Conjugation reactions on proteins have been used to access various post-translational modifications, for targeted delivery of drugs, for microscopy, and in studying receptor-ligand interactions. However, the ability to modify native proteins is constrained by the reactive functionalities of naturally occurring amino acids. This has driven research into the incorporation of unnatural amino acids (UAAs) into proteins. Research in this area has been motivated both by the possibility of increasing the breadth of chemical techniques for protein modification by introducing novel 'bio-orthogonal' reactive groups via UAA incorporation, as well as generating well-defined conjugates by the site-selective incorporation of these UAAs into proteins. The objective of this thesis is to both expand the diversity of UAAs for access to new metal-mediated reactions on proteins, as well as to utilise these reactions to reveal functional information about a range of biological systems. A brief introduction into current protein conjugation and UAA incorporation methods will be made in Chapter 1. In Chapter 2, the genetic incorporation of alkene-bearing UAAs into recombinant proteins expressed in both bacterial and mammalian systems is discussed. This technique is demonstrated to enable Ru-catalysed olefin cross-metathesis (CM) reactions on the resultant proteins. This work builds upon previously established methods to chemically incorporate CM handles onto proteins. The rational design of UAAs, as well as assays and modelling studies to screen them for recognition by the cellular incorporation machinery are discussed in detail. The expression of a range of alkene-tagged recombinant proteins, their complete characterisation, as well as the development of a more general protocol for on-protein CM is elucidated. In Chapter 3, the utility of UAA incorporation to probe mammalian cell translation systems is examined. Incorporation of an azide-bearing UAA, in addition to heavy stable-isotope labelled amino acids is used to uncover a previously unreported system of protein synthesis in mammalian cell nuclei, along with rapid metabolic degradation of the synthesised peptides. Various orthogonal methods for the detection of this system as well as possible reasons for its conservation are discussed. In Chapter 4, UAA incorporation and metal-mediated bioconjugation reactions are utilised in the development of a novel and generally applicable proteomics technique. This technique is used to determine quantitative changes in cell proteomes in response to external stimuli, and may be applied to systems to which traditional proteomics techniques cannot, such as ex vivo primary cells. Finally, in Chapter 5, further applications of UAA incorporation are discussed. Preliminary results are reported in efforts to use UAAs in the vibrational Raman microscopic imaging of biological systems, in generating HIV vaccines, and inducing T-cell stimulation.

In this thesis, the response of DNA and RNA to linear and torsional mechanical stress is studied using coarse-grained models. Inspired by single-molecule assays developed over the last two decades, the end-to-end extension, buckling and torque response behaviour of the stressed molecules is probed under conditions similar to experimentally used setups. Direct comparison with experimental data yields excellent agreement for many conditions. Results from coarse-grained simulations are also compared to the predictions of continuum models of linear polymer elasticity. A state diagram for supercoiled DNA as a function of twist and tension is determined. A novel confomational state of mechanically stressed DNA is proposed, consisting of a plectonemic structure with a denaturation bubble localized in its end-loop. The interconversion between this novel state and other, known structural motifs of supercoiled DNA is studied in detail. In particular, the influence of sequence properties on the novel state is investigated. Several possible implications for supercoiled DNA structures in vivo are discussed. Furthermore, the dynamical consequences of coupled denaturation and writhing are studied, and used to explain observations from recent single molecule experiments of DNA strand dynamics. Finally, the denaturation behaviour, topology and dynamics of short DNA minicircles is studies using coarse-grained simulations. Long-range interactions in the denaturation behaviour of the system are observed. These are induced by the topology of the system, and are consistent with results from recent molecular imaging studies. The results from coarse-grained simulations are related to modelling of the same system in all-atom simulations and a local denaturation model of DNA, yielding insight into the applicability of these different modelling approaches to study different processes in nucleic acids.

