This thesis investigates the mechanisms behind natural silk spinning by integrating rheology, spectroscopy and small angle scattering to better understand this process and to guide our efforts towards mimicking Nature’s ways of producing high performance fibres. As a result of natural selection, arthropods such as spiders and moths have evolved the ability to excrete silk proteins in a highly controlled manner. Spun from liquid feedstocks, silk fibres are used ex vivo to build structures with mechanical properties currently unmatched by industrial filaments. As yet, relatively little attention has been directed to the investigation of spinning under biologically relevant conditions. To better understand how and why silk is spun, this thesis bridges the gap between liquid silk flow properties and structure development. To directly connect the two, I have developed and deployed novel experimental platforms that combine infrared spectroscopy and small angle scattering with rheology. This approach has clarified long-standing ambiguities on the structural root of silk’s apparently complex flow properties. Small angle scattering revealed the length scales involved in the flow induced solidification under a range of spinning conditions. Mo reover, infrared spectroscopy offered a unique perspective into silk’s formation process immediately after excretion. In a similar manner to the post-extrusion tuning of the properties of partly solidified spider silk filaments, this thesis has revealed that silkworm silk fibres are far from completely formed once excreted. One might describe the filaments of mulberry silkworm as seeded molten polymers that form its hydrogen bonding network and crystallises slowly on site. Consequently, it enlightens that post-spinning conditions are equally paramount for silkworm silk, giving an explanation for the relatively poorer mechanical properties. The comparison of silks from a range of species, allowed this hypothesis to be extended to wild silkworm silk. My insights into spinning had the fortuitous repercussion of facilitating silk fibre solubilisation leading to the development of better artificial silk feedstocks flowing like native silks. With these findings, I believe we are now in an improved position to conceive artificial fibres with properties rivalling those of Nature.

RNA-dependent RNA polymerases (RdRps) play a vital role in the life cycle of RNA viruses, being responsible for genome replication and mRNA transcription. In this thesis viral RdRps (vRdRps) of dsRNA bacteriophage phi6 (phi6 RdRp) and Severe Acute Respiratory Syndrome (SARS) coronavirus [non structural protein 12 (NSP-12)] are studied. For SARS polymerase NSP-12, a library-based screening method known as ESPRIT (Expression of Soluble Protein by Random Incremental Truncation) was employed in an attempt to isolate domains of NSP-12 that express solubly in Escherichia coli (E. coli) and are thereby suitable for structural studies. This experiment identified for the first time in a systematic fashion, conditions under which the SARS polymerase could be solubly expressed at small scale and allowed mapping of domain boundaries. Further experiments explored different approaches for increasing expression levels of tractable fragments at large scale. Bacteriophage phi6 RdRp is one of the best studied vRdRps. It initiates RNA synthesis using a de novo mechanism without the need for a primer. Although formation of the de novo initiation complex has been well studied, little is known about the mechanism for the transition from initiation to elongation (i.e. extension of an initiated dinucleotide daughter strand). In the phi6 RdRp initiation complex the C-terminal domain (CTD) blocks the exit path of the newly synthesised dsRNA which must be displaced for the addition of the third nucleotide. The crystal structure of a C-terminally truncated phi6 RdRp (P2T1) reveals the strong non-covalent interactions between the CTD and the main body of the polymerase that must be overcome for the elongation reaction to proceed. Comparing new crystal structures of complexes of both wild-type (WT) and a mutant RdRp (E634 to Q, which removes a salt-bridge between the CTD and main body of the polymerase) with various oligonucleotides (linear and hairpin), nucleoside triphosphates (NTPs) and divalent cations, alongside their biophysical and biochemical properties, provides an insight into the precise molecular details of the transition reaction. Thermal denaturation experiments reveal that Mn2+ acquired from the cell and bound at the phi6 RdRp non-catalytic ion site sufficiently weakens the polymerase structure to facilitate the displacement of the CTD. Our crystallographic and biochemical data also indicate that Mn2+ is released during this displacement and must be replaced for the elongation to proceed. Our data explain the role of the non-catalytic divalent cation in vRdRps and pinpoint the Mn2+-dependent step in viral replication. In addition, by inserting a dysfunctional Mg2+ at the non-catalytic ion site for both WT and E634Q RdRps we captured structures with two NTPs bound within the active site in the absence of Watson-Crick base pairing with template and could map movements of divalent cations during preinitiation through to initiation. Oligonucleotides present on the surface of phi6 RdRp allowed mapping of key residues involved in template entry and unwinding of dsRNA; these preinitiation stages have not been observed previously. Considering the high structural homology of phi6 RdRp with other vRdRps, particularly from (+)ssRNA hepatitis C virus (HCV), insights into the mechanistic and structural details of phi6 RdRp are thought to be relevant to the general understanding of vRdRps.

