DNA gyrase, a type II topoisomerase, catalyses the introduction of negative supercoils into closed-circular DNA, using the energy from ATP hydrolysis. The reaction mechanism involves the breakage of one DNA double strand (the DNA gate) and the passing of another DNA strand (the passage helix) through that break and finally the re-sealing of the DNA gate. The strand-passage reaction was studied by the use of novel DNA substrates and by site-directed mutagenesis of one of the gyrase proteins. The DNA substrates were used to attempt to define the DNA segments used by the enzyme as the DNA gate and passage helix in a catenation reaction. This was achieved by using oligonucleotides to form partial duplex regions in single-stranded DNA. A high-affinity gyrase cleavage site from the plasmid pBR322 was cloned into M13mpl8 and generated both the single and double-stranded circular forms of the molecule (MAT1). It was shown that gyrase could form a specific DNA gate in a short duplex region in single-stranded MAT1 when quinolone drugs were present. This DNA gate was much smaller than that normally utilised by the enzyme. The catenation and decatenation reactions were examined in detail with normal duplex substrates; reactions using a non-hydrolysable ATP analogue gave different results to those previously reported for the eukaryotic homologue of gyrase, indicating a possible mechanistic difference between the enzymes. Conditions under which the partial duplex substrates would be catenated were not found. Site-directed mutagenesis was used to alter arginine residues thought to interact with the passage helix during the reaction cycle. Assays of the mutant protein revealed that supercoiling activity was markedly reduced, but that partial activities of gyrase, such as the ATPase and DNA cleavage reactions, were close to wild-type levels.

The variable heavy domains of heavy chain antibodies (VHH) possess unique characteristics which make them an attractive alternative to conventional antibody fragments for detailed NMRP based structural analysis. The small size offers an advantage over Fabs and the enhanced stability compared to scFvs is also beneficial. In addition, the elongated complementarity determining regions (CDRs) associated with VHH fragments may facilitate the characterisation of a more diverse range of antibody-antigen interactions as well as providing templates for studying the motion of CDRs. The work described in this thesis illustrates the feasibility of using VHH fragments to gain structural insight into antibody-antigen binding. The successful generation of isotopically labelled protein allowed the acquisition of high quality NMR spectra from which the solution structures of two free VHH fragments were determined. The solution structures revealed significant differences in the conformational dynamics at the antigen-binding site with both flexible and structured CDR loops being identified. The binding sites of both the VHH and the antigen were mapped by NMR minimal shift analysis. The work discussed in the latter chapters focuses on the structure of VHH 67, its interactions with ILP6 and how information gained from this antibody-antigen interaction may be applicable to drug discovery. The structure determination and binding site mapping showed that binding occurred through the long, structured CDR3 loop and also identified that VHH 67 bound to ILP6 through a novel binding site. This new binding site was confirmed by X-ray crystallography and the functional effects of the VHH 67:ILP6 interaction were ascertained. The relationship between binding affinity and the dynamics of CDR3 was also investigated.

Tropomyosin determinants for actin binding have not been identified completely and the nature and position of residues involved in thin filament dynamics has not been established. To date a number of Tropomyosin mutations have been linked to several muscle diseases including cardiomyopathies and skeletal muscle myopathies. In this thesis, we aimed to investigate the following tropomyosin mutations R90G, E163K, R167G, E240K, R244G and M2811 which have been shown to cause several severe skeletal muscle myopathies. We used various structural, biochemical and kinetic methods to assess the impact of these mutations on tropomyosin structure and biochemical properties. Fluorescence emission spectroscopy, and transient kinetics were used to assess the effect of these mutations on the equilibrium distribution and kinetics of transitions between different thin filament regulatory states. Overall the data demonstrated that: 1) all tropomyosin mutations except (E163K, E240K, M2811 and R90GR167G) affected the thermal stability of tropomyosin but not the a-helical coiled coil structure. 2) The size of the cooperative unit n was reduced by all tropomyosin mutations. 3) Tropomyosin mutations did not affect the proportion of thin filaments in the blocked state (at low Ca2+). 4) Tropomyosin mutations did affect the maximum observed rate constant of thin filament transition between the ON and OFF states. 5) Several tropomyosin mutations have affected tropomyosin-troponin binding affinity but none of the mutations had any effect on the actin binding affinity. Overall these results provide insight into the mechanism by which tropomyosin bind actin and troponin, tropomyosin related thin filament cooperativity and allosteric transitions.

