Fibrillins are modular, disulphide-rich glycoproteins that assemble into microfibrils in the extracellular matrix (ECM). These microfibrils are critical structural elements of many non-elastic and elastic connective tissues. They also regulate the availability of transforming growth factor-β signalling molecules in the ECM. Defects in microfibrils are associated with acquired and inherited connective tissue disorders. In particular, mutations in the human FBN1 gene, which encodes fibrillin-1, are associated with a spectrum of diseases, including Marfan syndrome (MFS). One of the proposed initial steps in microfibril assembly is the interaction between the N- and C-terminal regions of fibrillin monomers. The minimal regions of human fibrillin-1 required for an interaction in vitro were previously identified: the four N-terminal domains, from the fibrillin unique N-terminal (FUN) domain to the third epidermal growth factor-like (EGF) domain (FUN-EGF3), and the three C-terminal calcium-binding EGF-like (cbEGF) domains (cbEGF41-43). Here, fragments corresponding to these regions were produced and shown to interact in pull-down and surface plasmon resonance assays. In addition, the structure of the FUN-EGF3 fragment was determined using nuclear magnetic resonance spectroscopy. This showed the novel structure of the FUN domain and the interdomain interfaces in this region of fibrillin. Combining structural and sequence conservation data may help to identify regions of FUN-EGF3 important for binding to cbEGF41-43. Here, the interaction was probed by site-directed mutagenesis. However, substituting individual residues in FUN-EGF3 with alanine did not abrogate binding to cbEGF41-43. Three MFS-associated residue substitutions were also introduced into the FUN-EGF3 fragment. While they did not abolish the interaction with cbEGF41-43, they did cause misfolding. Two of these substitutions, N57D and W71R, also resulted in the defective secretion of a larger N-terminal fragment by fibroblast cells, suggesting a potential mechanism of disease pathogenesis. Although specific residues involved in the N-C interaction were not identified here, the FUN-EGF3 structure will be vital for understanding the molecular surfaces involved in microfibril assembly and growth factor binding.

Transition metals such as copper, zinc and nickel are required in many enzymatic processes that require redox changes. When transition metal concentration exceeds a certain threshold, their redox and metal binding properties make these elements extremely toxic. Bacteria regulate the cellular concentration of these important, yet toxic, elements using elaborate homeostatic systems. One such mechanism is the chemiosmotic extrusion of copper by the Cus system in the Gram-negative bacterium Escherichia coli. This work studies the regulation of the Cus system in response to copper and silver ions. Copper is an essential cofactor required in many enzymatic processes. But its redox properties can lead to toxicity. Silver is chemically similar to copper, but is not bioactive and its presence in cells can lead to extreme cytotoxicity. Transcription from cusCFBA genes is controlled by the CusR/CusS TCS in response to elevated levels of copper or silver in the periplasmic space of E. coli. Extracellular signals are transduced into the cell through phosphotransfer reactions between the prototypical histidine kinase CusS and the response regulator CusR. Copper sensing by the periplasmic domain of CusS is proposed to initiate signal transduction in the Cus system. Despite the frequency with which bacteria employ histidine kinases to sense their environment, signal recognition and incorporation by the protein is not well understood. The goal of this research is to investigate the role of CusS in regulating metal homeostasis in E. coli and characterize the periplasmic domain of the protein to determine its metal binding properties. The experiments described in this work reveal that the CusS is essential for copper and silver resistance and regulates expression from the cusCFBA promoter region. Signal recognition occurs by direct metal binding by the periplasmic domain of CusS. Metal binding causes a change in the secondary structure of the domain and its tendency to dimerize is enhanced under these conditions. The possibility of signal attenuation by interaction with the metallochaperone CusF is also discussed. These data help construct a model for signal transduction in the Cus system and help characterize, for the first time, a metal-responsive sensor histidine kinase in E. coli.

