No description available.

My research during this interdisciplinary investigation was to primarily focus on the development of novel single cell sampling and analysis methods. Using micromanipulation, microsampling and mass spectroscopic analysis techniques to determine the concentration of biologically relevant molecules quantitatively from living fungal cells, as well as to study the uptake of externally applied compounds. Evaluation of a range of on-line separation techniques, including Capillary Liquid Chromatography (CapLC), nanoLC and capillary electrophoresis (CE) was made for suitability to be coupled to a range of mass spectrometers for the analysis of fungal cytoplasm. Techniques for microsampling at the single cell level from the model organism, <i>Neurospora crassa</i>, and transfer of the sample from under the microscope for analysis by mass spectrometry, following on-line separation, were developed. Capillary electrophoresis electrospray mass spectrometry (CE-ESI-MS) was used to measure intra cellular concentrations of trehalose, quantitatively, with mechanisms developed for detection of external contamination of the samples taken from viable, actively growing cells of <i>Neurospora crassa</i>, by the use of laser induced fluorescence (LIF).  The measured concentration for intra-cellular disaccharide was determined to be 1.3mM by the methods developed and described during this thesis. In addition, topical applications of fungicide were made, with intra-cellular measurements taken by single cell analysis. Following application of 14.8μM azoxystrobin, the fungicide under investigation at saturation concentration in water, intra-cellular concentrations were measured at 9.9μM after 5 minutes.

The current theories of the membrane electrical properties of the giant algal cells of the Characeae are reviewed. The experimental techniques for the determination of such properties are discussed and a comparison between these techniques, for both the D.C. and A.C. case, are made. In the latter case it was necessary to obtain the time dependent solution of the A.C. cable theory and the numerical behaviour of this solution. From this comparison it is proposed that symmetric external current injection together with the 0.42L technique is a very suitable method for these studies. Using this method the membrane electric parameters of Nitella translucens were determined. The effects of external pH, 4-dinitrophenol and A.C. of different frequencies on these parameters were studied. The effects of external pH and the presence of DNP on the vacuolar pH were also determined. It was found that the experimental observations favoured the Spanswick theory for the Characean membrane electric properties, but modified to account for the membrane resistance being pH independent. The observed effects of DNP favoured the mechanism for the action of DNP proposed by Duncan and Croghan. The observed A.C. frequency dependence of the membrane resistance and experiments on the punch-through effect are inconsistent with the Coster double fixed charge model of membrane structure.

Fourier transform ion cyclotron mass spectrometry (FT-ICR MS) has been used to study a number of metalloproteins. The combination of high-resolution and mass-accuracy, along with fragmentation techniques such as CID, IRMPD and ECD, is shown to be a powerful method to probe metalloproteins and metallodrug interactions. The inherent high resolving power and mass measurement accuracy of the FT-ICR MS also enables the oxidation state of the metal present in a metalloprotein to be identified from the isotopically resolved spectra. Carbonic anhydrase II (29 kDa) is an enzyme that catalyses the reversible hydration of carbon dioxide and contains one Zn²⁺ ion. To investigate non-covalent protein-drug interactions, the inhibitor acetazolamide was added to a sample of carbonic anhydrase II. Nozzle-skimmer CID, IRMPD and titration methods were used to probe the strength of interaction of the carbonic anhydrase-acetazolamide complex. It was found that lower charge-states bind more strongly than higher charge-states suggesting that the protein unfolds when more protons are bound. FT-ICR MS was used to investigate the nature of adducts between the platinum anti-cancer drug cisplatin, and SOD. The structure showed a cisplatin adduct at His19 of beSOD, which had retained the chloride ligands; these are usually lost in aqueous conditions. After incubation with cisplatin, the mono-platinated species was the most prominent adduct observed. The use of ¹⁵N-labelled cisplatin led to unambiguous assignment of the two ammonia ligands being retained after binding to beSOD, supporting established cisplatin solution behaviour. Mass spectra of heSOD were more complex than those for beSOD due to phosphate buffer adducts and the loss or substitution of the chloride ligands. ECD fragmentation spectra of the isolated mono-platinum adduct of beSOD were used in order to localise the site of modification.

