The work embodied in this thesis pertains to an attempt to understand better, the molecular basis of protein-carbohydrate recognition. For this purpose a systematic study was undertaken, not only of the energetics of lectin-sugar interactions, which serve as molecular recognition prototype of protein-carbohydrate interactions, but also of the complex effects of solvent water molecules surrounding both the species in solution state. The systems chosen for investigation include the specific recognition of sugars by lectins from diverse families, leguminosae and moraceae. The following salient aspects of the molecular recognition process constitute the focus of this thesis: • Effect of site specifically modified, deoxy-, fluorodeoxy-, or methoxy- substituted D-galactopyranoside binding to lectins. Isothermal titration calorimetric (ITC) investigations of the binding of these sugars to a model lectin permitted the correct prediction of the architecture of the primary binding site in the absence of x-ray crystal or NMR structure of the combining site (Ref. 7). The study provided the only unambiguous means of a site specific mapping of the hydrogen-bond donor- acceptor relationship of the monosaccharide within the primary combining site of the lectin. • Novel features of lectin-sugar recognition. Molecular interactions and forces contributing to the stabilization of the saccharides in the primary combining site of lectins. Binding of site specifically modified fluoro- substituted D- galactopyranosides to WBA I led to the demonstration of the involvement of C- F««»H-0 hydrogen bonds in stabilizing the saccharide within the combining site of lectin (Ref. 7). Implication of the novel C-H«**O hydrogen bonds at the specificity determining C-4 position in enabling the methoxy- substituted D- galactopyranoside to be stabilized within the primary binding site of galactose specific lectins WBA I and jacalin. • Development of a novel coupled osmotic-thermodynamic approach for investigating the role of water molecules in determining the specificity of lectin- sugar interactions. The results obtained led to the first direct demonstration of a differential uptake of water molecules accompanying the specific process of recognition of sugars by lectins (Ref 2) • On the origin of enthalpy-entropy compensation, a ubiquitous phenomenon accompanying the thermodynamics of several ligand binding reactions in aqueous solutions in general and the molecular recognition involving all known lectin-sugar interactions, in particular. The results provide the first unequivocal solution state proof of water reorganization as the source of enthalpy-entropy compensation (Ref 3). A new diagnostic test of a true osmotic effect in molecular recognition phenomena was proposed (Ref. 2) and validated (Ref. 3). As an introduction, Chapter 1 is a comprehensive review of literature that touches upon the diverse properties of lectins and our present understanding of their multifarious roles and applications, which has led to their christening, perhaps appropriately, as molecules that mediate the 'social' functions of cells and tissues. Although a challenge it is still, to decipher the "glycocode", it is apparent that the fundamental basis of the recognition function of lectin-sugar interactions is the initial specific binding of the saccharide molecule by the globular proteinaceous lectin molecule. It is imperative, therefore, that an incisive investigation of the origin of specificity of the binding reaction as well as the solvent effects influencing both the interacting species be undertaken for a better understanding of the complete molecular recognition process. Towards this end is introduced in Chapter 1 our present understanding of the results on lectin-sugar interactions from two complementary approaches viz structural, including X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, as well as thermodvnamic ones, which have provided important information on the architecture of the combining sites, the dynamic modes of saccharide recognition and forces involved therein. Despite a detailed knowledge available from such methods, a structure-energetics correlation has persisted as a current challenge of the field. Towards achieving this goal, studies on the energetics of the recognition of sugars by lectins were undertaken, with an aim to better understand the origin of specificity of lectin-sugar interactions. This thesis attempts to provide new insights on some of the possible lacunae precluding structure-energetics correlation and suggests ways to overcome them. Chapter 2 deals with ITC investigation of the effect of deoxy-, fluorodeoxy-, and methoxy- substitutions on the binding of monosaccharides to the primary combining site of the lectin WBA I isolated from the mature seeds of the leguminosae family member Psophocarpus tetragonolobus as well as the moraceae lectin jacalin. These studies provide valuable information on the hydrogen-bond donor-acceptor relationships within the combining site of the lectins wherein the sugar molecule is liganded with the amino-acid residues of the lectin. This study is relevant for understanding the origin of specificity of monosaccharide binding within the primary combining site of the lectins. It has recently become apparent that there is a predisposition in three-dimensional space, of the donor-acceptor pairs within the sugar binding site of the lectins. Hence