The control of the flagellar motor in Rhodobacter sphaeroides was investigated. Unlike most flagellar motors which are controlled by reversing the direction of rotation, the R. sphaeroides motor is controlled via a stop-start mechanism. Advanced optical microscopy was employed alongside genetic, biochemical, and behavioural techniques. High-resolution measurements of rotating beads on flagellar stubs revealed that the R. sphaeroides motor is similar to its E. coli counterpart, rotating counterclockwise at comparable torques/speeds (1,300 pNnm/rad at stall torque), and exhibiting transient step changes in speed. The mean stop duration, mean stop frequency (number of stops per s), and run bias (fraction of time spent rotating) of wild-type at steady-state were 0.66 ± 1.01 s, 0.31 ± 0.19 s-1, and 0.80 ± 0.20, respectively. Manipulating signal inputs to the motor genetically, or by exposing cells to chemotactic stimuli revealed that (i) without chemotactic stimulation the motor rotates continuously, (ii) phosphorylated CheYs are required to stop the motor, and (iii) the chemotaxis system cannot control the speed of rotation of the motor (termed chemokinesis) as previously reported. Complementation studies revealed that CheY3, CheY4, and CheY5 are functionally equivalent. The copy numbers per cell of important CheYs were found to vary greatly under the conditions tested (<1,000, ~3,000, ~60,000 for CheY3, CheY4, and CheY6 respectively). To determine how CheY-P binding causes the motor to stop, external force (viscous flow or optical tweezers) was applied to chemotactically stopped motors. CheY-P binding might either cause the torque-generating units to disengage from the rotor, analogous to a clutch, or trigger the rotor to jam, analogous to a brake. The rotor resisted re-orientation during a chemotactic stop implying that the motor was held in a locked state. The value of torque resisting forward motion (keeping it locked) was estimated to be 2-3 x stall torque (2,500-4,000 pNnm/rad). Furthermore beads attached to flagellar stubs stop at fixed angles for several seconds, showing no large-scale Brownian motion. Step analysis revealed that these stop events occur at 27-28 discrete angles around the motor, which most likely reflect the periodicity of the rotor (i.e. copies of FliG). This represents the first experimental resolution of steps in the rotation of a wild-type bacterial flagellar motor with a full complement of torque-generating units.

The ubiquitin proteasome system targets proteins to the proteasome where they are degraded. Substrate recognition and processing prior to degradation take place at the 19S regulatory particle of the proteasome. A polyubiquitin chain, linked through isopeptide bonds formed between the C-terminal G76 and K48, is the signal responsible for delivery to the proteasome. Because chains linked via any of the seven lysine residues of ubiquitin exist in vivo and encode signals unrelated to protein degradation it is crucial for cells to avoid crosstalk between these different pathways. Several ubiquitin receptors related to proteasomal degradation have been identified but the selectivity between the different ubiquitin chains has not been assessed quantitatively while avoiding artefacts attributed to GST-dimerisation. By employing isothermal titration calorimetry, analytical ultracentrifugation and nuclear magnetic resonance, discrimination between K48- and K63-linked diubiquitin was established for the S. pombe proteasomal receptor Rpn10 and the shuttle protein Rhp23. The same methods allowed us to propose a discriminatory model for Rpn10. The crystal structures of the 19S regulatory particle subunits Rpn101-193 and Rpn121-224 have been determined and possible protein-protein interaction sites were identified by surface conservation and electrostatics analysis. Rpn12 surface residues were identified that had a negative effect on Rpn10-binding. This interaction was studied by surface plasmon resonance, fluorescence anisotropy and nuclear magnetic resonance. These experiments revealed a binding site on Rpn10 that is exclusively occupied by either ubiquitin or Rpn12 and for the first time demonstrated the interaction of a ubiquitin interacting motif with a protein other than ubiquitin.

