The complement system is regulated by protein-protein interactions. The binding sites within the regulatory proteins appear to be spread over two-to-four modular units (called CCPs), making it important to understand the relative arrangement of the modules. One- and two-module fragments from membrane cofactor protein (MCP), Vaccinia complement protein (VCP) - a viral mimic known to suppress the activity of complement - and complement receptor type 1 (CR1) were studied. A structural refinement of a fragment containing central CCP modules from VCP (which is composed of four modules) has been performed utilising restraints derived from the residual dipolar couplings (RDCs). The RDCs, cross-validated with the structural content of relaxation rates, provided a proof for structural differences between the NMR and crystal structures. Possible preferred arrangements of the modules have been described. The dynamic description derived from relaxation rates has shown that the link between the two central CCPs of VCP is highly flexible, more so than that between the two terminal ones. An indication of a difference in the level of inter-modular mobility between two CCP module-pairs from CR1 has also been obtained. The sensitivity of structure and stability of CCP modules to the presence of neighbouring modules is discussed. The local motions present at specific residues in each of the studied CCPs from MCP and VCP have been characterised in terms of time-scale and relative amplitude, and a partial description of local motions in the pairs of CCPs from CR1 was also obtained. The residues within the known and proposed binding sites of these proteins share dynamic properties with larger secondary structure elements in which they are located. Most of the residues implicated in binding appear to lie within structured parts of the protein.

To date two human proteins have been identified that can serve as the receptor for measles virus, namely membrane cofactor protein (MCP) and signalling lymphocyte activation molecule (SLAM). It is thought that a more through understanding of the structure and behaviour of the interacting regions of these receptors, will facilitate a more detailed understanding of the pathogenesis of MV. This thesis describes efforts to clone, express and characterise the relevant fragments of these receptors. MCP carries a functionally critical N-glycan on its second module (MCP2) and the crystal structure of MCP12 (expressed in mammalian cells) implies a structural role for this carbohydrate moiety. MCP12 and MCP2 were successfully cloned and expressed as secreted proteins in <i>Pichia pastoris</i> at levels sufficient for biophysical characterisation. Proton and ¹⁵N Nuclear magnetic resonance spectroscopy (NMR) studies, differential scanning calorimetry and mass spectrometry were employed to investigate the recombinant proteins. Both the natural sequences of MCP12 and MCP2 fragments expressed in <i>P. pastoris</i> were hyperglycosylated and therefore these recombinant fragments were investigated in both glycosylated and deglycolylated forms in order to study the effect of the glycans on module 2 of MCP, and with a view to detailed structural and dynamic studies using NMR. The data obtained with the <i>P. pastoris-</i>expressed fragments are not inconsistent with the hypothesis that the N-glycan on module 2 of the wild-type MCP is required for this molecule to be properly folded.  After extensive investigations, it was concluded that the inability of <i>P. pastoris </i>to attach the natural glycan is a barrier to more detailed structural studies. In an effort to produce MCP2 with a better defined glycosylation profile, efforts were made to express, this fragment in insect cells which are known to produce a ‘more natural’ glycosylation profile on proteins. Moreover, the MCP2 fragment was also expressed in <i>E. coli, </i>in order to produce a form of MCP2 that had never been glycosylated.

This thesis describes the reassignment of original two- and three-dimensional NMR data, acquisition of additional data sets and subsequent recalculation of DAF₂₃ solution structure. Additional data included acquisition of three new sets of residual dipolar couplings (RDCs); these provide additional restraints for structure calculation. New resonance assignment highlighted both wrong and missing assignments in the original work. New structures of two conformers are presented. Their intermodular orientations are not congruent with original solution structures, but with the crystal structures; this observation is analysed and discussed. Dynamics of DAF₂₃ are described using re-acquired ¹⁵N relaxation data and the model-free method of analysis. DAF₂₃ shows very limited intermodular movement, with consistent orientation. Key interface residues to not appear to be moving on timescales different to the rest of the modules. Conflicting interpretation of mutagenesis data is now resolved; key residues at the 2/3 interface are important in stabilising structure, not solvent exposed and directly involved in binding as previously thought. This thesis also describes the purification of DAF fragment DAF₁₂ and DAF₂₃₄, to a suitable quality for study by NMR spectrometry. Use of a strong cation exchange column and a gradient elution provided the level of purification required.

