, density functional, and self-consistent reaction field theories. Since electrons and nuclei are the elementary particles in the realm of chemistry, an internally consistent set of absolute standard values within chemical accuracy is supplied for all three chemical potentials of electrons and protons in aqueous solution. As a result, problems in referencing chemical data are circumvented, and a uniform thermochemical treatment of electron, proton, and proton-coupled electron transfer reactions in solution is enabled.</p><p>The formalism is applied to the primary and secondary radiation damage to DNA bases, e.g., absorption of UV light to yield electronically excited states, formation of radical ions, and transformation of nucleobases into mutagenic lesions as OH radical adducts and 8-oxoguanine. Based on serine phosphate as a model compound, some insight into the direct DNA strand break mechanism is given.</p><p>Psoralens, also called furocoumarins, are a family of sensitizers exhibiting cytostatic and photodynamic actions, and hence, they are used in photochemotherapy. Molecular design of more efficient photosensitizers can contribute to enhance the photophysical and photochemical properties of psoralens and to reduce the phototoxic reactions. The mechanisms of photosensitization of furocoumarins connected to their dark toxicity are examined quantum chemically.</p>

The realm of molecular physical chemistry ranges from the structure of matter and the fundamental atomic and molecular interactions to the macroscopic properties and processes arising from the average microscopic behaviour. Herein, the conventional electrodic problem is recast into the simpler molecular problem of finding the electrochemical, real chemical, and chemical potentials of the species involved in redox half-reactions. This molecular approach is followed to define the three types of absolute chemical potentials of species in solution and to estimate their standard values. This is achieved by applying the scaling laws of statistical mechanics to the collective behaviour of atoms and molecules, whose motion, interactions, and properties are described by first principles quantum chemistry. For atomic and molecular species, calculation of these quantities is within the computational implementations of wave function, density functional, and self-consistent reaction field theories. Since electrons and nuclei are the elementary particles in the realm of chemistry, an internally consistent set of absolute standard values within chemical accuracy is supplied for all three chemical potentials of electrons and protons in aqueous solution. As a result, problems in referencing chemical data are circumvented, and a uniform thermochemical treatment of electron, proton, and proton-coupled electron transfer reactions in solution is enabled. The formalism is applied to the primary and secondary radiation damage to DNA bases, e.g., absorption of UV light to yield electronically excited states, formation of radical ions, and transformation of nucleobases into mutagenic lesions as OH radical adducts and 8-oxoguanine. Based on serine phosphate as a model compound, some insight into the direct DNA strand break mechanism is given. Psoralens, also called furocoumarins, are a family of sensitizers exhibiting cytostatic and photodynamic actions, and hence, they are used in photochemotherapy. Molecular design of more efficient photosensitizers can contribute to enhance the photophysical and photochemical properties of psoralens and to reduce the phototoxic reactions. The mechanisms of photosensitization of furocoumarins connected to their dark toxicity are examined quantum chemically.