Precise regulation of cell cycle events by the Cdk-control network is essential for cell proliferation and the perpetuation of life. The unidirectionality of cell cycle progression is governed by several critical irreversible transitions: the G1-to-S transition, the G2-to-M transition, and the M-to-G1 transition. Recent experimental and theoretical evidence has pulled into question the consensus view that irreversible protein degradation causes the irreversibility of those transitions. A new view has started to emerge, which explains the irreversibility of cell cycle transitions as a consequence of systems-level feedback rather than of proteolysis. This thesis applies mathematical modelling approaches to test this proposal for the Mto- G1 transition, which consists of two consecutive irreversible substeps: the metaphase-to-anaphase transition, and mitotic exit. The main objectives of the present work were: (i) to develop deterministic models to identify the essential molecular feedback loops and to examine their roles in the irreversibility of the M-to-G1 transition; (ii) to present a straightforward and reliable workflow to translate deterministic models of reaction networks into stochastic models; (iii) to explore the effects of noise on the cell cycle transitions using stochastic models, and to compare the deterministic and the stochastic approaches. In the first part of this thesis, I constructed a simplified deterministic model of the metaphase-to-anaphase transition, which is mainly regulated by the spindle assembly checkpoint (the SAC). Based on the essential feedback loops causing the bistability of the transition, this deterministic model provides explanations for three open questions regarding the SAC: Why is the SAC not reactivated when the kinetochore tension decreases to zero at anaphase onset? How can a single unattached kinetochore keep the SAC active? How is the synchronized and abrupt destruction of cohesin triggered? This deterministic model was then translated into a stochastic model of the SAC by treating the kinetochore microtubule attachment at prometaphase as a noisy process. The stochastic model was analyzed and simulation results were compared to the experimental data, with the aim of explaining the mitotic timing regulation by the SAC. Our model works remarkably well in qualitatively explaining experimental key findings and also makes testable predictions for different cell lines with very different number of chromosomes. The noise generated from the chemical interactions was found to only perturb the transit timing of the mitotic events, but not their ultimate outcomes: all cells eventually undergo anaphase, however, the time required to satisfy the SAC differs between cells due to stochastic effects. In the second part of the thesis, stochastic models of mitotic exit were created for two model organisms, budding yeast and mammalian cells. I analyzed the role of noise in mitotic exit at both the single-cell and the population level. Stochastic time series simulations of the models are able to explain the phenomenon of reversible mitotic exit, which is observed under specific experimental conditions in both model organisms. In spite of the fact that the detailed molecular networks of mitotic exit are very different in budding yeast and mammalian cells, their dynamic properties are similar. Importantly, bistability of the transitions is successfully captured also in the stochastic models. This work strongly supports the hypothesis that uni-directional cell cycle progression is a consequence of systems-level feedback in the cell cycle control system. Systems-level feedback creates alternative steady states, which allows cells to accomplish irreversible transitions, such as the M-to-G1 transition studied here. We demonstrate that stochastic models can serve as powerful tools to capture and study the heterogeneity of dynamical features among individual cells. In this way, stochastic simulations not only complement the deterministic approach, but also help to obtain a better understanding of mechanistic aspects. We argue that the effects of noise and the potential needs for stochastic simulations should not be overlooked in studying dynamic features of biological systems.