there appears to be a stereochemical basis of distinguishing the recognition of the donor group vis-a-vis that of the acceptor group and that their spatial disposition determines the specificity of the saccharide recognition. Unambiguous assignment of which of the groups within the hydrogen bonded pairs is a donor and which one is the acceptor assumes greater importance. The ITC measurements of the binding of deoxy-, flurodeoxy-and methoxy-derivatives of D-galactopyranoside (oc-D-Gal) to the basic lectin from winged bean Psophocarpus tetragonolobus, WBA I revealed that each of the ligands bind to WBA I with the same stoichiometry of one per subunit (29 kDa) of WBA I. The binding enthalpies for various derivatives were essentially independent of temperature and showed complementary changes with respect to binding entropies. Replacement of the hydroxyl group by fluorine or hydrogen on C3 and C4 of the galactopyranoside eliminated their binding to the lectin, consistent with C3-OH and C4-OH acting as hydrogen bond donors. The affinity for C2 derivatives of galactose decreased in the order: GalNAc>2MeOGal>2FGal=Gal>2HGal which suggests that both polar and non-polar residues surround the C2 locus of galactose, consistent with the observed high affinity of WBA I towards GalNAc, where the acetamido group at C2 position is probably stabilized by both non-polar interactions with the methyl-group and polar interactions with the carbonyl group. The binding of C6 derivatives followed the order: Gal>6FGal>D-Fuc»6MeOGal=L-Ara indicating the presence of favourable polar interactions with a hydrogen bond donor in the vicinity. Based on these results the hydrogen bond donor-acceptor relationship of the complexation of methyl-a-D-galactopyranoside with the primary combining site of WBA I was proposed (Ref. /), which was subsequently validated by the crystal structure of methyl-a-D-galactopyranoside complexed with WBA I. This chapter also describes the results from ITC studies on the binding of monosaccharides and disaccharides to the lectin jacalin isolated from the mature seeds of the moraceae family member Artocarpus integrifolia. The novel observation about the existence of C-F*«*H-0 and C-H**»O hydrogen bonds in lectin-sugar interactions is also discussed in this chapter. Chapter 3 is a description of the detailed investigation on the role of water molecules in influencing the energetics of lectin-sugar recognition. A novel coupled osmotic-thermodynamic approach was developed to dissect the role of water molecules in determining the recognition of the sugars by lectins. For this purpose, the model system of mannotriose-concanavalin A was used because atomic level structural information on these complexes were available. The work described in this chapter, is the first solution state evidence for the role of water molecules in the specific interaction of carbohydrates with a legume lectin, concanavalin A (Con A) (Ref. 2). Sugar binding to Con A was accompanied by linear changes in the logarithm of binding constants as a function of neutral osmolyte strength, and were described by well defined negative slopes characteristic for each sugar. As these changes were independent of the chemical nature of the osmolyte used, the results were rationalized in terms of a true osmotic effect. It was demonstrated that the specific recognition of the branched trimannoside (3,6-di-0-(a-D-mannopyranosyl)~a-D-mannopyranoside), the individual dimannosidic arms (3-<9-(a-D-mannopyranosyl)-a-D-mannopyranoside, and 6-0-(a-D-marmopyranosyl)-a-D-mannopyranoside) and the monomeric unit D-mannopyranoside by Con A was accompanied by differential uptake of water molecules; 1,3 and 5 respectively. We also observed a conservation of the compensatory behaviour of binding enthalpies and entropies in the presence as well as absence of osmolytes. This provided the first definitive evidence that water-reorganization plays a direct role in effecting the phenomenon of enthalpy-entropy compensation in protein-ligand interactions in general and lectin-sugar interactions in particular, and that the specificity of lectin-sugar recognition is characterized by a differential uptake of water molecules. Chapter 3 also describes the first experimental identification of the origin of enthalpy-entropy compensation (EEC), a ubiquitous phenomenon accompanying the thermodynamics of multifarious biomolecular recognition processes. By coupling direct microcalorimetry with osmotic stress technique, an experimental handle was devised to test the hypothesis that solvent reorganization could be the source of EEC. The results provided an unequivocal demonstration that an osmotic change in water activity alone, at the same temperature and pH, is sufficient to result in the conservation of EEC during the molecular recognition of specific ligands by macromolecules belonging to thermodynamically diverse and unrelated systems, a compelling evidence that the primary source of EEC in aqueous solutions is attributable to reorganization of solvent water molecules, thus validating the test for the role of water reorganization as a source of EEC (Ref. 3). This provides the first definitive evidence for the notion that there is a direct involvement of water molecules in originating the EEC effect. Despite the generality of the results it is urged that several systems be subjected to a vigorous application of the coupled osmotic-thermodynamic