Thus thesis reports the high-resolution structures of four different heme-containing enzymes: two dioxygenases (tryptophan 2,3-dioxygenase and an indoleamine 2,3-dioxygenase), and mutant forms of flavocytochrome <i>b₂</i> and nitric oxide synthase. We have solved the x-ray crystal structure of TDO from <i>Xanthomonas campestris </i>in the apo-form (at 2.7 Å resolution) and with ferric heme at the active site (at 2.6 Å resolution). Two 1.6 Å and 1.8 Å resolution structures of the catalytically active, ferrous form of the enzyme in binary complex (respectively with the substrate L-tryprophan and with the substrate analogue 6-fluoro-tryptophan) have also been determined. These data allow important insights into substrate recognition, defining the substrate specificity. The structure shows that the enzyme is a tetramer with a heme cofactor and a substrate molecule bound at each active site. A second, possibly allosteric, L-Trip-binding site is also identified at the tetramer interface. The active site is fully formed only in the binary complex, showing that TDO is an induced-fit enzyme with significant structural changes on the binding of substrate. The structure of TDO in complex with L-Trp revealed that histidine 55 hydrogen bonds to substrate, helping to correctly position it in the active site and possibly being implicated as a catalytic base during the reaction. Histidine 55 was replaced by alanine (H55A) by site-directed mutagenesis. The crystal structure of TDO H55A has been solved to 2.15 Å resolution in a new crystal form, in the presence of the substrate L-Trp. The different crystal form stabilizes the N-terminal region of the H55A mutant enzyme, which is better defined than in wild-type TDO.

Production of polyamines and nucleoid-associated proteins is tightly regulated and restructures the nucleoid-associated proteins is tightly regulated and restructure the nucleoid under environmental conditions that induce DNA damage into an even more highly condensed conformation. These ‘stressful’ conditions can cause the specific methylation sequence of DNA to be lost, which leaves the DNA open to self-attack by restriction enzymes. One such enzyme is EcoKI, a type I restriction enzyme that protects the bacterial cell by destroying foreign invading DNA. Upon loss of specific methylation, EcoKI could potentially destroy the host DNA and kill the bacteria. This damaging restriction is alleviated due to partial proteolysis of EcoKI by C1pXP, although a reduced ability to destroy incoming foreign DNA is maintained. However, this method of alleviation does not exist for all type I enzymes, implying that additional restriction alleviation is required to protect bacteria. In this thesis, it has been found that the condensed structure of DNA produced by the polyamine spermidine and the nucleoid-associated protein StpA, as well as the non-specific DNA-binding of the ligand YOYO, dramatically inhibit EcoKI ATP hydrolysis an restriction activities. These results show that condensation may be a method used by bacteria to protect the nucleoid from self­-attack by EcoKI under DNA-damaging conditions, and therefore forms a second mechanism of restriction alleviation. Such a condensed DNA structure may inhibit access of the enzyme to its binding site as well as inhibiting the physical ability to translocate DNA. This is in contrast to invading foreign ‘naked’ DNA in the cytoplasm, which adopts a more open conformation, and therefore forms an ideal substrate for EcoKI translocation and restriction.

The work presented here probes a variety of non covalently interacting biological systems by electrospray mass spectrometry (ESIMS). Two systems were studied in depth. Cyclophilin A (CypA), an important protein in the immune system which forms a strong non covalent complex with Cyclosporin (CsA). Conditions required to preserve the complex between CypA and CsA were investigated. The optimised conditions were used to analyse a variety of CsA peptides analogues and synthetic mimics’ affinity for CypA, culminating in the screening of a combinatorial library of potential CypA inhibitors. Calmodulin (CaM) is an acidic ubiquitous calcium binding protein, which undergoes a large conformation change upon binding four Ca²⁺ions. Ca²⁺loaded CaM interacts with nitric oxide synthase (NOS) enzymes, which in turn control the production of the signalling molecule and cytotoxin NO. The interactions between CaM and the FMN region of nNOS are probed by ESIMS and the 46 kDa complex formed by CaM:nNOS has been retained in the gas phase. This is shown to be exclusively selective for CaM.4Ca²⁺ Further characterisation of this system is afforded by examining a complex of CaM with a 22 residue synthetic peptide which is the proposed CaM binding site of nNOS. The strength of the interaction was examined using collision induced dissociation at a variety of charge states. Affinities of magnesium, strontium, lead, and cadmium for the calcium binding sites of CaM have also been investigated by ESIMS. All of these divalent cations behave differently with respect to CaM. Analysis of non covalent interactions by ESIMS is shown to be a powerful technique which compliments other physical structural analysis techniques and opens new avenues to study effects of toxic metal ion binding.