Fluorescens Lifetime Imaging Microscopy (FLIM) and fluorescence lifetime spectroscopy have been used to characterise the photophysics of different mutants of Green Fluorescent Protein (GFP) and to show evidence of Förster Resonance Energy Transfer (FRET) between different mutants in both model systems and in biologically significant cell samples. Two different imaging systems have been used and compared, a time gated intensified CCD camera (tgiCCD) system and a time and space correlated single photon counting (TSCSPC) system. GFP has been cloned into Escherichia coli and investigated by FLIM and lifetime spectroscopy, both in vivo and in vitro, to show how the fluorescence lifetime of the protein changes as the microenvironment of the protein, and hence the fluorophore, changes. In conjunction with collaborators, cancer cell signalling was investigated by looking for FRET between molecules tagged with different GFPs in fixed mammalian cells. When no consistent energy transfer was observed between GFP and YFP, or between GFP and RFP, in the biologically important cells, a model system was devised.  This model system consisted of the mutant enhanced Cyan Fluorescent Protein (CFP) expressed in fixed cells both alone and as an engineered FRET construct with enhanced Yellow Fluorescent Protein (YFP). This system showed a reproducible FRET signal. Comparison of the results from the CCD camera system, frequently used by biologists to undertake FLIM, and the higher resolution TSCSPC detector highlighted a phenomenon that potentially undermines work that relies on average lifetime results. There is a difference in the rate, or amount, of energy transfer from different conformers of the CFP to the YFP acceptor. This results in an overestimation in the separation distances calculated by FRET between this donor –acceptor pair.

Biotin synthase (BS) catalyses the final sulfur incorporation step in the biosynthesis of biotin. Its chemical mechanism, primary amino acid sequence and tertiary place it in the Radical-SAM-dependent superfamily of >600 proteins. The gene encoding <i>E. coli</i> biotin synthase (<i>bio</i>B) has been expressed as a histidine-fusion protein (6HisBS) and the recombinant protein purified in a single step using immobilised metal-affinity chromatography (IMAC). In addition, a number of single and double amino acid mutations of the conserved cysteine residues within the ‘cys box’ were constructed and purified using similar methodology. Wild-type and mutant 6HisBS proteins were compared using mass spectrometry (LC-ESI-MS) and UV-visible spectroscopy to probe characteristics altered by the mutation. Biotin synthase was analysed for its ability to bind the cofactor pyridoxal 5’-phosphate (PLP). Two single point mutations (K490Q and K49R) of a putative PLP-binding residue, Lys 49, were isolated and compared with wild-type BS using a combination of biochemical and mass spectrometry techniques. Each protein was subjected to enzymatic proteolysis and peptide modification analysed by means of MALDI-ToF MS, ESI-QTOF MS and MS/MS. We identified a unique Lys49-containing fragment of the 6HisBS which corresponds to the PLP modified peptide. In contrast, this modified peptide is absent in the mutant proteins. This observation is further confirmed by MS/MS sequencing of this MS observed species which shows CID of both the peptide backbone and covalently attached PLP moiety. Biotin synthase was prepared in the presence of excess PLP and its interaction with cysteine monitored with UV-visible spectroscopy. We observed marked changes in the UV-vis profile of biotin synthase under these conditions which is consistent observations of PLP-dependent cysteine desulferase enzymes.

The most successful method to date for predicting protein tertiary structure from primary sequence data is homology modelling based on alignment with similar sequences of known structure. The use of a variety of computing methods to identify the best similarities is discussed. Model building based on alignments and the construction of libraries of side chain conformations is described. The application of sequence alignment modelling to the structure prediction of EcoKI type I DNA methyltransferase is shown in the context of corroborative laboratory experiments. Finally, a method is presented which incorporates sequence alignment with secondary structure prediction. A program - sss_align - which incorporates this method, was used to make blind fold recognition predictions as part of an international collaborative exercise in the critical assessment of methods of protein structure prediction ('CASP2'). It was shown by this and other assessment methods that sss_align will detect similarities between sequences which exhibit as little as 15% identity.