An Arthrobacter sp. which is capable of utilising L-threonine as the sole source of carbon and nitrogen for growth, has been isolated from soil by elective culture. The metabolic pathway by which this organism converts L-threonine into more common intermediary metabolites has been elucidated, and the properties of two key enzymes of the pathway have been studied. L-Threonine was shown to be oxidised in an NAD-dependent reaction catalysed by an inducible L-threonine dehydrogenase. Incorporation studies, in which [U-14c] L-threonine was added to the medium of organisms growing at the expense of threonine, or incubated with cell-free extracts of such organisms, indicated that in the Arthrobacter sp. the decarboxylation of AOB was of no physiological importance. Results showed that AOB was instead cleaved in a CoA-dependent reaction to yield acetyl-CoA and glycine. The enzyme which catalysed this reaction, aminoacetone synthase, was detected in cell-free extracts of threonine-grown organisms, and was inducibly formed only during growth on L-threonine; in particular it was not present in cells grown on acetate plus glycine. The cleavage reaction was reversible, and the activity of aminoacetone synthase was routinely assayed by the condensation of acetyl-CoA and glycine, and the formation of aminoacetone via AOB. Further incorporation studies, using [2-14c] acetate and [2-14c] glycine, showed that the acetyl-CoA and glycine derived from threonine were metabolised via the tricarboxylic acid plus glyoxylate cycles, and via the "folate-dependent "serine pathway" respectively (Sagers and Gunsalus, 1961). Extracts of threonine-grown organisms were shown to be rich in two key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, and in the component enzymes of the "serine pathway", viz. the "glycine cleavage system", L- serine hydroxymethyltransferase and L-serine dehydratase. L-Threonine dehydrogenase and aminoacetone synthase were partially purified from crude extracts of threonine-grown organisms, in order that their properties could be investigated. Certain preparations of L-threonine dehydrogenase were completely dependent upon the provision of bivalent cations for enzyme activity - this requirement being best fulfilled by Zn2+. Inhibitor studies confirmed the dependence upon Zn2+, and showed that the enzyme possessed one or more free thiol groupes which were essential for enzyme activity. The results of studies with purified aminoacetone synthase suggested that the enzyme was dependent upon pyridoxal 5'-phosphate for activity. By using the two purified enzymes it was convincingly demonstrated that both were required for the conversion of threonine into glycine and acetyl-CoA. The collaborative and sequential action of L-threonine dehydrogenase and aminoacetone synthase provides a novel route for the catabolism of threonine. This route differs fundamentally from that postulated by Elliott (1959, 1960), in which L-threonine is oxidised to aminoacetone, and this in turn metabolised to pyruvate via methylglyoxal and D-lactate. The pathway operating in the Arthrobacter sp. also provides a key metabolic role for aminoacetone synthase, an enzyme which had previously been thought to function solely in a somewhat speculative cyclical route for the oxidation of glycine (Elliott, 1959, 1960), by catalysing the production of aminoacetone from acetyl-CoA and glycine. A mechanism for the concerted action of L-threonine dehydrogenase and aminoacetone synthase which does not involve the production of free AOB is proposed and discussed.

A general procedure was laid down for estimating precise molecular weights by ultracentrifugation equilibrium using schlieren optical system. The phase plate diaphragm was checked against the knife edge of the schlieren optical system, and it was observed that the results using both the diaphragms are quite similar. The temperature indicating and control system of the MSE ultracentrifuge was also checked by running sedimentation velocity experiments at a wide range of temperatures, and it was found that the results of such experiments on s20,w are consistent, and are independent of temperature to the range studied. When similar experiments were performed on the Beckman Model E, the results were temperaure dependent. The new methods (Rowe and Rowe Method II and III) yielded excellent results. The method II provides satisfactory results for ideal systems using presmoothed data of concentration or concentration gradient as a function of distance from the axis of rotation. The method III provides acceptable results of Mo from data of a single sedimentation equilibrium experiment for both ideal and non-ideal systems. For both the methods to work it is essential to smooth the data with the help of fourier series, which is devised by incorporating it in the programs used by the digital computer. Procedures were also developed to determine Mo values by using hand smoothed data (instead of fourier smoothed data) with the desk computer. these procedures have made it possible to get comparable results of MO from a single photograph taken at equilibrium with any optical system without the cost of digital computer. Finally it is concluded that the new methods can be used for the estimation of precise values of molecular weights from a data of single experiment obtained by using any optical system.