Neisseria meningitidis is an obligate human pathogen causing over 1000 cases of meningococcal disease within the U.K., 10 % of which result in long-term disability or fatality. 10-70 % of the population carry N. meningitidis in their nasopharynx, the natural reservoir of this bacterium, as a commensal. The host-pathogen interactions of this species are complex and a greater understanding of the molecular mechanisms involved in pathogenesis and immune evasion is required. Three aspects of N. meningitidis pathogenesis were explored in this study. One mechanism of immune evasion which promotes serum resistance of N. meningitidis is recuitment of complement factor H through domains 6 and 7 (fH₆₇) by factor H binding protein (fHbp). In this study, mouse fH₆₇ was recombinantly expressed and purified. fHbp did not bind mouse fH₆₇ at physiologically relevant protein concentrations. The structure of mouse fH₆₇ was solved, showing differences in domain orientation and surface chemistry compared to the human version of this protein, potentially accounting for the host specificity of this interaction. Type IV pili (T4P) are crucial adhesins of N. meningitidis, the fibre of which is composed of thousands of copies of PilE. A method was developed to recombinantly produce large quantities of this protein from a variety of meningococcal strains and the structure was solved of one PilE protein. Subsequent analysis was performed with the PilE proteins investigating their interaction with the putative pilus receptor CD46 and human epithelia as well as their immunogenicity. A method was also established to produce PilC, the proposed tip-assocoated adhesin of T4P. ZapE has recently been identified as an important protein in pathogen colonisation, functioning as an ATPase linked to Z-ring formation in bacterial cell fission. Both N. meningitidis and E. coli ZapE were recombinantly produced. The domain boundaries were mapped and ATPase activity was confirmed. No interaction was seen with FtsZ but DNA binding and modulation was observed by shift assays, the exact function of which remains to be elucidated in future studies.

DNA translocases are molecular motors that use energy from nucleotide triphosphate (NTP) hydrolysis to move along, pump, remodel or clear DNA. Unlike helicases, double-stranded DNA (dsDNA) translocases do not unwind DNA; their action has no net product apart from inducing supercoils as a result of groove-tracking, which has hampered their characterisation. Many dsDNA translocases appear to have biased directionality. However, the inherent symmetry of dsDNA requires that translocase activity is regulated by specific sequences or through modulation by interaction partners. FtsK is a highly conserved bacterial cell-division protein, localised to the dividing septum, that coordinates chromosome segregation with cytokinesis. It is responsible for the resolution of chromosome dimers by activating the tyrosine recombinases XerCD bound to the 28bp chromosomal site dif. The C-terminal domain of FtsK (FtsKC) is a dsDNA translocase (speed ~5 kb/s, stall force ~60 pN) most closely related to superfamily 4 helicases and is active as a hexameric ring. A winged-helix subdomain at the C-terminus of FtsKC, FtsKgamma, binds to specific 8 bp sequences, KOPS, that are polarised in the bacterial chromosome from the origin to towards dif. FtsKgamma also interacts with XerD, activating it for catalysis. Studies of FtsK translocation have differed over whether KOPS act as a loading or a reversal sequence for FtsK. In Chapter 2, I use a continuous ensemble assay for dsDNA translocation to show that FtsK initiates rapidly at KOPS, with loading dependent on FtsKgamma. Translocation requires moderately cooperative ATP binding, while ATP hydrolysis has a more relaxed cooperativity. I have determined the ATP coupling efficiency of translocation to be ~1.6 bp/ATP, in line with theoretical estimates. Though FtsK probably strips most proteins from DNA, I show in Chapter 3 that FtsK stops translocating when it encounters XerCD bound to dif. The interaction is most likely a specific down-regulation, but surprisingly does not depend on FtsKgamma or on the catalytic or synaptic activity of XerCD. In Chapter 4, I show some preliminary structural data of FtsKC bound to dsDNA, with the aim of determining the first high resolution structure of a ring dsDNA translocase bound to nucleic acid.

