This thesis examines the tolerance and effects of unnatural amino acid (Uaa) incorporation into proteins in Escherichia coli using an expanded genetic code. Uaa incorporation was used to alter or install new properties in the target proteins, superfolder Green Fluorescent Protein (sfGFP) and cytochrome b562. Chapter 3 deals with the technical aspects of Uaa incorporation including orthogonality of the machinery and yield. Substitution of residues in sfGFP for unnatural analogues of tyrosine was shown to be a valuable way of altering the properties of the protein. Variants were generated with red-shifted fluorescence and altered excitation spectra. The majority of this work focused on the Uaa, p-azido-L-phenylalanine (azPhe) as it has a number of properties that would be desirable for use in proteins such as photoreactivity and selective reactivity with alkynes. By incorporating azPhe into key residues of sfGFP, variants were created that could be controlled using light (Chapter 4). Light-dependant fluorescence activation, deactivation and switching were demonstrated in vitro and in live cells. The molecular basis for these changes was investigated by a combination of spectroscopy and X-ray crystallography in Chapter 5. The photoreactivity of azPhe was exploited for a different purpose in Chapter 6. Proteins were used as an alternative to synthetic cages for studying low temperature phenyl azide photochemistry. Here, two radicals (anilino and triplet phenyl nitrene) were successfully caged and detected on photolysis, with the radical observed dependant on the protein environment. Finally, in Chapter 7 the selective reactivity of azPhe was used to create proteins capable of site-specific modification (via Click chemistry). The position of azPhe on the protein surface was shown to have a significant effect on reaction yield and kinetics. Modification was used to install proteins with novel properties such as red-shifted fluorescence emission and the ability to bind to non-biological materials like graphene.

No description available.

While the existence of protein was first described by Berzelius and Mulder back in 1838 and a single empirical formula noted (C400H620N100O120P1S1) (Vickery, 1950, Brand, 1946), early protein-based research was limited to the analysis of proteins which could be easily purified in large quantities, such as those obtained from blood, egg whites and those obtainable from slaughterhouses, such as digestive and metabolic enzymes (Chapman, 2005). Indeed, despite the development of recombinant deoxyribonucleic acid technologies in the 1970s (enabling protein expression) and the increasing sensitivity of techniques which enable the identification and sequencing of proteins separated by gel electrophoresis (Patterson and Aebersold, 2003), it was not until the late 1980s, with the description of soft biomolecule ionisation that large scale proteomic analyses were undertaken, based upon the use of mass spectrometry (Guerrera and Kleiner, 2005). While early mass spectrometry-based proteomic analyses focussed on the systematic identification of a great number of proteins within a single organism, the field of proteomics is now becoming increasingly quantitative (Baak et al., 2005), enabling the relative comparison of protein expression patterns between phenotypes, but also the targeted absolute quantification of specific proteins. During this project, a stable isotopically labelled internal standard based absolute quantitative technique, first described by Gerber and co-workers in 2003 (S. A. Gerber et al., 2003), was applied to the absolute quantification of three families of multiple protein isoforms. This area of research is of particular scientific interest as it is thought that up to 95% of human multi-exon genes may be subject to alternative splicing, making alternative splicing the rule, not the exception (Pan et al., 2008a). Indeed alternative splicing has also been implicated as both a cause and a consequence of disease. This technique should therefore enable both the confirmation of disease, based upon the identification of a set of phenotype specific protein biomarkers, but also the mapping of a disease’s progression (Venables, 2004). During this study, stable isotopically labelled internal standard peptides were selected for the absolute quantification of 11 confirmed protein isoforms, and two predicted protein isoforms. In addition, a separate MRM based LC-MS acquisition method was developed for the absolute quantification of each of the three families of protein isoforms (A-Raf, PDE4B and SERCA2) within a single analysis, and finally, these acquisition methods were applied to the absolute quantification of a range of immunoprecipitated, exogenously expressed protein isoforms. This project was, however, hindered by the sensitivity of the mass spectrometers available for use, preventing these acquisition methods from being applied to the absolute quantification of the endogenous levels of protein expression. While beyond the scope of this project, the further development of this quantitative technique should enable future researchers to: (i) Quantify each endogenously expressed protein isoform within a family of multiple protein isoforms. (ii) Assess any changes in the expression of each isoform in a range of cellular states, and (iii) Assess how a targeted drug treatment may affect the expression ratio of these protein isoforms.