Peptides have attracted increasing attention as therapeutics in recent years, at least partially as a consequence of the widespread acceptance of protein therapeutics; but also as possible solutions to problems such as short half-life and delivery of molecules, and as therapeutics in their own right. The current work presents three projects that involve applications of peptides in a therapeutic environment. The first project studies the use of ER retaining peptides and CPPs (Cell penetrating peptides) in enhancing the effective concentration of DNJ (1-deoxynojirimycin), an α-glucosidase inhibitor, in cells. DNJ constructs with ER retaining peptides (6-[N-(1-deoxynojirimycino)]-hexanoyl-KDEL and 6-[N-(1-deoxynojirimycino)]-hexanoyl-KKAA) and CPPs (6-[N-(1-deoxynojirimycino)]-hexanoyl-TAT and 6-[N-(1-deoxynojirimycino)]-hexanoyl-MAP) were synthesised and analysed for their inhibitory activity against α-glucosidase I and II in vitro. The constructs were then analysed in a cell-based assay to determine their inhibitory activity on α¬-glucosidase-mediated hydrolysis of N-linked oligosaccharides. FITC-labelled ER retaining peptides were also synthesised to determine the internalisation and trafficking of the constructs by FACS and IF-microscopy. While none of the DNJ-constructs showed higher cellular inhibition than NB-DNJ (N-butyl DNJ; Miglustat), the CPP construct 6-[N-(1-deoxynojirimycino)]-hexanoyl-TAT showed comparable activity and the ER retaining construct 6-[N-(1-deoxynojirimycino)]-hexanoyl-KDEL showed a small but significant increase in activity following long-term administration. The second project focuses on beauveriolides, a cyclic depsipeptide family shown to have activity as ACAT inhibitors and thus a possible treatment for Alzheimer’s disease by the decrease in the production of Amyloid β (Aβ). A published total synthetic method was improved by the use of a cross-metathesis to reduce the total synthesis by 5 steps and increase its flexibility to allow the production of analogues. The synthesised beauveriolide III was used in attempts to develop an IF-FACS-based assay to measure the intracellular concentrations of Aβ. However, the location of γ-secretase in the used cell-line meant that levels of intracellular Aβ were not sufficient to track any decrease caused by ACAT inhibition. The third project involves the design of a cyclic peptide that could block the binding site for the influenza virus in the host cell. The cyclic peptide (cGSGRGYGRGWGVGA) was developed from a comparative study of four different sialic acid-binding proteins and synthesised by solution cyclisation of the linear peptide synthesised by traditional solid phase peptide synthesis (SPPS). An in silico study showed that the cyclic peptide allowed overlap with the binding site of Hemagglutinin. A 1H NMR titration determined the dissociation constant of the cyclic peptide to sialic acid. The KD corresponded to a low binding affinity, however the observed binding seemed to be specific and caused by a single bound conformation.

JMJD6 or PSR (phosphatidyl serine receptor) was initially proposed to be a membrane receptor involved in apoptotic cell clearance by recognition of apoptotic cells. However, sequence analyses implied the presence of a jelly roll or double stranded beta helix (DSBH) structural domain in PSR/JMJD6 and similarity with JmjC family of enzymes which are involved in chromatin regulation. Subsequently, PSR was renamed as JMJD6 and was reported to be a histone arginine demethylase. Previous work from our group has shown that JMJD6 is a lysine hydroxylase that interacts with nuclear proteins including CROP and U2AF65 which are involved in mRNA splicing. Peptide screening and cell based assays led to the conclusion that JMJD6 catalyses lysine hydroxylation of splicing regulatory proteins containing arginine serine rich domains (SR proteins) including U2AF65 and Luc7like-2. Studies were carried out to investigate the putative arginine demethylation activity of JMJD6 using MS analysis of histone peptides and luminescence-based assays. New substrates from SR proteins were identified by immunoprecipitation of JMJD6 expressed in human cell lines followed by LC-MS/MS analysis and MALDI-MS based assays of synthesised peptide substrates. Work then focussed on studying the mechanism of lysyl-hydroxylation from substrate and enzyme perspective. A crystal structure of seleno-methionine labelled JMJD6 was obtained and it provided insights into the JMJD6 active site and its substrate interactions. Based on this data, single point variants of JMJD6 were prepared and their substrate binding properties were studied by MALDI-MS and 2OG turnover assays. Collagen lysyl-hydroxylases are also 2OG dependent oxygenases. Efforts to investigate the stereochemistry of JMJD6 catalysed hydroxylation, employing NMR and amino acid analyses were carried out. These studies led to the interesting finding that the C-5 stereochemistry of hydroxylysine in LUC7L2 peptide is opposite (2S,5S-hydroxylysine) to that present in collagen (2S,5R-hydroxylysine). It was found that JMJD6 undergoes autocatalytic self-hydroxylation. Lysine residues from both recombinant JMJD6 and that from HeLa cells at endogenous level were identified to be hydroxylated by amino acid and LC-MS/MS analyses. JMJD6 has a strong tendency to form aggregates and gel electrophoresis always reveals multimeric bands of various JMJD6 constructs. Characterisation and identification of oligomeric states of JMJD6 was carried out using Electron Microscopy. Studies were initiated to identify possible inhibitors by screening a set of 2OG analogues. The results from this preliminary inhibition studies have identified the tricarboxylic acid (TCA) cycle intermediates, succinate and fumarate to be JMJD6 inhibitors and form a basis of further studies aimed at identifying selective inhibitors.