An investigation into the conformational stability of the small globular protein ubiquitin (Ub) is described. Solid phase peptide synthesis has been successfully applied to the construction of both mammalian ubiquitin, as well as to a ubiquitin analogue (Ubdes-Core) lacking the protein's pronounced hydrophobic core. In each case the synthetic protein was purified to homogeneity using a combination of gel filtration, dialysis and ion exchange chromatography. Synthetic ubiquitin was found to be fully active in a ubiquitin specific <i>in vitro</i> protein conjugation assay, whereas Ubdes-Core was found to be completely inactive. The structure of these two proteins has been studied in some detail. Synthetic ubiquitin possesses an identical crystal and NMR derived solution structure to its natural counterpart. Removal of the hydrophobic core from ubiquitin results in a substantial loss in conformational stability (3.7 kcal mol<SUP>-1</SUP>) and consequently Ubdes-Core is unable to adopt the ubiquitin native fold. This leads to the conclusions that hydrogen bonding contributions are not in themselves sufficient to stabilize the native conformation of ubiquitin. The preparation of a 128 residue precursor protein comprising ubiquitin fused to a C-terminal extension protein (CE-52) is presented along with the CEP52 fragment alone. The compounds were prepared by solid phase peptide synthesis and purified using a combination of gel filtration, ion exchange and HPLC. Two-dimensional NMR analysis of the UbCEP52 precursor revealed, significantly, that it possesses no well defined tertiary structure. However, the protein was found to be processed <i>in vitro</i> to mature ubiquitin and CEP52. A possible novel function of ubiquitin in unfolded C-terminally fused conujugates is suggested.

The following thesis details the structural and thermo-dynamic analysis of melittin, mutant tryptophan cage constructs and a series of truncated murine β-defensin 14 derivatives in solution, <i>in vacuo </i>and <i>in silico. </i>These experiments are used to identify correlations between their solvated and desolvated structural isoforms and, in doing so, investigate the role of mass spectrometry in the field of structural proteomics. The integrated experimental approach described in this thesis employs an array of analytical techniques including charge partition calculations, capillary-induced thermal degradation, gas phase hydrogen/deuterium exchange and collision induced dissociation to obtain a consensus view of the structural organisation of each analyte. Supporting evidence is presented from antimicrobial assays, molecular modelling and dynamics calculations, circular dichroism and fluorescence spectroscopy and ion mobility mass spectrometry. In addition, a novel gas inlet system is detailed which allows gas phase ion/molecule reactions, such as hydrogen/deuterium exchange, to be performed within the quadrupole ion traps of a Finnigan LCQ Classic. Significant differences are observed between the solvated and desolvated structures of most of the analyte ions examined, unless they exhibit a very stable structural fold. This suggests an important role for mass spectrometry in the field of structural proteomics, but, one that is limited to the comparative study of different solvation states, the analysis of biologically important membrane interactions and the analysis of peptides and proteins that are expressed or secreted into hydrophobic environments.

Design, development and implementation of a new Ion Mobility capable Mass Spectrometer – the MoQToF – are presented. The instrument is a Micromass Q-ToF modified to include a temperature regulated drift cell. Initial testing of the instrument to measure cross sections of well characterised proteins (cytochrome C, ubiquitin and lysozyme) in a range of charge states is described, showing the apparatus to perform well in comparison with values reported by others on analogous instruments. Ion mobility data is presented on a number of novel systems from small peptides to large proteins. The largest volume of work focuses on the study of β-defensins and related peptides. Β-defensins are small anti-microbial peptides that form a vital part of the innate immune system of all mammals, they are characterised by the presence of six conserved cysteine residues connected via disulphide bonds. Characterisation of these bonds (number and topology) using mass spectrometry based techniques is presented. The ion mobility data presented here probes the influence of these disulphide bonds on conformational flexibility. The mode of action of β-defensins is not known, two techniques designed to further understanding of this are described here.  Firstly, a mass spectrometry based technique in which the interaction between a defensin, DEFB107, and an artificial membrane is studied using hydrogen deuterium exchange revealing the N-terminal section of the peptide to interact favourably with the lipid bilayer. Secondly, a heparin binding assay is described revealing a relationship between heparin binding strength and anti-microbial activity. This interaction is further studied using the MoQToF and molecular modelling.