The arsenite oxidase (Aio) catalyses the two-electron oxidation of arsenite to arsenate. The enzyme, purified and characterised from five mesophiles and one psychrotolerant bacterium, is a heterodimer with a large catalytic subunit (AioA) containing a molybdopterin guanine dinucleotide cofactor and 3Fe-4S center, and a small subunit (AioB) that contains a Rieske 2Fe-2S center. To understand the properties of a thermophilic arsenite oxidase, the enzyme from <i>Thermus thermophilus</i> str. HB8 was enriched and partially characterised. Because of the difficulties with enzyme purification and yield, the enzyme was heterologously expressed in <i>Escherichia coli</i>, purified and its properties compared to the native enzyme. The enzyme, also a heterodimer, was found to have maximal specific activity at 70^oC. This was 10^oC below and at least five-times less active than measurements taken from the study of the native enzyme. Comparisons with the heterologously expressed mesophilic arsenite oxidase from <i>Alcaligenes faecalis</i> were also made. The temperature profile of the <i>A. faecalis</i> enzyme was comparable to its mesophilic homologue from Rhizobium sp. NT-26, displaying an optimum temperature of 65^oC. Mechanisms thought to contribute to the thermostability of the HB8 Aio were investigated using a homology model of the enzyme, which was constructed using the X-ray crystal structures of the arsenite oxidases from <i>A. faecalis</i> and NT-26. A major stabilising factor in the model appeared to be the substitution of charged/polar residues with non-polar/hydrophobic residues of the interior surface/core of the enzyme. Studies of the electron transfer pathway were conducted using an electrochemical approach and the heterologously expressed NT-26 arsenite oxidase and its native physiological electron acceptor, a cytochrome c₅₅₂. The cytochrome was immobilised on an alkanethiol-modified gold electrode and used to mediate electrons between the enzyme and the electrode. This is the first electrochemical investigation of the arsenite oxidase in partnership with its native, physiological electron partner. A catalytic optimum of pH 6.5 was found and substrate Km of 277 µM. The “wiring” of the enzyme and its co-substrate (cytochrome c₅₅₂) to an electrode in this way also highlighted its potential use in a wider context as a biosensor.

Nck and WASP/N-WASP play essential roles in the signalling networks that control Arp2/3 dependent actin polymerisation in a variety of contexts. These include functions downstream of the PDGF, Met and T cell receptors, in endocytosis and in the formation of invadopodia and podosomes. Vaccinia virus exploits a similar signalling network to enhance its cell-to-cell spread. During viral egress newly assembled virus particles fuse with the plasma membrane and activate Src and Abl family kinases. This leads to phosphorylation of a vaccinia protein, A36, and recruitment of a complex of Nck, Grb2, WIP and N-WASP, which activates the Arp2/3 complex to induce the polymerisation of actin tails. The aim of this thesis was to elucidate the exact role of WIP in Nck and N-WASP signalling and furthermore, to understand the connectivity and interplay between the proteins in this important and conserved signalling network. I found that WIP, or the related protein WIRE, is essential for the induction of actin tails during vaccinia virus infection. I determined that interactions of WIP with the second SH3 domain of Nck and the WH1 domain of N-WASP are crucial for Arp2/3 dependent actin polymerisation. Moreover, in the presence of WIP, the interaction of Nck and N-WASP is dispensable for the actin-based motility of vaccinia virus. Furthermore, in the absence of Grb2, the second SH3 domain of Nck is critical for actin tails formation. My data demonstrates that WIP forms an essential link between Nck and N-WASP that is required to promote Arp2/3 dependent actin polymerisation.

Experimentation in shaken microplate formats offers a potential platform technology for the evaluation and optimisation of cell culture conditions. Provided that the results obtained are reliable, and indicative of large scale performance, it should be possible to obtain process design data early and cost effectively. This work describes a detailed engineering characterisation of liquid mixing and gas-liquid mass transfer in microwell systems and their impact on suspension cell cultures. Furthermore, an initial attempt at scaling a microwell culture to shake flasks and a 5-L stirred-tarik reactor is made. For suspension cultures of murine hybridoma cells producing IgGl, 24-well plates have been characterised in terms of power dissipation (P/V) (via CFD). Fluid flow patterns and oxygen transfer rate as a function of shaking frequency and liquid fill volume. Predicted ka values varied between 1.3 and 29 h_1 mixing time, quantified using decolourisation of iodine, varied from 1.7 s to 3.5 h while the P/V ranged from 5 to 35 W m-3. CFD simulations of the shear rate predicted hydrodynamic forces will not be lethal to cells. High shaking speed (> 250 rpm) was shown to be detrimental to cell growth, while a combination of low shaking speed and high well fill volume (120 rpm. 2000 //l) resulted in oxygen limited conditions. Using matched average energy dissipation as a basis for scale translation, cell growth and antibody titre were found to be similar in a 24-well plate. 250 ml shake flask and 5-L stirred-tank reactor. Overall this work has demonstrated that cell culture performed in shaken microwell plates can provide data that is both reproducible and representative of larger-scale cultures. Linked with automation this provides a route towards the high throughput evaluation of robust cell lines under realistic process conditions.