This thesis examines the dynamic mechanical properties of natural silk fibres, mainly from silkworm species Bombyx mori (B. mori) and spider species Nephila edulis, using dynamic mechanical thermal analysis, DMTA. The aim is not only to provide novel data on mechanical properties of silk, but also to relate these properties to the structure and morphology of silk. A systematic approach is adopted to evaluate the effect of the three principal factors of stress, temperature and hydration on the properties and structure of silk. The methods developed in this work are then used to examine commercially important aspects of the ‘quality’ of silk. I show that the dynamic storage modulus of silks increases with loading stress in the deformation through yield to failure, whereas the conventional engineering tensile modulus decreases significantly post-yield. Analyses of the effects of temperature and thermal history show a number of important effects: (1) the loss peak at -60 °C is found to be associated the protein-water glass transition; (2) the increase in the dynamic storage modulus of native silks between temperature +25 and 100 °C is due simply to water loss; (3) a number of discrete loss peaks from +150 to +220°C are observed and attributed to the glass transition of different states of disordered structure with different intermolecular hydrogen bonding. Excess environmental humidity results in a lower effective glass transition temperature (Tg) for disordered silk fractions. Also, humidity-dynamic mechanical analysis on Nephila edulis spider dragline silks has shown that the glass transition induces a partial supercontraction, called Tg contraction. This new finding leads to the conclusion of two independent mechanisms for supercontraction in spider dragline silks. Study of three commercial B. mori cocoon silk grades and a variety of processed silks or artificial silks shows that lower grade and poorly processed silks display lower Tg values, and often have a greater loss tangent at Tg due to increased disorder. This suggests that processing contributes significantly to the differences in the structural order among natural or unnatural silks. More importantly, dynamic mechanical thermal analysis is proposed to be a potential tool for quality evaluation and control in silk production and processing. In summary, I demonstrate that DMTA is a valuable analytical tool for understanding the structure and properties of silk, and use a systematic approach to understand quantitatively the important mechanical properties of silk in terms of a generic structural framework in silk proteins.

There is currently a need for vaccine adjuvants that are effective for eliciting Th1-type CD4 and CD8 T cell responses when formulated with protein and peptide-based subunit vaccines. Some of the most promising adjuvants in this regard are combined small molecule Toll-like receptor-7/8 agonists (TLR-7/8a). However, poor pharmacokinetic properties have precluded TLR-7/8a for use in vaccines. In this thesis, polymer carriers were used to control pharmacokinetics and to modulate activity of TLR-7/8a for use as vaccine adjuvants. Combinatorial synthesis and in vivo structure-activity studies were used to evaluate how properties of Polymer-TLR-7/8a conjugates (Poly-7/8a) influence innate immune activation in lymph nodes that drain the site of vaccine administration. The most striking finding was that particle formation by Poly-7/8a strongly enhances the magnitude and duration (>14 days) of innate immune activation in lymph nodes by restricting agonist biodistribution and promoting uptake by dendritic cells. Particle-forming Poly-7/8a optimized for activity were found to induce only local innate immune activation (not systemic) and were effective for eliciting Th1-type CD4 and CD8 T cells that mediated protection against infectious challenge. Based on the importance of particle formation for activity of Poly-7/8a, thermo-responsive Poly-7/8a were developed that exist as single water-soluble macromolecules in solution but undergo temperature-driven particle formation in vivo. In conclusion, polymer carriers of TLR-7/8a represent a versatile and effective platform for modulating innate immune activity and warrant further investigation as a class of adjuvants for vaccines.

