Cheng, Tat Cheung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 181-188). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

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

by Patrick, Tik-wan Law. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 144-155). / Acknowledgements --- p.i / Contents --- p.ii / Abstract --- p.vi / Abbreviations --- p.viii / List of figures --- p.ix / List of tables --- p.xiv / Chapter Chapter One: --- Introduction --- p.1-17 / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- The Human genome project --- p.2 / Chapter 1.3 --- The EST approach --- p.3 / Chapter 1.4 --- Significance of the EST approach --- p.3 / Chapter 1.5 --- Human heart cDNA sequencing --- p.5 / Chapter 1.6 --- Significance of the human adult heart EST project --- p.7 / Chapter 1.7 --- Ribosomal proteins --- p.8 / Chapter 1.7.1 --- The ribosomal constituents --- p.8 / Chapter 1.7.2 --- Eukaiyotic ribosomal proteins --- p.10 / Chapter 1.8 --- Mammalian ribosomal proteins --- p.11 / Chapter 1.8.1 --- Evolution of mammalian ribosomal proteins --- p.11 / Chapter 1.8.2 --- Significance of mammalian ribosomal proteins --- p.12 / Chapter 1.9 --- Possible functional roles of ribosomal protein --- p.14 / Chapter 1.10 --- Nomenclature of ribosomal proteins --- p.16 / Chapter 1.11 --- The theme of the thesis --- p.17 / Chapter Chapter Two: --- Materials and Methods --- p.18-49 / Chapter 2.1 --- Cycle sequencing --- p.18 / Chapter 2.1.1 --- Plating out the cDNA library --- p.18 / Chapter 2.1.2 --- Amplification of the cDNA clones by PCR --- p.19 / Chapter 2.1.3 --- Purification and quantitation of the PCR product --- p.20 / Chapter 2.1.4 --- Cycle DNA sequencing --- p.20 / Chapter 2.2 --- Cloning of hrpL29 in pUC 18 cloning vector --- p.21 / Chapter 2.2.1 --- Amplification of the phage by plate lysate --- p.21 / Chapter 2.2.2 --- Amplification of the insert by PCR --- p.22 / Chapter 2.3 --- Screening for hrpL29 transformant --- p.22 / Chapter 2.3.1 --- Mini-preparation of plasmid DNA (Sambrook et al，1989) --- p.22 / Chapter 2.3.2 --- Large scale preparation of plasmid DNA --- p.24 / Chapter 2.4 --- Primer design for cloning of an intron of hrpL29 --- p.26 / Chapter 2.5 --- Isolation of the intron of hrpL29 by PCR --- p.26 / Chapter 2.6 --- Restricted endonuclease digestion --- p.27 / Chapter 2.7 --- Purification of DNA from the agarose gel --- p.27 / Chapter 2.8 --- Dephosphorylation of linearized plasmid DNA --- p.29 / Chapter 2.9 --- DNA ligation --- p.29 / Chapter 2.10 --- "Preparation of competent bacterial cells for transformation (Hanahan,1985)" --- p.30 / Chapter 2.11 --- Plasmid DNA Transformation --- p.31 / Chapter 2.12 --- Unicycle DNA sequencing by T7 polymerase (Pharmacia) --- p.32 / Chapter 2.13 --- Synthesis of radiolabelled DNA probe --- p.33 / Chapter 2.14 --- "Oligonucleotide synthesis, deprotection and purification" --- p.34 / Chapter 2.14.1 --- Oligonucleotide synthesis --- p.34 / Chapter 2.14.2 --- Deprotection and purification of oligonucleotides --- p.35 / Chapter 2.15 --- Southern analysis --- p.36 / Chapter 2.15.1 --- "Isolation of genomic DNA from leukocytes (Ciulla et al,1988)" --- p.36 / Chapter 2.15.2 --- Restricted digestion and fractionation of genomic DNA --- p.37 / Chapter 2.15.3 --- Southern transfer of DNA onto a membrane support --- p.37 / Chapter 2.15.4 --- Prehybridization of the Southern blot --- p.40 / Chapter 2.15.5 --- Hybridization of the Southern blot --- p.40 / Chapter 2.16 --- Northern analysis --- p.41 / Chapter 2.16.1 --- "Isolation of total RNA by using the AGPC-RNA method (Chomczynski and Sacchi,1987, modified)" --- p.41 / Chapter 2.16.2 --- Separation of total RNA by electrophoresis and transfer onto a membrane support --- p.43 / Chapter 2.16.3 --- Prehybridization of the Northern blot --- p.46 / Chapter 2.16.4 --- Hybridization of the Northern blot --- p.47 / Chapter 2.17 --- First strand cDNA synthesis (Pharmacia) --- p.48 / Chapter 2.18 --- PCR of the first strand cDNA --- p.48 / Chapter Chapter Three: --- Results --- p.50-113 / Chapter 3.1 --- Partial sequencing of adult human heart cDNA clones --- p.50 / Chapter 3.2 --- DNA homology searching by using the program BLASTN --- p.52 / Chapter 3.2.1 --- Catalogue of the 502 ESTs of the cardiovascular system --- p.54 / Chapter 3.2.2 --- Classification and frequency of the human adult heart cDNA clones --- p.63 / Chapter 3.3 --- Submission of the cDNA sequences to NCBI --- p.64 / Chapter 3.4 --- Pattern of gene expression in the human adult cardiovascular system --- p.66 / Chapter 3.5 --- "Sequence determination of hrpL29 (Law et. al., 1996)" --- p.72 / Chapter 3.5.1 --- Cycle Taq sequencing of hrpL29 --- p.72 / Chapter 3.5.2 --- Subcloning of the hrpL29 cDNA insert into the pUC18 DNA cloning vector --- p.75 / Chapter 3.5.3 --- Unicycle T7 sequencing of hrpL29 --- p.77 / Chapter 3.6 --- Sequence alignment and comparison of hrpL29 with other known sequences in the databases --- p.79 / Chapter 3.7 --- The primary structure of hrpL29 --- p.83 / Chapter 3.8 --- Results of RT-PCR and PCR --- p.88 / Chapter 3.9 --- Genomic analysis of hrpL29 --- p.92 / Chapter 3.9.1 --- Isolation of the first intron of hrpL29 --- p.92 / Chapter 3.9.2 --- Southern analysis of hrpL29 --- p.97 / Chapter 3.10 --- Northern analysis of hrpL29 --- p.103 / Chapter 3.10.1 --- Tissue distribution of hrpL29 mRNA in rat tissues --- p.103 / Chapter 3.10.2 --- Time course of hRPL29 expression in mouse heart --- p.106 / Chapter 3.10.3 --- Time course of hRPL29 expression in mouse brain --- p.110 / Chapter Chapter Four: --- Discussion --- p.114-139 / Chapter 4.1 --- Characterization of the ESTs --- p.114 / Chapter 4.2 --- Significance of the heart EST project --- p.116 / Chapter 4.3 --- Redundancy of the EST sequencing --- p.118 / Chapter 4.4 --- The importance of frequent database searching --- p.119 / Chapter 4.5 --- The importance of an efficient comparison algorithm --- p.120 / Chapter 4.6 --- Human ribosomal protein L29 (hRPL29) --- p.122 / Chapter 4.7 --- Internal duplication in hRPL29 --- p.124 / Chapter 4.8 --- Primary structure analysis of hRPL29 --- p.126 / Chapter 4.9 --- RT-PCR and PCR of the first strand cDNA with primers using the C095-ATG and dT primer --- p.128 / Chapter 4.10 --- Southern analysis of hrpL29 --- p.128 / Chapter 4.11 --- Northern analysis of hrpL29 --- p.133 / Chapter 4.11.1 --- Tissue distribution of the mRNA species of hrpL29 --- p.133 / Chapter 4.11.2 --- Time course of hRPL29 expression in mouse heart and brain --- p.134 / Chapter 4.12 --- Possible functional role of hRPL29 --- p.135 / Chapter 4.13 --- Further aspects --- p.137 / Appendix --- p.140-143 / References --- p.144-155