Malaria is a disease of global importance, causing hundreds of thousand of deaths a year. The majority or deaths are caused by Plasmodium falciparum, a parasite transmitted by the mosquito Anopheles. Its pathogenicity largely results from an ability to transform infected erythrocytes by creating knob-like structures that result in endothelial adhesion. Two major components of these knob structures have been identified as P. falciparum erythrocyte membrane protein 1 (PfEMP1) and knob-associated histidine rich protein (KAHRP). The extracellular fragment of PfEMP1 is responsible for antigenic variability and cytoadherence while its intracellular domain (ATS) connects to the cytoskeleton via interactions with other plasmodium-encoded proteins. In addition, perforin-like proteins (PLPs) with a MACPF domain have been identified in the genome of Plasmodium. PLPs are highly conserved and are expressed in various life-cycle stages of the parasite. They are believed to form pores in membranes of the host cell but their structure is yet unknown. The aim of the work in this thesis was to obtain new information about the structure and role of malaria proteins, thus giving a better understanding of the disease and its possible treatment. Studies of numerous designed constructs of the ATS family were carried out using biophysical methods including high resolution NMR and CD. These revealed that ATS domains are mainly unstructured with a relatively small folded core, consisting of a bundle of &alpha;-helices. Surprisingly, no evidence could be found for ATS binding to KAHRP in solution conditions although previous pull-down data had indicated an interaction. Bioinformatics analysis and yeast-two-hybrid data suggested, however, that there is a conserved protein interaction epitope on the central flexible part of ATS. It was shown, using fluorescence anisotropy measurements, that this part of ATS associates with a parasite protein containing a PHIST (Plasmodium helical interspersed sub-telomeric) domain. Expression constructs of the PLP protein family were designed and manufactured, with the aim of enabling structural studies of this putative pore protein.

Transient receptor potential (TRP) channels are expressed throughout the central nervous system and have a unique ability to detect a wide range of stimuli including changes in voltage, temperature, pH, lipid environment, small molecule agonists, and mechanical stress. While it is known that TRP channels contain the same six transmembrane helix (S1-S6), tetrameric architecture as voltage-gated channels, the degree to which functional and structural analogies are relevant remains poorly understood. This thesis describes a multidisciplinary approach toward understanding the structure and function of TRP channel transmembrane domains by focusing on the S1-S4 transmembrane helices of the TRPV1. This focus is inspired by the voltage-sensor domain (VSD) of the S1-S4 helices of voltage-gated channels, for which a range of studies show functional and structural independence. While some TRP channels are voltage-sensitive, their S4 helix does not contain the positive string of amino acids of canonical VSDs. However, the S1-S4 helices are functionally significant as the binding site of small molecule ligands in both TRPV1 and TRPM8 (for capsaicin and menthol, respectively). The question of TRP channel transmembrane domain modularity is addressed in this thesis by expression and purification trials as well as radioligand-binding assays. It is demonstrated that the S1-S4 and S1-S6 helices of TRPV1 can be properly inserted, overexpressed, and show signs of stability upon detergent-extraction from Saccharomyces cerevisiae membranes. However the TRPV1 S1-S4 and S1-S6 helices do not show wildtype (WT)-like binding in [³H]-RTX binding assays. These results indicate that the TRPV1 transmembrane domains are likely structural but not functional domains. The S. cerevisiae expression system remained promising for the overexpression of TRP transmembrane domains as well as the production of functional, though not stable upon detergent-extraction, WT TRPV1. This WT TRPV1 was subsequently found to functionally bind both RTX, used in ligand binding assays, as well as the double-knot toxin (DkTx), targeted to the pore domain (the S5-S6 helices). An effect of DkTx on RTX binding affinity demonstrates an allosteric interaction indicative of a possible tighter packing between the two transmembrane domains than is seen in voltage-gated channels containing the canonical VSD. Computational approaches additionally allowed for the investigation of the intramembrane capsaicin binding site in the TRPV1 S1-S4 helices, crucial to the initial motivations of this study. While the literature locates the capsaicin binding site to the TRPV1 S1-S4 helices, a `binding pocket' has yet to be defined, with regards to the orientation of bound capsaicin and its access route to the site via the bilayer. Using molecular dynamics (MD) simulations the preferred location of capsaicin within the bilayer is defined, as well as the elucidiation of capsaicin flip-flop between bilayer leaflets as a key event prior to TRPV1 binding. A transient binding was also observed between a homology model of the TRPV1 S1-S4 helices and capsaicin, possibly encouraging the idea that the S1-S4 helices still contain a partial binding site, though of too low affinity to be observed in the binding experiments performed here.