No description available.

No description available.

A cytochrome <I>c</I><SUB>3</SUB> has been purified from <I>Shewanella </I>sp. NCIMB400 and the gene encoding it, <I>cycA</I> has been cloned and sequenced along with some flanking sequence. The mature protein is 86 amino acids in length and contains four covalently bound haem groups, with a molecular weight of 11780 Da. The electronic absorption spectrum is characteristic of a low-spin c-type cytochrome with an α-peak at 551nm. A redox titration of the four haems, yielded of values at -60 mV and -200 mV, within the range observed for cytochromes <I>c</I><SUB>3</SUB>. Resonance Raman spectra of the protein contain bands characteristic of low-spin haems, consistent with bis-histidine ligation for all haems. The deduced amino acid sequence from the <I>cycA</I> gene is 86 residues long for mature cytochrome <I>c</I><SUB>3</SUB> and has an additional 25 amino acid, periplasm directing presequence. The predicted sequence contains four, CXXCH haem binding motifs. This is consistent with the presence of four <I>c</I>-type haems in the mature protein. A total of eight histidines in the amino acid sequence is consistent with four bis-histidine ligated haems. Two additional reading frames, in the same orientation, lie on either side of the <I>cycA</I> gene. The reading frames show sequence similarly with two cytoplasmic proteins and are clearly unrelated to cytochrome <I>c</I><SUB>3</SUB>. Flavocytochrome <I>b</I><SUB>2</SUB> (L-lactate : cytochrome <I>c</I> oxidoreductase (E. C. 1.1.2.3.)) is a homotetrameric enzyme from the mitochondria of <I>Saccharomyces cerevisiae. </I>Each monomer consists of an N-terminal cytochrome domain and a C-terminal flavin domain, joined by a short flexible peptide. The haem domain has been identified as being mobile from the crystal structure of the <I>Saccharomyces cerevisiae </I>enzyme and a nuclear magnetic resonance study of <I>Hansenula anomala </I>flavocytochrome <I>b</I><SUB>2</SUB>. The rate of flavin to haem electron transfer observed in the wild-type enzyme is ˜ 1500 s<SUP>-1</SUP>. This rate has is lower than expected considering the distance between the cofactors is ˜10Å.

The first part of this work is dedicated to technical issues and system developments; the design and construction of an open microscope which forms the base for an optical tweezers set-up, is described. Subsequently the creation of a generic optical tweezers set-up is presented. This set-up was used in combination with two photon fluorescence to investigate the deteriorating of the quality of the trap due to optical aberrations. It is shown that using adaptive optics (i.e. a deformable membrane), the aberrations are partially corrected. Furthermore, it was demonstrated that using the deformable membrane one can achieve the axial-position control of the trapped particle. The same open microscope was used in combination with a ferroelectric liquid crystal spatial light modulator to construct multiple trap tweezers. The fast switching speeds of the ferroelectric device, compared to the conventional nematic systems, is shown to enable very rapid reconfiguration of trap geometries, controlled, high speed particle movement, and the firs tweezers array multiplexing. The second part of the work focuses on biophysical applications. A commercial microscope combined with fluorescence imaging is used to analyse the stretching and unwinding of DNA, as well as DNA condensation. Stretching and unwinding of polymers under flow is a very important phenomenon associated with the rheological properties of dilute polymer solutions. Scaling theory predicts two broad regimes for the overall shape of the deformed DNA molecule: the ‘trumpet’ regime at low flow velocities and a ‘stem and flower’ regime at high flow velocities. I studied the DNA shape at different velocities using optical trapping of single DNA molecules tethered on polystyrene beads. The results show clearly the two theoretically predicted regimes of ‘trumpet’ and ‘stem and flower’.