Many bacteria use inorganic nitrogen and sulfur compounds for energy metabolism. These compounds are often toxic and so bacteria must adapt to survive their deleterious effects. Bacteria use specific proteins in order to metabolise, sense and detoxify these compounds. In this thesis protein interactions with inorganic nitrogen and sulfur compounds are examined at the mechanistic level. Intermediates in the Sox sulfur oxidation pathway are covalently attached to a cysteine on the swinging arm of the substrate carrier protein SoxYZ. An interaction between the Sox pathway enzyme SoxB and the carrier protein SoxYZ is demonstrated. A crystal structure of a trapped SoxB-SoxYZ complex at 3.3 Å resolution identifies two sites of interaction, one between the SoxYZ carrier arm and the SoxB active site channel and the other at a patch distal to the active site. The presence of a distal interaction site suggests a mechanism for promiscuous specificity in the protein-protein interactions of the Sox pathway. Using biophysical methods it is shown that SoxB distinguishes between the substrate and product forms of the carrier protein through differences in interaction kinetics and that the carrier arm-bound substrate group is able to out-compete the adjacent C-terminal carboxylate for binding to the SoxB active site. The thiosulfate dehydrogenase TsdA has an unusual His/Cys coordinated heme. TsdA catalyses oxidative conjugation of two thiosulfate molecules to form tetrathionate. Mass spectrometry and UV/visible spectroscopy are used to identify an S-thiosulfonate reaction intermediate which is covalently attached to the cysteine heme ligand. A catalytic mechanism for TsdA is proposed using a crystal structure of TsdA at 1.3 Å resolution alongside site-directed mutagenesis of active site residues. Nitric oxide is produced by the mammalian immune response to kill bacterial pathogens. Part of the killing mechanism occurs through the reaction of nitric oxide with protein-bound iron-sulfur clusters. However, the same type of reaction is also exploited by nitric oxide-sensing bacterial proteins. An infrared spectroscopy approach is developed to detect the products of iron-sulfur protein nitrosylation. Using this methodology it is shown that the presence of trace O2 strongly impacts which products are formed in these nitrosylation reactions. These observations are of physiological relevance because bacteria are often exposed to NO under aerobic conditions during an immune response.

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.

Allergic reactions are mainly mediated by the interactions between the IgE and its ligands, amongst them CD23 and CD21 in what is termed the IgE network. CD23 is involved in upregulating IgE expression by forming a trimolecular complex with CD21 and IgE on the B-cell surface, resulting in the specific activation of IgE-positive B cells. CD21 also interacts with C3d and is a bridge between the innate and the immune system. A crystal structure of the interaction has been solved (Szakonyi et al., 2001) but was controversial because it contradicted previous biochemical analyses. The aims of this thesis were to use various biophysical techniques to study the interactions between the molecules in the IgE network and its possible inhibition. Part 1: Characterisation of a phage display-derived peptide that inhibit IgE binding to CD23 A peptide was previously derived using phage display technology and tested for binding ability to CD23 using SPR and ITC. Subsequent NMR experiments were performed to identify the binding site, followed by characterization of its derivatives. Crystallisation of CD23 with the peptide and soaking with its truncated tripeptide, NWP, were also attempted. Part 2: Characterisation of CD23 and its interaction with its ligands X-ray crystallography was undertaken to solve the structure of derCD23 in complex with a phage display-derived peptide (Part1) followed by crystal soaking with a truncated tripeptide, NWP. However, a reproducible, high-resolution wild type derCD23 structure was determined at 1.9 Å. A comparison of the binding behaviour between the monomeric derCD23 and a trimeric CD23 construct was carried out in order to see the effect of oligomerisation upon IgE binding. Using the known interaction map as well as a crystal structure, the possible interacting residues between CD23 and IgE were examined. The characterisation of the CD23/CD21 interaction was continued from previous efforts in order to confirm that the binding epitope