An experimental system is described in which rapid and extensive sporulation of the obligately anaerobic bacterium Clostridium pasteurianum ATCC 6013 was reproducibly obtained in batch culture using a simple, defined medium containing only salts, vitamins, glucose and ammonium ions. Electron microscopy was used to follow the progress of cytological changes during sporulation and to relate these to the time course of Incubation in the sporulation medium. The production of a refractile spore from a vegetative cell took 9 to 10 h at 37°C with the approximate times taken for the completion of each recognizable stage being as follows:- from septation to the completion of engulfment, 2 h; from engulfment to deposition of coat material, 3 h and from the first appearance of spore coat material to deposition of spore cortes, 2.5 h. Approximately 80% of the organisms produced mature, refractile spores and the degree of synchrony with which sporulation was accomplished compared very favourably with the best reported for aerobic sporulating bacteria. This C. pasteurianum system is therefore eminently suitable for use in studies of the biochemical changes associated with sporulation of an obligate anaerobe. A preliminary Investigation was made of some of the biochemical events associated with sporulation of C. pasteurlanum. Glucose was not a repressor of sporulation. in fact, high (3-4% w/v) initial concentrations were required for optimal sporulation in the defined minimal medium. A striking and characteristic early event of sporulation was swelling of the cell brought about by the extensive deposition of granulose (an amylo- pectin-like polymer) in the sporangial cytoplasm. Even so, glucose uptake and the production of acetate and butyrate proceeded continuously throughout sporulation, suggesting that the energy required for sporulation was derived from continuous fermentation of exogenous glucose. A somewhat surprising finding was that sporulation proceeded without the production of detectable levels of extracellular proteinase, though very low intracellular proteolytic activity was present during both growth and sporulation. Furthermore, the extensive protein turnover which is associated with sporulation in aerobic bacteria was not detected in C. pasteurianum. Another departure from the pattern of events normally associated with sporulation of Bacillus species was that C. pasteurianum apparently produces no antibiotic during spore formation. The exosporium of C. pasteurianum, discovered during the electron microscopic examination of the sporulation process proved to possess several interesting features, some of which were investigated in more detail. The presence of a large opening at its base - not a usual feature of exosporia, was confirmed by demonstrating that ferritin molecules or bacteriophage particles could gain entrance to the peri-sporal space of autolytically liberated spores. When the three-dimensional structure of the spore was examined using the critical-point drying technique, it was found that the spore was slung within its exosporium by a system of 'suspensors' which were not readily detectable by other methods of specimen preparation. A more detailed investigation of the nature of the exosporium showed it to possess a simple, homogeneous structure consisting of superposed sheets of identical, possibly doughnut-shaped, subunits which were arranged in a regular, close packed hexagonal lattice with a nearest neighbor separation of 5 nm. In surface view a subordinate, hexagonal pattern of 'holes' placed 10 nm apart was also discernible. The apparently paracrystalline structure of the exosporium suggested that during sporulation its synthesis might proceed by a process of spontaneous self-assembly. The presence of an opening in the exosporium posed the question of whether this might facilitate exit of the emergent germling cell. Examination of spore germination and outgrowth by electron microscopy revealed that whereas both spore coat and spore cortex underwent lytic digestion during this process, the exosporium remained essentially intire. However, escape of the germling cell was not impeded, since this invariably took place via the open end of the exosporium.

The nucleotide sequences of genes, cat, encoding chloramphenicol acetyltransferases in Shigella flexneri and Streptomyces acrimycini were determined following subcloning of the determinants in bacteriophage M13. The predicted open reading frames, 213 and 219 amino acids respectively, demonstrate 30% or greater homology with one another and with the published sequences of other variants. Homology is most evident within the region flanking an essential histidyl residue (His-195) which is believed to function as a general base during catalysis. The 5' and 3' non-coding DNA of the Enterobacterial gene (cat) contains sequences equivalent to the E. coli consensus transcriptional initiation and termination motifs. Oligonucleotide site-directed mutagenesis of cat was utilised to test the hypothesis that the His-195 imidazole interacts, via a hydrogen-bond, with the carboxylate anion of an acidic amino acid - a process defined as tautomeric stabilisation. Five highly conserved aspartyl and glutamyl residues were replaced by asparagine or glutamine and the catalytic competence and substrate affinities of the mutant enzymes were determined. The K values for each substrate, chloramphenicol and acetyl-CoA, were essentially unchanged in the mutants but in one (Asp-199 replaced by Asn) the turnover coefficient, k, was diminished over 700-fold relative to wild type indicating that Asp-199 has an important, though non-essential, role in catalysis and is therefore a strong candidate for the tautomeric stabilisation of His-195. Inactivation of both the Asn-199 mutant and wild type enzyme by the affinity reagent 3-bromoacetyl chloramphenicol results in specific alkylation of the 3-nitrogen of the His-195 imidazole. This suggests that Asp-199 modulates the catalytic competence of His-195 by increasing the basicity of N-3 rather than by ensuring productive orientation of the imidazole. Plasmid and Ml 3 vectors were constructed to facilitate future studies of chloramphenicol acetyltransferase employing site-directed mutagenesis.