Ubiquitin is a small globular protein (76 amino acids, MW 8565 Da) which occurs in both eukaryotes and prokaryotes. It is involved in many biological processes, its function to 'tag' other proteins, to signal degradation. Ubiquitin is an ideal model for structural studies since it has a well defined secondary and tertiary structure and is stable to changes in temperature, pH and to a wide range of solvents. Synthetic techniques have been used to incorporate unnatural amino acids into the hydrophobic core of ubiquitin, the aim being to observe the structural defects incurred by these changes. Two analogues of ubiquitin have been under investigation. The first analogue has the unnatural amino acids, 2S, 4S, 5-Fluoroleucine substituted into two leucine positions which, from studies of the native protein were directed towards each other, across the hydrophobic core. The structure of this fluorinated analogue has been determined using both <SUP>1</SUP>H and <SUP>19</SUP>F, 1D and 2D NMR methods. The NMR solution structure reveals that the fluoro-methyl groups adopt preferred conformations within the protein core. The second analogue incorporates unnatural aminobutyric acid and norvaline into positions on the α-helix, at positions i, i +4. These unnatural amino acids replace valine and isoleucine, therefore eliminating β-branching at these specific positions and also giving a net loss of two methyl volumes from the hydrophobic core. The structure has been determined using <SUP>1</SUP>H 2D NMR and detailed comparisons made to the X-ray crystallographic structure of the analogue. The solution structure reveals that the substituted residues adjust their positions to compensate for the lost methyl volume.

No description available.

Nucleosides are transported across the inner membrane of <i>E. coli</i> by at least two separate energy-driven systems known as NupC and NupG. These systems are encoded by the genes <i>nupC</i> and <i>nupG</i> respectively. CTN1 (a mammalian transporter isolated form the jejunum) is a homologue of NupC. CNT1 is able to transport the nucleoside analogue AZT, used to combat HIV infection. CNT1 and NupC share substrate specificity and they have amino acid sequence identity of 27%, particularly in their C-terminal region. With the completion of the <i>E. coli</i> genome project paralogues of NupC and NupG have been found, designated as YeiM and YeiJ, and XapB and YegT respectively. To functionally characterise the putative nucleoside transporter-encoding genes <i>xapB, yegT, yeiM </i>and <i>yeiJ,</i> mutants were constructed which the coding sequence of these genes had been deleted from their chromosome. From this work it was established that all four putative nucleoside transporters XapB, YegT, YeiM and YeiJ were able to transport adenosine in the assays at similar levels as the positive control. Furthermore in kinetic analysis of the transporters it was possible to determine Km values for the four transporters with adenosine as substrate, which were in the same order of magnitude as mammalian nucleoside transporters and that shown for xanthosine uptake by XapB. The mutant strains were used to construct a bioassay, in which their ability to transport nucleoside analogues was assessed. AZT was used as model substrate. The use of multiple mutants in experiments involving nucleoside uptake as a sole carbon source further established the fact that the collection of genes studied in this project was indeed nucleoside permeases. Preliminary experiments gave an insight in developing a high throughput bioassay which can be developed further in order to produce a useful tool for nucleoside analogue drugs assessment.

Two soluble, novel cytochromes <i>c </i>have been discovered in the purple phototrophic bacterium <i>Rhodobacter sphaeroides. </i> They have been designated Diheme cytochrome c (DHC) and <i>Sphaeroides </i> heme protein (SHP). DHC is a 16 kDa, low-spin diheme protein and SHP is a 12 kDa high-spin, monoheme protein, which is capable of binding oxygen. The crystal structure of SHP indicates that the sixth heme ligand is asparagines, which is the first known report of an amide as the sixth heme ligand in a wild-type protein. Upon reduction of the SHP heme, this asparagines swings away, rendering ferrous SHP as penta-coordinate and able to bind small ligands, such as CO and NO, as well as O₂. CN^- can bind to ferric SHP. The complete crystal structure of DHC has been solved and as well as indicating two bis-His ligated hemes, a lack of β-sheet character and a structurally unique second heme domain, it also shows pronounced acidic patches surrounding both heme regions, which complement the basic patch surrounding the SHP heme region. This provides evidence of an electrostatic attraction between the proteins. DHC and SHP are encoded on the same operon, which also encodes a membrane-bound cytochrome <i>b</i>. Thus DHC could be the natural electron donor for SHP, which could function as the terminal electron acceptor in a novel respiratory pathway. The midpoint reduction potentials of the hemes in DHC are -340 ± 4 and -210 ± 4 mV. The midpoint reduction potential of the SHP heme is -105 ± 2 mV. This indicates that electron transfer from DHC to SHP is thermodynamically favourable. DHC and SHP also bind very tightly to each other (<i>K_d</i> = 200 nM) at low salt 10 mM KC1). At higher salt concentrations, binding becomes weaker, again indicating an electrostatic interaction. Phenotypic studies on SHP have not yet uncovered its function but have ruled out its involvement in respiration on various inorganic substrates and in response to oxidative and nitrosative stress.

