Thesis (Ph. D.)--University of Sydney, 2004. / Title from title screen (viewed 14 May 2008). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the chool of Molecular and Microbial Biosciences, Faculty of Science. Includes bibliographical references. Also available in print form.

Thesis (Ph. D.)--Oregon State University, 2006. / Printout. Includes bibliographical references (leaves 127-136). Also available on the World Wide Web.

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

Proefschrift Rijksuniversiteit Groningen. / Datum laatste controle: 15-12-1997. Met lit. opg., bibliogr. - Met samenvatting in het Nederlands.

No description available.

Antibodies are capable of high specificity interactions with a virtually infinite range of substrates (antigens). This property has lead to a number of scientific and medical applications. The extreme variability is essentially confined to 6 hypervariable loops or 'CDRs' which constitute the antigen combining site. To make intelligent modifications to antibody affinity and specificity, by methods such as site directed mutagenesis, requires an understanding of the relationship between primary sequence and three dimensional structure of the combining site. A new 'combined algorithm' which makes use of both knowledge-based and ab initio (conformational search) modelling approaches is presented. It is routinely and reliably able to predict the conformation of all six CDRs and requires no arbitrary decisions by the user. All known protein structures are searched for loops of conformation similar to known antibody structures. These are positioned onto the conserved framework and the loops are processed into a form suitable for conformational search using the program CONGEN (Bruccoleri and Karplus, Macromolecules 18(1985),2767--2773). The midsection of each loop is deleted and reconstructed by conformational search. The conformations generated are screened using a solvent-modified potential and, from the low energy conformations, a final choice is made on the basis of structurally determining residues. The method presented provides a route by which to model modifications to known antibody structures or to model complete antibody combining sites - either in combination with other less computer intensive methods, or alone. The procedure has been tested by the individual modelling of the 6 CDR's of two antibodies, in the presence of the crystal structure of the other 5 loops. In addition, it has been applied to modelling CDR's which are difficult to model by other methods and to the construction of a complete antibody combining site. In all cases the algorithm performed very well.

Experiments facilitating the engineering of the PETNR active site to accommodate a range of non natural enone substrates with substituents localised on the α and β carbons of the unsaturated bond are described. In order to facilitate the high throughput purification of PETNR libraries poly histidine (PETNRHis) and biotin (PETNRBio) tagged PETNR variants were generated. High throughput protocols were developed for the automated generation, purification and screening of libraries in a 96 well format. Protocols were optimised and trialled using blocks consisting of PETNRHis WT only and characterised in terms of intra block variation. A range of single site saturation mutagenic libraries were generated at positions in the active site consisting of T26, Y68, W102, H181, H184, Y186, Q241 and Y351. Sequencing results indicated randomised libraries with the occasional instance of bias evident. Expression and purification in a 96 well format was monitored by SDS PAGE and protein quantitation. Library activity was quantified and demonstrated to retain varying degrees of activity with the model substrate 2-cyclohexenone. Following this verification of the experimental protocol libraries were screened against a range of substrates analogous to substrates demonstrated to be active with PETNRWT but incorporating substituents at the α and β carbons. 'Hits' generated from these screening reactions were studied further by the determination of the specific activity and quantitation of substrate/product from biotransformation reactions. From these screening experiments totalling 3,600 individual reactions, 35 were identified as potential hits, of these 8 proved to be genuinely improved variants. Substituents at the β carbon were demonstrated to compromise the activity of the WT enzyme most severely. Positions 68, 102, and 351 were demonstrated to play an important role in the accommodation of substituents at the α carbon whilst residues 26 and 351 are important for the β carbon. The best variants demonstrated up to 9 fold improvements in poor substrates which represented rates in excess of those observed for model substrates.

Copper-containing nitrite reductase (NiR) is a homotrimer of two cupredoxin domains and catalyzes the single electron reduction of NO2- to NO during dissimilatory denitrification. To investigate the evolution of NiR, methods of mutagenic library generation and high-throughput variant screening from E. coli colonies were developed. These methods allow for facile screening of 105 mutants for folding efficiency or substrate specificity. Initial proof of principle studies yielded several variants that oxidized the artificial substrate ο-dianisidine up to 8 times faster than wild type NiR, suggesting that this methodology has the potential to engineer NiR to acquire other reductase functions. A crystal structure was solved for a putative multicopper oxidase (MCO) and NiR homologue from Arthrobacter sp. (AMMCO) to 1.8 Å resolution. The overall folds of AMMCO and NiR are very similar (r.m.s.d. of 2.0 Å over 250 Cα atoms); Like NiR, AMMCO is a trimer with type-1 Cu sites in the N-terminal domain of each monomer; however, the active site of AMMCO contains trinuclear Cu site characteristic of MCOs instead of a the mononuclear type-2 Cu site found in NiR. Detailed structural analysis supports the theory that two-domain MCOs similar to AMMCO were intermediaries in the evolution of NiR and the more common three-domain MCOs. The physiological function of AMMCO remains uncertain, but genomic, crystallographic and functional analysis suggests that the enzyme is involved in metal regulation. Considering the extensive similarity between AMMCO and NiR, particularly at the active site, engineering a trinuclear cluster into NiR appears feasible with a modest number of alterations to the polypeptide chain. With the aid of my newly developed high-throughput screening technique and site-directed mutagenesis, the mononuclear NiR active site was remodelled into a trinuclear Cu site similar to that of MCO. A crystal structure of this variant was solved to 2.0 Å and the presence of three copper atoms at the engineered cluster was confirmed by Cu-edge anomalous diffraction data. Although the trinuclear copper cluster is present and catalyzes the reduction of oxygen, achieving rates of catalysis seen in native MCOs has proven more difficult. With the framework provided, further engineering NiR into a robust MCO is likely to provide further insights into the structural basis of oxygen reduction by trinuclear copper sites. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The research carried out within this thesis was aimed at the development and implementation of a biocatalytic route towards Ticagrelor, a platelet-aggregation inhibitor. A bio-retrosynthetic consideration of the target compound yielded different possible strategies, which were analysed in terms of enantioselectivity and efficiency. The ultimate goal was to generate a biocatalyst specifically tailored to the starting material to yield the target compound in high optical purity and conversion. Different approaches to the chemoenzymatic generation of the cyclopropyl subunit (cf figure) in enantiomerically pure form were proposed and tested. The lipase from Thermomyces lanuginosus proved to be the most selective and active enzyme tested and was used as a model enzyme, initially yielding an E of 76 at a conversion of 50% after 48h. Through both reaction engineering and rational protein design approaches the time to attain 50% conversion could be reduced to 24 h while the enantioselectivity of the process increased to 100. Moreover, in a rational protein design approach different residues in the lid of the lipase were identified through analysis of the resolved crystal structures and subsequently mutated in order to investigate the influence of these residues on the overall performance of the lipase towards the target biotransformation. Mutations on Asn88 resulted in the inactivation of the enzyme while an Asp57Asn mutation resulted in a more active enzyme. Ultimately, this research has contributed to making the synthetic route towards Ticagrelor more environmentally sustainable, diminishing the need for the use of toxic, unsustainable and sterically demanding auxiliaries, as well as the amount of waste produced. The principles of green chemistry have been applied to the case studied. The synthetic route towards a key fragment of Ticagrelor has been significantly shortened using a biotransformation with an enzyme that can be recycled and employed in catalytic quantities.

No description available.