Ribosomal proteins (RPs), synthesized in the cytoplasm, need to be transported from the cytoplasm to the nucleolus (a nuclear compartment), where a single molecule of each RP assembles with rRNAs to form the large and small ribosomal subunits. The objectives of this research were to identify nuclear/nucleolar localization signals (NLSs/NoLSs; generally basic motifs) that mediate the transport of Arabidopsis RPL23aA, RPL15A and RPS8A into the nucleus and nucleolus, and to study transcriptional regulation and subcellular localization of RPs. While all previous research has shown that nucleolar localization of proteins is mediated by specific basic motifs, in this study, I showed that a specific number of basic motifs mediated nucleolar localization of RPL23aA, rather than any specific motifs. In this protein, single mutations of any of its eight putative NLSs (pNLSs) had no effect on nucleolar localization, however, the simultaneous mutation of all eight completely disrupted nucleolar localization, but had no effect on nuclear localization. Furthermore, mutation of any four of these pNLSs had no effect on localization, while mutation of more than four increasingly disrupted nucleolar localization, suggesting that any combination of four of the eight pNLSs is able to mediate nucleolar localization. These results support a charge-based system for the nucleolar localization of RPL23aA. While none of the eight pNLSs of RPL23aA were required for nuclear localization, in RPS8A and RPL15A, of the 10 pNLSs in each, the N-terminal two and three NLSs, respectively, were absolutely required for nuclear/nucleolar localization. Considering the presence of only a single molecule of each RP in any given ribosome, which obligates the presence of each RP in the nucleolus in equal quantities, I studied transcriptional regulation of Arabidopsis RP genes and the subcellular localization of five RP families to determine the extent of coordinated regulation of these processes. Variation of up to 300-fold was observed in the expression levels of RP genes. However, this variation was drastically reduced when the expression level was considered at the RP gene family level, indicating that coordinate regulation of expression of RP genes, coding for individual RP isoforms, is more stringent at the family level. Subcellular localization also showed differential targeting of RPs to the cytoplasm, nucleus and nucleolus, together with a significant difference in the nucleolar import rates of RPS8A and RPL15A. Although one could expect coordinated regulation of the processes preceding ribosomal subunit assembly in the nucleolus, my results suggest differential regulation of these processes.