The 1,4-benzodiazepin-2-one motif has been taken as a privileged skeleton for making antitrypanosomal agents. A library of over sixty 1,4-benzodiazepin-2-one derivatives has been synthesised employing novel synthetic routes. These derivatives were characterised spectroscopically, by mass spectrometry, and by combustion analysis. Five derivatives were characterised, in the solid state, by single crystal X-ray crystallography. Biological assays of the library of compounds against Trypanosoma brucei brucei (T. b brucei) revealed a range of trypanocidal activities. A first generation library activity showed biological activity as low as 6.25 μM (minimum inhibitory concentration, MIC value). Structure activity relationships in this work revealed that an aromatic substituent at the C3 and N1 positions of the 1,4-benzodiazepin-2-one are important for improved bioactivity. In order to improve biological activity, putative P-2 transporter motifs were exploited in the 1,4-benzodiazepin-2-ones. Structural activity relationships indicate that the inclusion of a guanidine moiety, a putative P-2 transporter motif, can improve the biological activity of these molecules. In vitro screening of these compounds showed a range of antitrypanosomal activities against T. b.brucei, with a number in the low micromolar range (MIC ≥ 0.78 μm) including (S)-1-(4-((3-benzyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-1-yl)methyl)phenyl)guanidine, (S)-1-(3-((3-benzyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-1-yl)methyl)phenyl)guanidine, (S)-1-(4-((3-benzyl-5-cyclohexyl-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-1yl)methyl)phenyl)-guanidine and (S)-1-(1-benzyl-5-cyclohexyl-3-isopropyl-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-7-yl)guanidine)phenylquanidine.

The application of a hybrid instrument combining Evanescent Wave Cavity Ring-Down Spectroscopy (EW-CRDS) with electrochemical and fluidic methods is described. The electrochemical/fluidic methods were used to induce a surface process, the effects of which were subsequently monitored in situ and in real time with exquisite spectral sensitivity and excellent temporal resolution by EW-CRDS. The well-defined manner in which the surface processes were initiated allowed the extraction of kinetic rate constants by fitting the EW-CRDS data to mathematical models of the surface process coupled to convection-diffusion. The investigations described include: the study of the thermodynamics and kinetics of the adsorption of tris(bipyridine)ruthenium(II) ([Ru(bpy)3]2+) to polypeptide films using EW-CRDS with chronoamperometry; the real-time electrochemistry of cytochrome c immobilised on silica by EW-CRDS with chronoamperometry; the kinetics of adsorption and DNA-assisted desorption of 5,10,15,20-tetra(N-methylpyridinium-4-yl)porphyrin at the silica-water interface using EW-CRDS with an impinging jet flow cell; and the monitoring the adsorption of cationic phospholipid vesicles at the silica-aqueous interface and the interaction of 5,10,15,20-Tetraphenyl-21H, 23H-porphine-p,p′,p″,p′′′-tetrasulfonic acid tetrasodium hydrate with the resulting bilayer also using EW-CRDS with an impinging jet flow cell. The work described in this thesis provides a platform on which EW-CRDS can be used to study dynamics at biointerfaces, such as the association of ions, peptides, proteins and drugs with phospholipid bilayers, the electron transfer between redox enzymes in a biomimetic environment, and the lateral diffusion of protons, ions and proteins at biomembranes. Such studies are essential to the understanding of many important cellular processes in addition to the development and optimisation of a number of bio-inspired technologies.