Gene regulation is vital to the success of all living organisms. Understanding this complex process is crucial to our knowledge of how cells function and how in some cases they can lead to debilitating or even fatal disease. In this thesis I focus on a set of DNA-binding proteins known as transcription factors (TFs), proteins fundamental to the process of gene regulation at the level of transcription. I develop assays and techniques for the detection and quantitation of TFs in vitro and in vivo as well as a method for TF encapsulation and release. The advantages of the TF detection assays in this thesis are made possible through the use of single-molecule (sm) fluorescence. This methodology enables detection of individually labeled molecules allowing discrimination of sample heterogeneities inaccessible with ensemble techniques. Here I present two different TF assays based on two sm observables: relative probe stoichiometry and Förster resonance energy transfer (FRET). The first assay design, based on stoichiometry, detects TFs using TF-dependent coincidence of two distinctly labelled DNA ‘half-sites’. I demonstrate sensitive detection (~ pM) in solution and on surfaces, multiplexed detection of multiple TFs, and detection in cell lysates. A kinetic model of the system is also developed, verified experimentally and used to quantify TF concentrations without the need for a calibration curve. The second assay design, based on FRET, is a novel approach to TF detection using TFmediated DNA bending. TFs are detected by bending the sensor and monitored with FRET at the single-molecule or ensemble level. I demonstrate TF detection in purifed form and expressed in cell lysates. As this sensor was designed for use in vivo, methods to hinder nuclease degradation are explored. For TF detection in vivo, I describe a successful strategy to internalise fluorescently labeled molecules into live E.coli. Viability and internalisation efficiency are characterised and ensemble measurements with FRET standards are demonstrated. Importantly, sm FRET measurements in vivo are achieved opening many exciting possibilities. The FRET based TF sensor is then internalised as a step towards real-time in vivo monitoring of TF concentrations. Finally a system based on DNA nanotechnology is presented for the non-covalent encapsulation and release of TFs. Such a system could be delivered into a cell to alter levels of gene expression using external stimuli as inputs. We believe these tools will generate valuable information in the study of prokaryotic gene expression as well as providing a potential commercial avenue towards diagnostics.

Protein-protein recognition plays a central role in the surveillance of self and non-self in the mammalian immune system and ultimately in cellular survival within the organism. Two systems of fundamental importance to the immune system are the Tumour Necrosis Factor (TNF) and the T cell receptor (TCR) families. High-throughput methods developed within the Oxford Protein Production Facility have been successfully applied to the production of members of the TNF receptor and ligand superfamilies for structural characterisation. The TNF receptor DR6 was successfully refolded from E.coli inclusion bodies using a rapid-dilution technique and yielded diffraction quality crystals. Data collected from these crystals will be used to obtain an x-ray crystallographic model of DR6. Vascular Endothelial Growth Inhibitor (VEGI) was produced as a soluble recombinant protein in E.coli, and formed a number of poorly diffracting crystals, it is hoped that further trials and optimization of conditions will lead to improved data quality. Lymphotoxin β receptor was produced in a Eukaryotic system. This has shed light on the complications posed by signal peptide cleavage and glycosylation on the production of protein for crystallization trials. TNF superfamily proteins are ideal targets for the design of novel therapeutic agents due to their involvement in a number of disease pathologies. Various methods of molecular docking and small molecule design were applied to the search for potential inhibitors of receptor binding for the TNF ligand proteins TRAIL and BAFF. A number of potential drug leads were identified from the National Cancer Institute drug database. The Natural Killer (NK) T cell restricted TCRs recognise CD1d-presented glycolipid. Determination of the crystal structures of the invariant NK TCR and the NK restricted TCRs 5E and 5B shows that these proteins adopt the canonical structures of class I MHC restricted TCRs. This suggests that the binding of CD1d-glycolipid by these receptors will conform to the same model of binding seen for the class I MHC restricted TCRs.