A very versatile chemioenzymatic synthesis of stereospecifically labelled amino acids first developed in our laboratory has been altered and improved so that only chemical steps are involved. Unlike the original synthesis, monodeuteriated compounds can be obtained so that isotope effects can be clearly identified. The synthesis has been used to prepare (2R,3R)-[3-2H1]-p-chloroalanine, (2R,3S)-[3-2H1]-p-chloroalanine, (2S,3R)[ 3}Hd-p-chloroalanine and (2S,3S)-[3-2Hd-p-chloroalanine. The first two compounds were incubated with D-amino acid aminotransferase, and the second two compounds were incubated with L-aspartate aminotransferase. The stereochemical outcome of these reactions indicated retention of stereochemistry in the processes, casting light on similarities between the evolutionary families of PLP-dependent enzymes. A synthesis of stereospecifically labelled (2S)-propargylglycine was attempted. The key intermediates (2S,3R)-[3-2H1]-N-2-nitrobenzenesulfonylaziridine-2-carboxylate and (2S,3S)-[3-2H d-N -2-nitrobenzenesulfonylaziridine-2-carboxylate were synthesised and regiospecific ring opening of the intermediates was achieved. Time did not permit full deprotection of these products

The interactions between proteins and peptides in aqueous solution have been investigated using a classical molecular dynamics procedure with a molecular mechanical representation of the potential energy surface. During post-processing of the trajectory an implicit solvation method has been applied in order to calculate the free energy of each of the complex, protein and peptide structures in solution, allowing the binding free energy of the protein-peptide complex to be evaluated. Entropic contributions have been estimated using classical ideal gas thermodynamics. A program has also been developed that systematically mutates each of the peptide residues to alanine and determines the effect on the binding free energy. The method has been applied to the interaction between the oncoprotein Mdm2 and the tumour suppressor peptide p53 and reasonable agreement has been found with previous theoretical and experimental studies. The method has also been extended to the interactions between IQN17, an engineered protein that represents a potential drug target in the HIV-1 gp41-mediated cellular fusion process, and several peptides that have been shown to inhibit cellular fusion. The key residues in the binding of each protein-peptide system have been identified and quantitative information regarding the factors influencing binding obtained and compared with available experimental data, demonstrating encour aging agreement with analogous alanine scanning experiments.

Protein comparative modelling (CM) is a predictive technique to build an atomic model for a polypeptide chain, based on the experimentally determined structures of related proteins (templates). It is widely used in Structural Biology, with applications ranging from mutation analysis, protein and drug design to function prediction and analysis, particularly when there are no experimental structures of the protein of interest. Therefore, CM is an important tool to process the amount of data generated by genomic projects. Several problems affect the performance of CM and therefore solutions for them are needed to increase its applicability. In this work different algorithms and approaches were tested with this aim, particularly to help in template selection and alignment, and some useful insights were obtained. First, this work describes the development of DomainFishing, a tool to split protein sequences into functionally and structurally defined domains and to align each of them to the available templates. The performance of our approach is benchmarked and some problems and possible developments are identified. When comparing different alignment procedures none of them is found to be consistently superior, suggesting that a combination of several could be an advantage. Driven by these ideas and the fact that selecting templates can be a difficult problem, a new modelling approach is designed and tested. This algorithm uses crossover, mutation and selection within populations of protein models generated from different templates and alignments to obtain recombinant structures optimised in terms of fitness. Despite our simple definition of fitness, the procedure is shown to be robust to some alignment errors while simplifying the task of selecting templates, making it a good candidate for automatic building of reliable protein models. In-house benchmarks of the method show its strengths and limitations. The method was also benchmarked during the fifth Critical Assessment of techniques for protein Structure Prediction (CASP5), in which its perfomance was encouraging both for comparative modelling and fold recognition targets, among the top 20 predictors. Finally, we present some data to support a possible evolutionary feedback mechanism between protein structure and gene structure, using human and murine genomic data, structural data from the Protein Data Bank and the protein recombination methodology. This may have some implications for understanding protein evolution and protein design, which are discussed.