Liquid extraction surface analysis (LESA) is an ambient ionisation technique which allows direct analysis of surfaces coupled with mass spectrometry. LESA mass spectrometry has been used successfully to analyse small molecules, but there are a limited number of examples where the approach has been applied to protein analysis. The work presented here aims to develop novel applications of LESA mass spectrometry of proteins. LESA mass spectrometry was used to analyse intact proteins from polymeric membranes. The rationale for these experiments was the potential application to analyse proteins electroblotted following polyacrylamide gel electrophoresis, i.e. top-down proteomics, and in air monitoring. The subsequent focus was dried blood spot (DBS) analysis. An automated LESA based trypsin digestion protocol was developed and coupled with liquid chromatography tandem mass spectrometry to enable DBS proteomics. i.e., untargeted global protein identification via a bottom-up approach. Approaches for DBS proteomics (in the absence of LESA) were explored further using conventional digestion procedures coupled with protein depletion. LESA was also applied for targeted analysis of proteins from DBS, to determine variants of alpha-1-antitrypsin. Finally, native LESA mass spectrometry was developed to analyse non-covalent complexes from dried surfaces. Native LESA mass spectrometry successfully identified the haemoglobin tetramer directly from DBS.

This thesis reports the Structure Activity Relationship study that was performed upon the 5-substituted-indole core as a means to identify Negative Allosteric Modulators of the human 5-HT3A receptor for the development of potential drugs for the treatment of IBS-d. Herein is reported the successful identification of a PAM to NAM switch and three novel NAMs which provide the basis for further study into the treatment of IBS-d and insight into the identity of the allosteric site of the human 5-HT3A receptor. The design, synthesis and testing of a novel fluorescent analogue of the orthosteric antagonist Quipazine is also described for the application of an improved competitive binding experiment without the need for radio-labelled ligands. Furthermore, the design and synthesis of novel diazirinyl-indoles for photo-affinity binding studies towards the identification of the allosteric site of the 5-HT3A receptor. Finally, the design and synthesis of novel BODIPY-BAPTA based fluorescent PET sensors for the detection of larger than usual ranges in concentration of cellular Ca2+ levels are described.

Chiral recognition, determination of enantiomeric excess and the separation of enantiomers are challenging problems for the chemist. This work has as its aim the design and syntheses of new electrochemical chiral receptors for neutral molecules. All the receptors prepared contain a ferrocene group as electroactive reporting unit. The differences among the receptors mainly relate to the binding site and the chiral group. The first type of receptor, presented in Chapter 2, consists of chiral ferrocene containing boronic acids that have been used to electrochemically sense aromatic and aliphatic chiral and achiral diols. The electrochemical determination of the enantiomeric excess of a mixture of two enantiomers of Binol performed with one of these boronic acids represents a new advance in supramolecular chiral sensing. In Chapter 3 the synthesis of ferrocene-containing chiral macrocycles of different sizes is described. The binding site is a cavity featuring a diamidopyridine moiety, with the chirality introduced through a Binol unit. These receptors showed low interaction with achiral cyclic ureas and chiral carboxylic acids. Chapter 4 describes the study of self-assembled monolayers onto gold surfaces. The monolayers are formed by ferrocene-containing amides of lipoic (thioctic) acid and, for the first time, isolipoic acid. The studies indicate that isolipoic acid could be an attractive anchor group for SAM formation when strong control over the chirality of the monolayer is required.

No description available.

Two staining protocols were formulated that enabled the detection of cellular stress at the single-cell level for Bacillus cereus. Both DiOC6(3) and RedoxSensor Green™ can be employed to detect perturbations in the energetic status of the cell at concentrations of 0.30 \mug.mL-1 and 3.0 \muM respectively. These methods can be employed for sensitive analysis of bacteria of both industrial and clinical interest. Flow cytometry was used throughout this work in order to assess the quality of recombinant Escherichia coli populations present within an agitated bioreactor. It was demonstrated in shake-flask culture that the cells could be grown to moderate cell densities (OD600nm 7 25) whilst producing measurable levels of antibody fragment. Despite being described in a patent which claims invention of a 100 % effective repression system (Hodgson et al., 2006), there was extensive evidence of promoter leakiness. Fab production was usually synonymous with cellular breakdown, however, a strategy based on simultaneous feeding and induction, before the exhaustion of the primary carbon source, yielded the highest concentration of Fab, 105 mg.L-1, with more than 50 mg.L-1 successfully targeted to the extracellular environment. Unlike all the previous cultures, this attainment also preceded the breakdown in the cellular structure.