of CD23 for CD21 lies within the C-terminus of CD23. Characterisation of the interactions of CD23/IgE/FcεRI was performed to examine these multimolecular interactions and possible regulatory mechanisms in mast cell degranulation. It was shown that CD23 can form multimeric complexes with IgE-Fc that bind to FcεRI with higher apparent affinity than IgE-Fc alone, which may lead to increases in mast cell degranulation. It was also found that the IgE bound on FcεRI still binds to CD23 although with a lower binding capacity, presumably due allosteric changes. The binding of CD23 with a monoclonal antibody IDEC-152 was also characterised using SPR and NMR spectroscopy. It was proposed that IDEC-152 might interfere with the trimerisation site of CD23 thus reducing its affinity for IgE. A thermofluor assay was developed and optimised for potential screening of compounds that bind to derCD23 using a qPCR machine, which may be useful to screen compounds that bind to CD23 as part of future drug discovery project. Crystallisation of the derCD23/CD21 and IgE/triCD23/CD21 complexes was also attempted as part of ongoing crystallisation projects. Part 3: The interaction between C3d and CD21 The interaction between C3d and CD21 is believed to be a bridge between the innate and adaptive immune response, and is thought to be pivotal in the initiation of autoimmune disease. Following from previous studies on this interaction, further characterisations were performed using NMR and ITC to confirm the involved sites on CD21 (SCR1-2) in binding to C3d. Several potential salt bridges have been identified so far, allowing a high-resolution docked structure of the C3d/CD21 complex.

Membrane proteins play a key role in numerous physiological processes such as transport, energy transduction in respiratory and photosynthetic systems, and signal transduction, and are of great pharmaceutical interest, comprising more than 60% of known drug targets. However, crystallisation of membrane proteins, and G protein-coupled receptors (GPCRs) in particular, still relies heavily on the use of protein engineering strategies, which have been shown to hamper protein activity. Here, a range of biophysical methods were used to study the structure and function of two membrane proteins, a prokaryotic peptide transporter, PepT_So and a GPCR, neurotensin receptor 1 (NTS1), using different membrane reconstitution methods to study the proteins in a native-like environment. Firstly, using the pulsed electron paramagnetic resonance (EPR) method of double electron-electron resonance (DEER) the conformation of PepT_So reconstituted into lipid bilayers was assessed and compared to previous structural data obtained from crystallography and modelling. The influence of the membrane potential and the presence of substrate on the conformational heterogeneity of this proton-coupled transporter were investigated. Secondly, NTS1 purification was optimized for biophysical study. Cysteine mutants were created and a labelling protocol was developed and optimized for fluorophore and nitroxide labelling studies. NTS1 was then studied by continuous-wave EPR, to assess the influence of ligand on local protein dynamics, and to assess the structure of a receptor segment known as helix 8, that was proposed to be an α-helix, but was only observed to be helical in one of the NTS1 crystallographic studies. Ensemble and single-molecule Förster resonance energy transfer (FRET), and DEER were combined to study the dimerisation behaviour of NTS1, showing novel dynamics of the interfacial associations. Finally, the signalling mechanism of NTS1 was also investigated using microscale thermophoresis (MST) to assess the affinity of the receptor for G protein in vitro in the absence of ligand, or in the presence of agonist or antagonist. MST measurements were performed in detergent and in nanodiscs of different lipid compositions, to assess the influence of the lipid environment on receptor function. In summary, this thesis demonstrates the potential of biophysical techniques to study various aspects of membrane protein structure and function in native-like lipid systems, complementing e.g. structural data obtained from crystallographic studies with functional data for membrane proteins in more native environments, as well as shedding light on protein dynamics. The work presented here provides novel insights into PepTSo transport, and in particular into NTS1 structure, signalling, and oligomerisation, opening up several avenues for future research.