approach proposed herein before constituting it as a proof. Suffice to say, it is perhaps heartening that at last one has a handle to test the role of water molecules in effecting EEC in the solution state and appreciate the diverse roles played by water molecules in mediating molecular recognition reactions. The proposal presented in Ref 2, that the strong isoequilibrium relationship of enthalpy with entropy during the recognition of saccharides by Con A studied under osmotic stress, be considered as diagnostic of a true osmotic effect was subsequently validated in a thermodynamically diverse and unrelated system of peptide recognition by monoclonal antibody, the results from which are discussed in an Appendix (A) to this thesis (Ref 4). That the stabilities of these lectins are not hampered in the presence of osmolytes was demonstrated using differential scanning calorimetry (DSC) (Ref 2). During the course of these DSC studies, we discovered an unusual feature in an animal galectin. Despite possessing the legume lectin fold, the 14-kDa S- type lectin exhibits multiple oligomeric states that are influenced profoundly by complementary ligands and surprisingly do not dissociate at the denaturation temperature. These results are discussed in an Appendix (B) to this thesis (Ref. 5). The general discussion and conclusions drawn from this work are summarized in chapter 4. Briefly, the following salient conclusions can be drawn from the work presented in this thesis: 1. Unambiguous assignment of hydrogen-bond donor-acceptor relationship at each of the hydroxyl group of the monosaccharide bound to the lectin belonging to different families has been demonstrated (Refs. 1,6). 2. First report of novel hydrogen bonds in lectin-sugar interactions such as C- F«MH-0 (Ref 1) and C-H^*O hydrogen bonds (Ref 6). 3. Unusual structural stabilities in a galectin with a fold similar to that in legume lectins but with starkly different thermodynamic stabilities (Ref 5). 4. We have demonstrated for the first time in solution state, that water molecules are involved in the specific recognition of sugars by concanavalin A (Ref 2). It appears that lectin-sugar recognition reactions are, in general, mediated by a net uptake of water molecules during the binding process (Ref 7). 5. We have provided the first experimental demonstration that reorganization of water molecules is the source of enthalpy-entropy compensation in molecular recognition processes (Ref 3). 6. We provide evidence for another facet in the recognition of antigens by antibodies, viz water release accompanying the binding reaction (Ref 4). The studies reported in this thesis provide the foundation for embarking on a systematic study not only of the origin of specificity of lectin-sugar recognition but also of the complex roles that water molecules play in mediating these molecular recognition processes. These specific binding reactions wherein non-linear thermodynamics predominates and precludes a direct structure-energetics correlation emphasize the need to account for the effect of solvent water molecules in lectin-sugar interactions in particular and, without any overemphasis, in molecular recognition processes in general.

Carnitine is an essential metabolite in the human body. It carries out several roles in human metabolism, including that in fatty acid metabolism. γ-Butyrobetaine hydroxylase (BBOX) is an Fe(II) and 2-oxoglutarate dependent oxygenase, which catalyses the final step of carnitine biosynthesis, i.e. hydroxylation of γ-butyrobetaine (GBB) to carnitine. Inhibition of BBOX has potential in the treatment for cardiovascular diseases. The work described in this thesis focussed on mechanistic and inhibition aspects of BBOX catalysis. Firstly, a set of analytical tools for BBOX activity measurements was developed. The synthesis of fluorinated substrate analogues provided the basis for development of two assays for use in vitro with the isolated protein and in lysates, with detection by fluorescence or ¹⁹F NMR, respectively. Furthermore, the use of ¹⁹F NMR to monitor protein-ligand interactions was exemplified with the work on metallo-β-lactamases. The developed fluoride-release assay was then used to screen a library of small molecules and led to recognition of scaffolds with potential applications as inhibitors. Further structure-activity relationship studies led to the identification of potent BBOX inhibitors, which were then evaluated for their activity in cells. The crystal structure of human BBOX with one of the lead inhibitors revealed that BBOX can undergo significant conformational changes, involving a movement of an active site loop. BBOX conformational flexibility may have a role in the GBB mediated substrate inhibition observed both with isolated protein and in cells. In addition to the mechanistic and functional studies, the potential of BBOX as a biocatalytic tool was examined. BBOX has been shown to catalyse a hydroxylation of the symmetrical dialkyl piperidine carboxylic acids, leading to formation of up to three stereocentres in one reaction. In the last part of this work properties of human BBOX were compared to BBOX from Pseudomonas sp. AK1, revealing differences in kinetic behaviour and substrate specificity. Novel substrates for bacterial BBOX were identified. Pseudomonas sp AK1 BBOX was shown to hydroxylate amino acid analogues leading to formation of 1,2-amino alcohols.