Ribosomes which conduct protein synthesis in all living organisms are comprised of two subunits. The large 60S ribosomal subunit catalyzes peptidyl transferase reactions and includes the polypeptide exit tunnel, while the small (40S) ribosomal subunit recruits incoming messenger RNAs (mRNAs) and performs proofreading. The plant 80S cytoplasmic ribosome is composed of 4 ribosomal RNAs (rRNAs: 25-28S, 5.8S and 5S in the large subunit and 18S in the small subunit) and 81 ribosomal proteins (r-proteins: 48 in the large subunit, 33 in the small subunit). RPS15a, a putative small subunit primary binder, is encoded by a six member gene family (RPS15aA-F), where RPS15aB and RPS15aE are evolutionarily distinct and thought to be incorporated into mitochondrial ribosomes. In vitro synthesized cytoplasmic 18S rRNA, 18S rRNA loop fragments, and RPS15a mRNA molecules were combined in electrophoretic shift assays (EMSAs) to determine the RNA binding characteristics of RPS15aA/-D/-E/-F. RPS15aA/F, -D and -E bind to cytoplasmic 18S rRNA in the absence of cellular components. However, RPS15aE r-protein tested that binds mitochondrial 18S rRNA. In addition, RPS15aA/F only binds one of three 18S rRNA loop fragments of helix 23 whereas RPS15aD/-E bind all three 18S rRNA helix 23 loop fragments. Additionally, RPS15aD and RPS15aE did not bind their respective mRNA transcripts, likely indicating that this form of negative feedback is not a post-transcriptional control mechanism for this r-protein gene family. Furthermore, the addition of RPS15a transcripts to the EMSAs did not affect the binding of RPS15aA/F, -D and -E to 18S rRNA helix 23 loop 4-6, indicating that rRNA binding is specific. Supershift EMSAs further confirmed the specificity of RPS15aA/F and RPS15aE binding to loop fragment (4-6) of 18S rRNA. Taken together, these data support a role for RPS15a in early ribosome small subunit assembly.

Ribosomes which conduct protein synthesis in all living organisms are comprised of two subunits. The large 60S ribosomal subunit catalyzes peptidyl transferase reactions and includes the polypeptide exit tunnel, while the small (40S) ribosomal subunit recruits incoming messenger RNAs (mRNAs) and performs proofreading. The plant 80S cytoplasmic ribosome is composed of 4 ribosomal RNAs (rRNAs: 25-28S, 5.8S and 5S in the large subunit and 18S in the small subunit) and 81 ribosomal proteins (r-proteins: 48 in the large subunit, 33 in the small subunit). RPS15a, a putative small subunit primary binder, is encoded by a six member gene family (RPS15aA-F), where RPS15aB and RPS15aE are evolutionarily distinct and thought to be incorporated into mitochondrial ribosomes. In vitro synthesized cytoplasmic 18S rRNA, 18S rRNA loop fragments, and RPS15a mRNA molecules were combined in electrophoretic shift assays (EMSAs) to determine the RNA binding characteristics of RPS15aA/-D/-E/-F. RPS15aA/F, -D and -E bind to cytoplasmic 18S rRNA in the absence of cellular components. However, RPS15aE r-protein tested that binds mitochondrial 18S rRNA. In addition, RPS15aA/F only binds one of three 18S rRNA loop fragments of helix 23 whereas RPS15aD/-E bind all three 18S rRNA helix 23 loop fragments. Additionally, RPS15aD and RPS15aE did not bind their respective mRNA transcripts, likely indicating that this form of negative feedback is not a post-transcriptional control mechanism for this r-protein gene family. Furthermore, the addition of RPS15a transcripts to the EMSAs did not affect the binding of RPS15aA/F, -D and -E to 18S rRNA helix 23 loop 4-6, indicating that rRNA binding is specific. Supershift EMSAs further confirmed the specificity of RPS15aA/F and RPS15aE binding to loop fragment (4-6) of 18S rRNA. Taken together, these data support a role for RPS15a in early ribosome small subunit assembly.