The Type Three Secretion Systems (T3SSs) of Gram-negative bacteria, including Shigella, Salmonella, and Enteropathogenic/Enterohaemorrhagic Escherichia coli (EPEC/EHEC), pass virulence factors directly into the host to mediate invasion. Prior to secretion down the narrow T3SS channel, effector proteins associate with chaperone proteins. The binding enables the T3SS to keep effectors soluble and partially unfolded for secretion. In the first part of this thesis, the association of one promiscuous chaperone, Spa15 of Shigella flexneri, with three of its cognate effectors has been studied. In addition to the role this plays in secretion, the binding of one particular substrate leads to Spa15 being involved in the regulation of the T3SS. The oligomerisation and impact of substrate binding upon Spa15 has been determined by crystallography and EPR. Once secreted, T3SS effectors subvert the host cytoskeleton for the benefit of the bacteria. Soluble homologues of Spa15 effectors from EHEC and Salmonella have been purified, and their interactions with host GTPases which lead to stress fibre phenotypes observed. The Twin Arginine Translocation (Tat) pathway provides a contrasting view of bacterial secretion. Instead of preventing folding in the cytoplasm, it is a criterion of transport that the protein be folded. One of the reasons for internal folding is the necessity to insert cofactors which could not be incorporated externally. In the second part of this thesis, a protein which exemplifies this necessity is studied. This is PhoD, the model protein for Tat export from Bacillus subtilis. PhoD is an alkaline phosphodiesterase expressed to scavenge phosphate in times of phosphate deficiency. The structure of PhoD has been solved, and the protein is shown to be able to cleave a component of its own cell wall. It uses an unusual catalytic site more reminiscent of the eukaryotic purple acid phosphatases than of other currently known alkaline phosphatases. Furthermore this site appears to require metal binding before export from the bacterial cytoplasm.

The 2OG oxygenases comprise a superfamily of ferrous iron dependent dioxygenases with multiple biological roles, including in hypoxia sensing, transcriptional control, and splicing control. It was recently proposed that 2OG oxygenases catalyse the hydroxylation of ribosomal proteins in prokaryotes (ycfD) and in humans (NO66 and MINA53), raising the possibility that 2OG oxygenases also control translation. The work described in this thesis concerned investigations on the biochemical and functional aspects of prokaryotic and mammalian ribosomal protein hydroxylases (ROX) in vitro and in cells. An efficient chromatographic system linked to mass spectrometric analysis (LC-MS) was developed for studying the masses of individual ribosomal proteins (>90% coverage of ribosomal proteome) to ±1 Da accuracy. It was demonstrated that ycfD catalyses the hydroxylation of R81 on L16 in E. coli, in a manner dependent on atmospheric oxygen levels. YcfD deletion results in growth phenotype at low temperatures and in minimal medium, and in decreased global translation rates in minimal medium; ycfD deletion does not affect translational accuracy and ribosome assembly. Furthermore, ycfD-deletion results in increased sensitivity to the antibiotics chloramphenicol and lincomycin. Consistent with a 2OG-oxygenase mediated mechanism of antibiotic resistance, chloramphenicol sensitivity of the E. coli wild-type strain could be increased by inhibiting the activity of ycfD by removing co-factors required for catalytic activity (Fe(II) and O2), and, at least in part, by using a ycfD inhibitor, IOX1, which inhibits ycfD with IC₅₀ of 38 μM in vitro. The therapeutic potential of a post-translational modification mediating antibiotic resistance provides an opportunity for medicinal targeting of ribosome-modifying enzymes, for example ycfD, which may be more ‘druggable’ than the ribosome itself. In co-treatment with an existing antibiotic, such as chloramphenicol, a small molecule inhibitor would achieve a potentiated antibiotic effect. Structural aspects of ROX hydroxylation were pursued by characterising a thermophilic ROX-substrate complex; a ycfD homologue was identified in the thermophilic bacterium Rhodothermus marinus and shown to be a thermophilic 2OG oxygenase ycfD_RM, acting on R82 of ribosomal protein L16_RM. The activity of ycfD_RM in cells was limited at high growth temperature and oxygen solubility