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disease resulting in episodic but progressive extraskeletal bone formation. FOP is caused by missense mutations in the cytoplasmic domain of the type I bone morphogenetic protein (BMP) receptor ACVR1, leading to dysregulated activation. Currently there are no available drug treatments and the structural mechanism of mutant activation is still poorly characterised. To address this, a number of BMP and TGFβ receptors, including FOP mutants of ACVR1 were cloned, expressed and purified for both structural and biophysical experiments. The arginine at the site of most recurrent FOP mutation, R206H, is common across all type I receptors except BMPR1A and BMPR1B which have a lysine at this site. The novel structure of BMPR1B differed to wild-type ACVR1 showing some of the conformational changes expected of the active conformation. However, a variety of disease related ACVR1 mutant structures, including ACVR1 R206H, revealed a surprisingly persistent inactive conformation in the kinase domain. Some conformational changes suggestive of activation were observed in the mutant Q207D affecting the ATP pocket, the β4–β5 hairpin and the activation loop. Additionally, the structure of the Q207E mutant showed a slight release of the regulatory glycine-serine rich domain from its inhibitory position. These subtle changes suggest that the mutant inactive conformation is destabilised and potentially more dynamic. In agreement, all of the ACVR1 mutants showed reduced binding to the inhibitory protein FKBP12. However, mutant phosphorylation of the substrate Smad1 was not constitutive, but dependent on the co-expression of the partner ACVR2, consistent with recent evidence from transgenic knock-out mice. A novel 2-aminopyridine inhibitor scaffold with favourable specificity for ACVR1 was identified using a fluorescence-based thermal shift assay. Further derivatives were characterised with improved potency and selectivity. The crystal structures of ACVR1 bound to these inhibitors showed exquisite shape complementarity, contributing to their favourable specificity. This work has increased the understanding of FOP-associated mutant activation and provided a novel starting scaffold for potential drug development.

Foi levado a efeito estudo sobre formas múltiplas de aldolase de músculo de coelho. A enzima foi purificada a pH 7,5 por eluição com substrato a partir de coluna de fosfocelulose. A enzima foi ainda cristalizada por diálise dessas preparações contra solução saturada de sulfato de amônio. Formas múltiplas de aldolase foram obtidas por fracionamento a diferentes pI por eletrofocalização em gradiente de Ampholine na faixa de pH entre 7,0 a 10,0. Nessas condições foram separados cinco híbridos resultantes da associação ao acaso das sub-unidades &#945; e &#946;, os quais foram analisados em estado de dissociação a partir de proteínas carboximetiladas e separadas por eletroforese em gel de poliacrilamida na presença de uréia 8M. Foi também estudado o aparecimento de sub-unidade &#945; e &#946; em músculo de coelhos de idades que variavam de 1 a 240 dias, verificando-se que em coelhos de 1 dia existia apenas a proteína &#945;4, surgindo sub-unidades &#946; já em animais de 10 dias. / Not available.

The proton dependent oligopeptide transporters (POT family) are members of the Major Facilitator Superfamily of secondary active transporter proteins. They use the transmembrane proton gradient to drive the uptake of di- and tripeptides into the cytoplasm. Members of the family are highly conserved in pro- and eukaryotic genomes, and in humans they are responsible for the oral absorption of many drug families, including -lactam antibiotics. Recently, the crystal structures of PepTSo and PepTSt, two prokaryotic homologues of the human proteins PepT1 and PepT2, captured the proteins in two distinct conformations, providing insight into the structural aspects of the transport mechanism. A protocol was designed for functional liposome reconstitution of POT proteins, and transport assays were conducted to characterise their substrate specificity, pH dependence and kinetic properties. Using site-directed mutagenesis, we identified binding site residues involved in peptide recognition and proton translocation, and distinguished between the two roles by comparing protein activity in proton- and peptide-driven conditions. We also investigated the roles of key residues in the conformational transitions that accompany the transport cycle, using data from biochemical assays, molecular dynamics simulations and modeling, as well as electron paramagnetic resonance measurements. In addition, several bacterial POT members were screened for crystallisation, in order to assess their stability and crystal diffraction quality in different detergents. Further work was performed with bacterial POT homologues YdgR and GkPOT, including binding studies using NMR spectroscopy and assaying drug transport in vivo and in vitro. Together, the data establish bacterial POTs as model systems for studying the mammalian oligopeptide transporters, and a mechanistic model for peptide transport is proposed.

