Leung Tak Yuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 57-63). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abstract --- p.ii / Abbreviations --- p.iii / Abbreviations of amino acids --- p.iv / Abbreviations of nucleotides --- p.iv / Naming system for TRP mutants --- p.v / Chapter Chapter 1 --- I ntroduction / Chapter 1.1 --- Hyperthermophile and hyperthermophilic proteins --- p.1 / Chapter 1.2 --- Hyperthermophilic proteina are highly similar to their mesophilic homologues --- p.2 / Chapter 1.3 --- Hyperthermophilic proteins and free energy of stabilization --- p.3 / Chapter 1.4 --- Mechanisms of protein stabilization --- p.4 / Chapter 1.5 --- The difference in protein stability between mesophilic protein and hyperthermophilic protein --- p.4 / Chapter 1.6 --- Ribosomal protein L30e from T. celer can be used as a model system to study thermostability --- p.9 / Chapter 1.7 --- Protein engineering of TRP --- p.10 / Chapter 1.8 --- Purpose of the present study --- p.12 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Bacterial strains --- p.13 / Chapter 2.2 --- Plasmids --- p.13 / Chapter 2.3 --- Bacterial culture media and solutions --- p.13 / Chapter 2.4 --- Antibiotic solutions --- p.13 / Chapter 2.5 --- Restriction endonucleases and other enzymes --- p.14 / Chapter 2.6 --- M9ZB medium --- p.14 / Chapter 2.7 --- SDS-PAGE --- p.14 / Chapter 2.8 --- Alkaline phosphatase buffer --- p.15 / Chapter 2.9 --- DNA agarose gel --- p.15 / Chapter 2.10 --- "Gel loading buffer, DNA" --- p.16 / Chapter 2.11 --- "Ethidium bromide (EtBr), lOmg/ml" --- p.16 / Chapter 2.12 --- Constructing mutant TRP genes --- p.16 / Chapter 2.12.1 --- Polymerase Chain Reaction (PCR) --- p.17 / Chapter 2.12.2 --- Gel electrophoresis --- p.19 / Chapter 2.12.3 --- DNA purification from agarose gel --- p.19 / Chapter 2.12.4 --- "Construction of R39A, R39M, K46A, K46M, E47A, E50A, R54A, R54M" --- p.19 / Chapter 2.12.5 --- "Construction of double mutant R39A/E62A, R39M/E62A" --- p.20 / Chapter 2.13 --- Sub-cloning --- p.21 / Chapter 2.13.1 --- Restriction digestion --- p.22 / Chapter 2.13.2 --- Ligation vector with mutant TRP gene insert --- p.22 / Chapter 2.13.3 --- Amplifying vector carrying mutant TRP gene insert --- p.22 / Chapter 2.13.4 --- Mini-preparation of DNA --- p.22 / Chapter 2.13.5 --- Preparations of competent cells --- p.23 / Chapter 2.13.6 --- Transformation of Escherichia coli --- p.24 / Chapter 2.13.7 --- Screening tests --- p.25 / Chapter 2.14 --- Over expression of mutant TRP --- p.26 / Chapter 2.14.1 --- Transformation --- p.26 / Chapter 2.14.2 --- Expression --- p.26 / Chapter 2.14.3 --- Cell harvesting --- p.27 / Chapter 2.14.4 --- Expression checking --- p.27 / Chapter 2.14.5 --- SDS-PAGE --- p.27 / Chapter 2.14.6 --- Staining the acrylamide gel --- p.28 / Chapter 2.15 --- Purification of mutant TRP protein --- p.28 / Chapter 2.15.1 --- Cells lysis --- p.28 / Chapter 2.15.2 --- Chromatography --- p.29 / Chapter 2.15.3 --- Concentrating TRP as protein stock --- p.31 / Chapter 2.16 --- Protein stability --- p.32 / Chapter 2.16.1 --- Chemical stability --- p.33 / Chapter 2.16.2 --- Thermal stability --- p.34 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Construction of mutant TRP genes --- p.36 / Chapter 3.1.1 --- PCR mutagenesis --- p.36 / Chapter 3.1.2 --- Sub-cloning of mutant TRP gene to express vector pET8c --- p.37 / Chapter 3.2 --- Expression and purification of mutant TRP --- p.38 / Chapter 3.3 --- Protein stability --- p.39 / Chapter 3.3.1 --- Free energy of unfolding --- p.39 / Chapter 3.3.2 --- Thermal stability --- p.43 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- "Effect of R39, K46, E62, E64" --- p.47 / Chapter 4.2 --- Double mutation at R39 and E62 --- p.50 / Chapter 4.3 --- Effect of R54 --- p.51 / Chapter 4.4 --- Effect of E47 and E50 --- p.53 / Chapter 4.5 --- Conclusion --- p.54 / References --- p.57 / Appendix --- p.64

Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1995. / Added t.p. with thesis statement inserted.

Strategies for the engineering of allosteric proteins, which are proteins that bind ligands at a specific site other than the reaction site and affect the reaction activity, are still being perfected. There have been allosteric proteins successfully engineered based on the hypothesis that the two allosterically related sites are distinct, modular domains and use trial and error to construct and test novel protein domain fusions for allostery. This work uses laboratory evolution to engineer the peptide binding affinity of the protein binding domain of the allosteric E. coli protease DegS. The protein binding domain is a PDZ domain (named for Postsynaptic density protein (PSD-95), Discs-large protein (Dlg), and Zonula occludens-1 (ZO-1)) that binds the C-terminus of unfolded outer membrane porins. Combinatorial libraries of PDZ domain variants were displayed anchored to the periplasmic membrane of E. coli. The cells were permeabilized and incubated with fluorescent peptide ligands. PDZ domains were screened by flow cytometry for binding to the target peptide ligands. The PDZ domain binding affinity was improved by 20-fold for the peptide ligand that represents the physiological ligand; and the PDZ domain binding affinity was expanded to accommodate a negatively charged residue in a novel peptide ligand. The E. coli anchored peripalsmic expression (APEx) methodology in conjunction with flow cytometry had not previously been used to modify the binding affinity of a PDZ domain. The selected PDZ domain variants were then fused to the wild-type DegS protease domain and analyzed to determine if allosteric activation was made more sensitive to the native ligand or altered to respond to the novel peptide ligand. Interestingly, the DegS fusion protein with the PDZ variant containing the most subtle mutations retained a degree of allostery for the physiological peptide ligand and obtained a degree of allostery for the novel activating peptide ligand. Other selected PDZ variants with additional and expected mutations in the ligand binding site did not respond allosterically to the peptide ligands and the respective DegS fusions were constitutively active, suggesting that the amino acid network linking the allosteric binding event to protease activity is intricately integrated. / text

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