was demonstrated as a likely limiting factor of ycfD_RM activity, thus identifiying a potential 2OG oxygenase oxygen sensor in prokaryotes. A crystal structure of ycfD_RM in complex with L16RM substrate fragment was determined to 3.0 Å resolution. Structural analyses suggested that ycfD_RM contains 30% more hydrophobic interactions and 100% more salt-bridge interactions than ycfD_EC, suggesting that these interactions are important for thermal stabilisation of ycfD_RM. The structures reveal key interactions required for binding of ribosomal proteins. Substantial structural changes were observed in the presence of the substrate fragment, which implies induced-fit binding of the L16_RM substrate. The work has informed further structural studies on the evolutionarily related human ROX, NO66 and MINA53, for which substrate structures have been obtained since the completion of the work. The LC-MS analysis of ribosomal proteins was extended to mouse and human cells to demonstrate that the human ROX homologue of ycfD, MINA53, hydroxylates the 60S ribosomal protein rpL27a in cells. It was demonstrated that rpL27a hydroxylation is widespread and found in all mouse organs analysed, as well as in cancer cell lines and in clinical cancer tissues. A partial or complete reduction of rpL27a hydroxylation was observed in a number of clinically identified MINA53 mutations from the COSMIC database of cancer mutations. Structural analysis suggested that mutations occur more frequently at structurally important regions of MINA53, including the βIV-βV insert in the core fold of MINA53. The identification of inhibiting clinical mutations suggests that rpL27a hydroxylation level could be used as a cancer mark, and in the future for selective inhibition by ribosomal antibiotics. The work presented in this thesis demonstrates that it is possible to selectively inhibit modified ribosomes; an inhibitor of unhydroxylated rpL27a could therefore, at least in principle, be active against the sub-set of tumours with inactivating mutation(s) of MINA53, but not normal tissue. Future work should therefore focus on identifying a selective inhibitor of unhydroxylated eukaryotic ribosomes which could be applied for treatment of cancers harbouring deactivating MINA53 mutations. The same approach could be applied to other ribosome modifications (to rRNA, ribosomal proteins, and ribosome-associate factors) that are different in cancer compared to normal cells.

The bacteria Streptococcus pyogenes produces a multitude of proteins which interact with and alter the functions of the host immune system. Two such proteins, Endoglycosidase S (EndoS) and Immunoglobulin G-degrading enzyme from S. pyogenes (IdeS) are able to specifically alter the effector functions of immunoglobulin G (IgG). EndoS is a glycoside hydrolase which removes the conserved <i>N</i>-linked glycan from IgG Fc whereas IdeS is a cysteine protease that cleaves the exible protein hinge of IgG. The activity of both proteins results in the reduced ability of IgG to elicit immune responses through Fc receptor binding and complement activation. Amongst other applications, both EndoS and IdeS are actively being explored as new therapeutics for IgG-mediated autoimmune diseases. Given the therapeutic potential of EndoS and IdeS, experiments were designed to investigate the structural and functional characteristics of these enzymes in an effort to understand their specficity for and activity against IgG. Here, bioinformatic and biophysical characterisation of EndoS identified subdomains outside of the catalytic domain which contribute to glycoside hydrolase activity. The substrate specificity of EndoS was also explored and showed that EndoS hydrolyses a broad range of glycans from the IgG scaffold. EndoS was also shown to have activity against alternative glycoprotein substrates, however, this non-specific activity was negligible in the context of whole serum. The effect of EndoS-mediated deglycosylation on the structure of the IgG Fc domain was explored using both X-ray crystallography and small-angle X-ray scattering. Small angle X-ray scattering was also used to characterise both EndoS and IdeS in complex with IgG Fc. Solution-state models of each complex were produced providing preliminary data towards how these enzymes interact with IgG. Overall, the results presented here contribute to our understanding of these enzymes which is of importance as they go forward into clinical applications.