Previous work, reported in the literature, has suggested that isolated catalytic domains from highly reducing iterative polyketide synthases (HR iPKS) should be able to perform their programmed function independently of the complete polyketide synthase (PKS). To test this hypothesis, the isolated enoyl reductase domain (ER) of squalestatin tetraketide synthase (SQTKS) was produced and tested in vitro with substrate mimics of intermediates of the biosynthesis of squalestatin tetraketide 54. Both the selectivity of the enzyme and the stereocontrol of the catalyzed reduction were investigated. Two assay methods were designed using UV absorption and LCMS to measure the initial rate and percentage turnover of the enzymatic reaction to test the substrate selectivity of the ER domain. The results of these assays demonstrated that substrate recognition was dependent on methylation of the substrate and in particular methylation at the 8-position was critical for preventing the enzymatic reduction from occurring. A second set of assays was designed to test the stereochemical outcome of the enzymatic reduction. These results demonstrate that although the transfer of ~-hydride from NADPH to the substrate is controlled, the isolated ER domain is unable to control the astereocentre. The results from this study have shown that the ER domain is able to recognize substrates without the complete PKS but for the reduction to be stereo controlled the complete PKS may be required. To better understand the results a computer model of the isolated ER domain was created and by examining the model four amino acids (F 1941, I 2001 , L 2146 and I 2147) that could control substrate recognition in the ER domain were highlighted. Also three other amino acids (R 1924, D 1925 and M 1927) that might be involved in controlling the stereochemistry of the a-position were also found in the active site of the ER domain.

No description available.

No description available.

No description available.

This thesis details the synthesis of self-immolative linkers based on 1 ,4-disubstituted-1 H-1,2,3- triazoles prepared via copper catalysed [3+2] azide-alkyne cycloaddition chemistry and their degradability as a function of pH. Their application to aqueous soluble polymer conjugates was also demonstrated. This thesis is divided into five chapters and an appendix. Chapter 1 provides an introduction to the theory of self-immolative elimination, and discusses the types of self-immolative linkers developed and their application to the controlled release of drug, sensor and fragrance molecules. The discussion has focused on the occurrence and use of self-immolative linkers in both biological and synthetic polymer conjugates and their release characteristics. Chapter 2 details the synthesis of a range of structurally diverse carbamate, carbonate and ether functionalised self- immolative triazoles bearing model fragrances or fragrance surrogates. A range of organic azide, and carbamate, carbonate and ether functionalised alkyne precursors were synthesised and methodologies optimised. Optimisation of the subsequent copper catalysed [3+2] azide-alkyne cycloaddition reaction was also conducted. Furthermore, a range of analytical standards were prepared for a comparative study aimed to identify, and differentiate between, degradation pathways. Chapter 3 depicts the degradation studies conducted on the self-immolative triazole linkers prepared . in Chapter 2. The acid and/or base sensitivity of these compounds were determined using a standardised methodology, and their labilities compared within, and across, each series. NMR spectroscopy was utilised to gather kinetic data with which structure-activity relationships could be highlighted and quantified. The analytical standards synthesised in Chapter 2 were utilised to positively identify reaction intermediates and product fragments. These analyses led to the proposal, and subsequent discussion of reaction mechanisms. Chapter 4 describes the synthesis of a range of polymers prepared by atom-transfer radical polymerisation featuring self-immolative triazole tethers. Firstly, a range of methodologies to obtain azide containing polymers were explored. Initiator, catalyst and solvent systems were optimised for each series. The copper catalysed [3+2] cycloaddition was examined using a range of synthesised polymers, and the lead material subjected to a degradation study. The 'staged-release' of multiple components under acidic conditions was demonstrated utilising analogous conditions to those developed in Chapter 3. Chapter 5 provides experimental details and analytical data for the small molecule compounds synthesised in Chapter 2, the degradation experiments conducted in Chapter 3 and polymeric materials synthesised in Chapter 4.

This work describes the development of a systematic methodology to overcome two of the difficulties commonly encountered when expressing eukaryotic domains in bacterial hosts, namely the failure to obtain folded protein in vivo and the low solubility of the expression product. This methodology made possible the production of samples of the first scavenger receptor cysteine rich (SRCR) domain of human CD5, /zCD5dl, with properties suitable for multidimensional NMR studies. The SRCR domains of /zCD5 express in bacteria as insoluble aggregates. The aggregates were purified in order to perform the folding in vitro. Optimal conditions for folding were found using a novel systematic screen based on a fractional factorial design. In vitro folding yields were assessed using RP-HPLC and non-denaturing PAGE. The attainment of /zCD5dl protein samples of sufficiently high concentration to perform multidimensional NMR was achieved by performing rational apolar-to-polar mutations selected by analysis of a multiple sequence alignment. Eight single residue mutants were engineered and expressed. Four of them had better in vitro folding yields than the wild type and a double mutant was constructed by combination of the best behaved single mutations. This double mutant was used to determine the structure of the domain. NMR experiments at 298 K showed that some regions of /zCD5dl undergo conformational exchange on a microsecond to millisecond timescale, hampering the assignment of the resonance signals. Increasing the temperature to 318 K was found to greatly enhance the quality of the NMR spectra and enabled the assignment of more than 95% of the resonances. The solution structure of /*CD5dl was determined using standard interproton distance and dihedral angle-restrained molecular dynamics protocols. Forty percent of the residues were found to be in structurally well-defined regions, including all of the regular secondary structure features found for other members of SRCR superfamily. The remaining residues of the polypeptide appear to be distinctly less well ordered.

No description available.