Correct functioning of the brain relies upon the precise connectivity between the billions of neurons that make up this crucial organ. Aberrations in the formation of these elaborate neural networks lead to neurodegenerative and neuropsychiatric disorders. A synapse-spanning molecular triad, involving members of the Neurexin, Cbln and ionotropic glutamate receptor delta families of proteins, is crucial for the accurate formation and proper function of synapses in the cerebellum. This trans-synaptic complex has been implicated in the molecular mechanisms behind motor control and motor learning, and furthermore individual members have been linked to diseases such as Alzheimer’s, autism spectrum disorders and schizophrenia. The major findings presented in this thesis include: crystal structures of the amino-terminal domains (ATD) of the two members of the ionotropic glutamate receptor delta (iGluR-Delta) family, functional characterisation of the effects of disrupting the ATD interface in one member of the iGluR-Delta family, a crystal structure of the C1q domain of Cbln1, biophysical analysis of the molecular interactions within the Neurexin-Cbln1-GluD2 trans-synaptic complex, as well as evidence for the domain arrangement of the ecto-domain of the iGluR-Delta proteins. Together, these data enhance our knowledge of the molecular details of this macro-molecular complex and provide evidence to support models for the mechanisms of their involvement in synapse formation and function, thereby making a contribution to the vast and medically relevant field of molecular neurobiology.

In the post-genomic era, recognition of the importance of sugars is increasing in biological research. For the precise analysis of their functions, homogeneous materials are required. Chemical synthesis is a powerful tool for preparation of homogeneous oligosaccharides and glycoconjugates. Glycosidases are potent catalysts for this purpose because they realize high stereo- and regio- selectivities under conditions benign to biomolecules without repetitive protection/deprotection procedures. A glycosynthase is an aritificial enzyme which is derived from a glycosidase and is devised for glycosylation reaction. To suppress the mechanistically inherent oligomerization side reaction of this class of biocatalysts, a glycosidase with plastic substrate recognition was engineered to afford the first α-mannosynthase. This novel biocatalyst showed low occurrence of oligomerized products as designed and was applied to prepare a wide range of oligosaccharides. Glycosidases are also valuable tools for glycan engineering of glycoconjugates, which is a pivotal issue in the development of pharmaceutical agents, including immunoglobulin G (IgG)-based drugs. EndoS, an endo-β-N-acetylglucosaminidase from Streptococcus pyogenes, natively cleaves N-glycans on IgG specifically. When the latent glycosylation activity of this enzyme was applied, the N-glycan remodelling of full-length IgG was successfully achieved for the first time and a highly pure glycoform was obtained using the chemically synthesized oxazoline tetrasaccharide as glycosyl donor. This biocatalytic reaction allows development of a novel type of antibody-drug conjugates (ADCs) in which drug molecules are linked to N-glycans site-specifically. For this purpose, glycans with bioorthogonal reaction handles were synthesized and conjugated to IgG. A model reaction using a dye compound as reaction partner worked successfully and the synthetic method for this newly designed ADC was validated. Glycan trimming of glycoproteins expressed from Pichia pastoris was performed using exoglycosidases to derive homogeneous glycoform. Jack Bean α-mannosidase (JBM) trimmed native N-glycans down to the core trisaccharide structure but some of the glycoforms were discovered to be resistant to the JBM activity. Enzymatic analyses using exoglycosidases suggested that the JBM-resistant factor was likely to be β-mannoside. In summary, this work advanced application of modified glycosidases for preparation of oligosaccharides and also demonstrated biocatalytic utility of glycosidases to produce biologically relevant glycoconjugates with homogeneous glycoforms.