The synthesis of a series of peptides spanning the entire sequence of the protein nerve growth factor (NGF) has been carried out using solid-phase peptide synthesis (SPPS). A twenty-two residue peptide sequence from the carboxyl-terminal region of NGF was found to inhibit the binding of native NGF to its known receptor, in a cell-free assay. This fragment may define an area in the native NGF molecule which is important for receptor interactions. The optimisation of this initial fragment found that the inhibitory effect was retained when the fragment was reduced to a fifteen residue, disulphide-linked peptide. Further attempts at optimising this smaller fragment have been carried out by substituting unnatural amino-acids into the sequence, as well as altering the conformation around the disulphide bond. Several of the smaller NGF peptides have been investigated by high-field nuclear magnetic resonance (N.M.R.). The complete assignment of several analogues of the fifteen residue, disulphide-linked peptide has been achieved. Analysis of the 'through-space' interactions indicated that these peptides adopt only random conformations in solution. As a prelude to a total NGF synthesis, the assembly of the well-characterised protein, hen egg white lysozyme (HL) has been attempted in order to test the current methods for approaching the synthesis of large, multi-cysteine peptides. Some evidence that the peptide chain was successfully assembled has been presented and the difficulties encountered in attempts to purify and fold the protein are discussed.

The gene encoding the <I>E. coli</I> flavodoxin NADP<SUP>+</SUP> oxidoreductase (FLDR) has been overexpressed in <I>E. coli</I> and purified to homogeneity. The molecular mass of FLDR apoprotein was determined as 27648 Da. The midpoint reduction potentials of the oxidised/semiquinone and semiquinone/hydroquinone couples of FLDR (-308mV and -268mV, respectively) were measured using redox potentiometry. FLDR was fully characterised kinetically both by steady state and pre-steady state techniques. Arginines (R144, R174 and R184) in the proposed NADPH binding site of <I>E. coli</I> flavodoxin NADP<SUP>+ </SUP>oxidoreductase (FLDR) were replaced by alanines and the mutant enzymes fully characterised and studied by pre-steady-state and the steady-state kinetics. From our studies R174 and R184 appear to interact with the adenosine ribose 2' phosphate, while R144 is more likely to stabilise NADPH binding by interaction with the nicotinamide ribose 5' phosphate. R174A and R184A are more efficient enzymes than wild-type or R144A with NADPH as substrate, consistent with the proposed phosphate-binding roles for these residues. Arginine residues R237 and R238 in the proposed binding site for FLDR redox partner flavodoxin, have been mutated to alanine. These mutant enzymes have been characterised by pre-steady-state and steady-state kinetics, UV-Vis spectrophotometry, CD and florescence. These mutants are less efficient electron transfer proteins. In a separate project it was attempted to identify genes associated with the antibiotic biosynthetic pathway of aristeromycin from <I>Streptomyces citricolor</I>. An aristeromycin-induced protein was isolated from <I>S. citricolor</I> purified to homogeneity and an N-terminal sequence was determined. From this an oligonucleotide was designed and used to probe <I>S. citricolor</I> chromosomal DNA. A 1000bp fragment of DNA was isolated and sequenced, and the presence of part of an ORF identified.

The product of Synechocystis PCC 6803 open reading frame slr0176, InrS (Internal nickel responsive Sensor) a member of the CsoR-RcnR metalloregulatory family, has been characterised in the course of this work and found to regulate the expression of nrsD, encoding a nickel export protein, in a nickel dependent manner. InrS occludes a previously unidentified cryptic promoter in a nickel resistance operon. In addition to Ni(II) InrS is also competent to bind and respond to Zn(II), Co(II) Cu(II) and Cu(I) in vitro as determined by metal binding studies & fluorescence anisotropy. The factors that favour a response to nickel but disfavour responses to zinc and copper in vivo have been explored including determining allosteric coupling free energies of Ni(II) and Cu(I) binding. The cognate and non-cognate metal binding affinities of InrS, ZiaR (zinc-sensing SmtB family member), Zur (zinc-sensing Fur family member) and CoaR (cobalt-sensing MerR family) have been determined through competition with a range of metal chelators and infer that selective sensing of zinc and nickel can be dictated by the relative affinities of the metalloregulators but selective cobalt sensing cannot. The primary metal coordination sphere of InrS has been investigated through a combination of site directed mutagenesis and metal binding studies. Three of four Ni(II) ligands constituting the square planar Ni(II) site have been identified and mutant variants with weakened Ni(II) affinities produced. InrS displays metal binding properties characteristic of the copper sensing sub-branch of this protein family thought to be a consequence of the primary coordination sphere. Results of this work have been used to refine predictions as to the metal(s) sensed by uncharacterised members of this protein family based on the predicted secondary rather than primary coordination sphere. An alternative allosteric network involving a secondary coordination sphere hydrogen bond in nickel sensing CsoR-RcnRs is proposed.