Tissue engineers – in order to develop therapies for the treatment of complex neurological injuries and diseases – attempt to recreate elaborate developmental mechanisms in vitro. Neuronal precursor cells are excellent candidates for the study of developmental operations such as cell adhesion, differentiation, and axonal pathfinding. Hyaluronan (HA) is a common polysaccharide that is found extensively throughout the neuronal extracellular matrix (ECM), and can be functionalised and crosslinked to form stable hydrogels that support growing neuronal cells. Hyaluronan hydrogels can be modified chemically and mechanically to mimic the ECM of the developing brain, awarding control over mechanisms such as differentiation and axonal pathfinding. This thesis is concerned with the functionalisation and characterisation of HA hydrogels, ultimately in order to simulate vital properties of the developing brain. Here we show that HA hydrogels can be finely tuned mechanically (by modulating stiffness and viscosity), and chemically, by the conjugation of peptides that mimic the neural cell adhesion molecule (NCAM). NCAM mimics and novel mimics of sialylated NCAM significantly influence the differentiation of NSPCs in 2D and 3D. HA hydrogels successfully support long term culture of neural cells in 3D, and encourage the formation and extension of neurites of several cell types including human, mouse and rat neuronal precursor and stem cells. These results demonstrate for the first time that novel NCAM mimicking peptides can be conjugated to well defined hydrogel matrices that influence intricate developmental behaviours in 3D. Understanding how neural cells form functional networks is essential for the development of clinical approaches that attempt to address the injuries and diseases that affect these systems.

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.

Kinks are functionally important structural features found in the alpha-helices of many proteins, particularly membrane proteins. Structurally, they are points at which a helix abruptly changes direction. Previous kink definition and identification methods often disagree with one another. Here I describe three novel methods to characterise kinks, which improve on existing approaches. First, Kink Finder, a computational method that consistently locates kinks and estimates the error in the kink angle. Second the B statistic, a statistically robust method for identifying kinks. Third, Alpha Helices Assessed by Humans, a crowdsourcing approach that provided a gold-standard data set on which to train and compare existing kink identification methods. In this thesis, I show that kinks are a feature of long -helices in both soluble and membrane proteins, rather than just transmembrane -helices. Characteristics of kinks in the two types of proteins are similar, with Proline being the dominant feature in both types of protein. In soluble proteins, kinked helices also have a clear structural preference in that they typically point into the solvent. I also explored the conservation of kinks in homologous proteins. I found examples of conserved and non-conserved kinks in both the helix pairs and the helix families. Helix pairs with non-conserved kinks generally have less similar sequences than helix pairs with conserved kinks. I identified helix families that show highly conserved kinks, and families that contain non-conserved kinks, suggesting that some kinks may be flexible points in protein structures.

The Protein Data Bank (PDB) is the definitive electronic repository for experimentally-derived protein structures, composed mainly of those determined by X-ray crystallography. Approximately 200 new structures are added weekly to the PDB, and at the time of writing, it contains approximately 97,000 structures. This represents an expanding wealth of high-quality information but there seem to be few bioinformatics tools that consider and analyse these data as an ensemble. This thesis explores the development of three efficient, fast algorithms and software implementations to study protein structure using the entire PDB. The first project is a crystal-form matching tool that takes a unit cell and quickly (< 1 second) retrieves the most related matches from the PDB. The unit cell matches are combined with sequence alignments using a novel Family Clustering Algorithm to display the results in a user-friendly way. The software tool, Nearest-cell, has been incorporated into the X-ray data collection pipeline at the Diamond Light Source, and is also available as a public web service. The bulk of the thesis is devoted to the study and prediction of protein disorder. Initially, trying to update and extend an existing predictor, RONN, the limitations of the method were exposed and a novel predictor (called MoreRONN) was developed that incorporates a novel sequence-based clustering approach to disorder data inferred from the PDB and DisProt. MoreRONN is now clearly the best-in-class disorder predictor and will soon be offered as a public web service. The third project explores the development of a clustering algorithm for protein structural fragments that can work on the scale of the whole PDB. While protein structures have long been clustered into loose families, there has to date been no comprehensive analytical clustering of short (~6 residue) fragments. A novel fragment clustering tool was built that is now leading to a public database of fragment families and representative structural fragments that should prove extremely helpful for both basic understanding and experimentation. Together, these three projects exemplify how cutting-edge computational approaches applied to extensive protein structure libraries can provide user-friendly tools that address critical everyday issues for structural biologists.