This thesis describes work undertaken on two aspects of eukaryotic pre-messenger RNA splicing. The first study investigated the use of oligonucleotide-limited transcription to produce model substrates for in vitro splicing reactions. It was found that the length of 3' terminal exon of the pre-mRNA could be reduced to four nucleotides, and possibly less, and both steps of splicing could still be observed. However at short 3'exon lengths, step 2 (exon ligation and release of lariat intron) was inefficient. This data correlated with previous studies of splicing complex formation on truncated pre-mRNAs and, in addition, suggested transient non-specific binding between the 3'exon and the splicing complex was required to activate the 3'splice site during step 2. The effect of changing the 3'exon sequence to either 5'- or 3'splice site sequences was also investigated. Mutation to a 5'splice site sequence had no effect on splicing efficiency at the authentic splice sites, while mutation to a 3'splice site resulted in enhanced 3'- 5'exonuclease degradation of the lariat-3'exon splicing intermediate.;The second study tested a number of models explaining the mechanism of developmental1y regulated alternative 3' end processing of murine immunoglobulin M pre-mRNA. The system contains two polyadenylation sites one of which is located within the 3' terminal intron. Competition has been proposed to occur between the two polyadenylation sites or between splicing of the terminal intron and polyadenylation at the site within the intron. The data produced suggest the competition is principally between the splicing reaction and use of the polyadenylation site within the intron. It was also found that the intron 3'splice site was sub-optimal due to a small stem-loop structure which sequesters the polypyrimidine tract and cleavage site. This is the first demonstration of a naturally occuring secondary structure modifying the in vivo efficiency of a splice site.

The multicatalytic proteinase (MCP) is a 700 kDa multisubunit complex which is found in eukaryotic cells. MCP is involved in ubiquitin-dependent non-lysosomal protein degradation and has been implicated in the processing of antigens by the major histocompatability complex class 1 pathway. The MCP purified from rat liver is composed of at least 16 distinct subunits of molecular masses between 22 and 34 kDa. MCP is known to possess at least three distinct peptidase activities, described as the 'trypsin-like', 'chymotrypsin-like' and 'peptidylglutamylpeptide hydrolase' activities. However, it is not known which subunits are responsible for the different peptidase activities. The main aims of my study were (1) to establish which subunit(s) possesses the active site responsible for 'trypsin-like' activity, (2) to identify the catalytic amino acid residues responsible for this activity and (3) to determine whether all MCP particles possess these components. 'Protection' experiments with the peptide aldehyde leupeptin, which is a specific reversible inhibitor of the 'trypsin-like' activity, have identified one or two subunits which possess thiol groups essential for 'trypsin-like' activity. These thiol groups are probably not involved in catalysis but may be located very close to the active site responsible for 'trypsin-like' activity. An active site specific peptidyl chloromethane which specifically inactivates the 'trypsinlike' activity, labels two distinct subunits of 23-24 kDa. One of these polypeptides is also labelled by an active site specific peptidyl diazomethane for the 'chymotrypsin-like' activity, in addition to three other distinct polypeptides of 28-23 kDa. Together these results suggest that one active site is responsible for 'trypsin-like' activity while four distinct active sites are responsible for 'chymotrypsin-like' activity. Affinity chromotography was carried out using an immobilised specific inhibitor of the 'trypsin-like' activity. The results of these studies suggest that all MCP particles purified from rat liver possess the catalytic component responsible for the 'trypsin-like' activity.

Quinolone drags are a clinically significant family of antibacterial compounds that are known to affect the activity of DNA gyrase, an essential bacterial enzyme involved in controlling the topological state of DNA, Gyrase holoenzyme, a complex of two A and two B subunits, can introduce negative supercoils into DNA using energy derived from the hydrolysis of ATP. Although addition of quinolones rapidly inhibits the supercoiling activity of gyrase, it was found that quinolone-dependent DNA cleavage was a slow process, leading to the suggestion that there may be two levels of interaction between quinolones and the gyrase-DNA complex. Rapid gel-filtration experiments have shown that stable quinolone binding requires the presence of both gyrase and DNA; no significant binding was found to either gyrase or DNA alone. Enzyme containing gyrase A protein with the mutation Ser83 to Trp (which is known to confer quinolone resistance) showed greatly reduced drug binding. It is concluded that efficiency of binding is primarily determined by the gyrase A subunits. Investigation of transcription by T7 and Escherichia coli RNA polymerases has found that quinolone-mediated stabilisation of a gyrase-DNA complex prevents passage of polymerase along the template. Inhibition of transcription required the presence of gyrase and quinolone together; RNA polymerase was unaffected by either quinolone or gyrase alone, implying that polymerase can normally pass or displace gyrase. In the presence of ciprofloxacin, gyrase was found to shield a region of about 26 bp of DNA from transcription by T7 RNA polymerase, with especially strong protection of a 20 bp core. Preliminary experiments performed using an in vitro DNA replication system suggest that DNA polymerases may be similarly interrupted by a gyrase-quinolone-DNA complex.