The Streptomyces avidinii protein streptavidin binds the small molecule biotin (vitamin H / B₇) with extraordinary stability, resulting in the streptavidin-biotin interaction being one of the strongest non-covalent interactions known in nature (K_d ~ 10^-14 M). The stable and rapid biotin-binding, together with high resistance to heat, pH and proteolysis, has given streptavidin huge utility, both in vivo and in vitro. Accordingly, streptavidin has become a widely used tool in many different biotechnological applications. Streptavidin has also been the subject of extensive research efforts to glean insights into this paradigm for a high-affinity interaction, with over 200 mutants of the protein reported to date. Despite the high stability of the streptavidin-biotin interaction, it can and does fail under certain experimental conditions. For example, streptavidin-biotin dissociation is accelerated by an increased temperature or lower pH (conditions often encountered in cellular imaging experiments), and by mechanical stress, such as the shear force arising from fluid flow (encountered when streptavidin is used as a molecular anchor in biosensor chips and arrays). This study details efforts made at increasing further the utility of streptavidin, by increasing the stability of biotin and biotin-conjugate binding. A rational site-directed mutagenesis approach was used to create 27 mutants, with eight of these mutants possessing higher-stability biotin-binding. The most stable biotin-binding mutant was named traptavidin and was extensively characterised. Kinetic characterisation revealed traptavidin had a decreased dissociation rate from biotin and biotin-conjugates when compared to wildtype streptavidin, at both neutral pH and pH 5. Atomic force microscopy and molecular motor displacement assays revealed the traptavidin-biotin interaction possessed higher mechanical stability than the streptavidin-biotin interaction. Cellular imaging experiments revealed the non-specific cell binding properties of streptavidin were unchanged in traptavidin. X-ray crystallography was also used to generate structures of both apo- and biotinbound traptavidin at 1.5 Å resolution. The structures were analysed in detail and compared to the published structures of streptavidin, revealing the characteristics of traptavidin arose from the mutations stabilising a flexible loop over the biotin-binding pocket, as well as reducing the conformational change on biotin-binding to traptavidin. Traptavidin has the potential to replace streptavidin in many of its diverse applications, as well as providing an insight into the nature of ultra-stable noncovalent interactions.

Understanding the factors influencing the ET characteristics of redox proteins confined at an electrochemical interface is of fundamental importance from both pure (fundamental science) and applied (biosensory) perspectives. This thesis reports on progress made in the emerging field of coupled electrochemical characterization and optical imaging in moving the analysis of redox-active films to molecular scales. More specifically the combination of cyclic voltammetry and wide-field Total Internal Reflection (TIRF) microscopy, here named ‘Fluorescent Cyclic Voltammetry’ (FCV), was applied to monitoring the response of surface-confined redox active proteins at submonolayer concentrations. The combined submicrometre spatial resolution and photon capture efficiency of an inverted TIRF configuration enabled the redox reactions of localized populations of proteins to be directly imaged at scales down to a few hundreds of molecules. This represents a 6-9 orders of magnitude enhancement in sensitivity with respect to classical current signals observed in bioelectrochemical analysis. Importantly, measurements of redox potentials at this scale could be achieved from both natural and artificially designed bioelectrochemical fluorescent switches and shed fundamental light on the thermodynamic and kinetic dispersion within a population of surface confined metalloproteins. The first three chapters of this thesis provide an overview of the relevant literature and a theoretical background to both the rapidly expanding fields of electroactive monolayers bioelectrochemistry and TIRF imaging. The initial design and construction of a robust electrochemically and optically addressable fluorescent switch, crucial to the applicability of FCV is reported in chapter 5. The generation of optically transparent, and chemically modifiable electrode surfaces suitable for FCV are also described. Chapter 6 describes the response of the surface confined azurin-based switch. Analysis of the spatially-resolved redox reaction of zeptomole samples in various conditions enables the mapping of thermodynamic dispersion across the sampled areas. In chapter 7 the newly developed FCV detection method was extended to investigate more complex bioelectrochemical systems containing multiple electron transferring redox centres and responding optically at different wavelengths. This approach provides a platform for spectral resolution of different electrochemical processes on the same sample. Finally in chapter 8 an electrochemical procedure is proposed for investigating the kinetic response of redox proteins using a fundamentally new methodology based on interfacial capacitance. In using variations in the surface chemistry to tune the rate of electron transfer, the approach was shown to be a robust and facile means of characterising redox active films in considerably more detail than possible through standard electrochemical methodologies. Ultimately, it can be applied to probe dispersion within protein populations and represents a powerful means of analysing molecular films more generally.

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.