Ribosomal proteins (RPs), synthesized in the cytoplasm, need to be transported from the cytoplasm to the nucleolus (a nuclear compartment), where a single molecule of each RP assembles with rRNAs to form the large and small ribosomal subunits. The objectives of this research were to identify nuclear/nucleolar localization signals (NLSs/NoLSs; generally basic motifs) that mediate the transport of Arabidopsis RPL23aA, RPL15A and RPS8A into the nucleus and nucleolus, and to study transcriptional regulation and subcellular localization of RPs. While all previous research has shown that nucleolar localization of proteins is mediated by specific basic motifs, in this study, I showed that a specific number of basic motifs mediated nucleolar localization of RPL23aA, rather than any specific motifs. In this protein, single mutations of any of its eight putative NLSs (pNLSs) had no effect on nucleolar localization, however, the simultaneous mutation of all eight completely disrupted nucleolar localization, but had no effect on nuclear localization. Furthermore, mutation of any four of these pNLSs had no effect on localization, while mutation of more than four increasingly disrupted nucleolar localization, suggesting that any combination of four of the eight pNLSs is able to mediate nucleolar localization. These results support a charge-based system for the nucleolar localization of RPL23aA. While none of the eight pNLSs of RPL23aA were required for nuclear localization, in RPS8A and RPL15A, of the 10 pNLSs in each, the N-terminal two and three NLSs, respectively, were absolutely required for nuclear/nucleolar localization. Considering the presence of only a single molecule of each RP in any given ribosome, which obligates the presence of each RP in the nucleolus in equal quantities, I studied transcriptional regulation of Arabidopsis RP genes and the subcellular localization of five RP families to determine the extent of coordinated regulation of these processes. Variation of up to 300-fold was observed in the expression levels of RP genes. However, this variation was drastically reduced when the expression level was considered at the RP gene family level, indicating that coordinate regulation of expression of RP genes, coding for individual RP isoforms, is more stringent at the family level. Subcellular localization also showed differential targeting of RPs to the cytoplasm, nucleus and nucleolus, together with a significant difference in the nucleolar import rates of RPS8A and RPL15A. Although one could expect coordinated regulation of the processes preceding ribosomal subunit assembly in the nucleolus, my results suggest differential regulation of these processes.

No description available.

Ma Hoi-Wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 113-120). / Abstracts in English and Chinese. / Acknowledgement --- p.I / Abstract --- p.II / 摘要 --- p.III / Content --- p.IV / Abbreviations --- p.X / List of Figures --- p.XII / List of Tables --- p.XIV / Chapter Chapter One --- Introduction --- p.1 / Chapter 1.1 --- Interactions that stabilize proteins --- p.1 / Chapter 1.2 --- Some common strategies of protein engineering to improve thermostability --- p.6 / Chapter 1.3 --- Ribosomal protein T. celer L30e as a study model for thermostability --- p.7 / Chapter 1.4 --- Extra proline residue is one of the insights by comparing the two proteins --- p.10 / Chapter Chapter Two --- Materials and Methods --- p.13 / Chapter 2.1 --- General Techniques --- p.13 / Chapter 2.1.1 --- Preparation of Escherichia coli competent cells --- p.13 / Chapter 2.1.2 --- Transformation of Escherichia coli competent cells --- p.14 / Chapter 2.1.3 --- Spectrophotometric quantitation of DNA --- p.14 / Chapter 2.1.4 --- Agarose gel electrophoresis --- p.14 / Chapter 2.1.5 --- DNA extraction from agarose gel electrophoresis using Viogene Gene Clean kit --- p.15 / Chapter 2.1.6 --- Plasmid DNA minipreperation by Wizard® Plus SV Minipreps DNA Purification System from Promega --- p.16 / Chapter 2.1.7 --- Polymerase Chain Reaction (PCR) --- p.17 / Chapter 2.1.8 --- Ligation of DNA fragments --- p.18 / Chapter 2.1.9 --- Sonication of pellet resuspension --- p.18 / Chapter 2.1.10 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.19 / Chapter 2.1.11 --- Native polyacrylamide gel electrophoresis --- p.20 / Chapter 2.1.12 --- Staining of protein in polyacrylamide gel by Coommassie Brillant Blue R250 --- p.22 / Chapter 2.1.13 --- Protein Concentration determination --- p.22 / Chapter 2.2 --- Cloning the Mutant Genes --- p.22 / Chapter 2.2.1 --- Site-directed mutagenesis --- p.22 / Chapter 2.2.1.1 --- Generation of full length mutant gene by megaprimer --- p.23 / Chapter 2.2.1.2 --- Generation of mutant gene by QuikChange® Site-Directed Mutagenesis Kit from Stratagene --- p.26 / Chapter 2.2.2 --- Restriction Digestion of DNA --- p.27 / Chapter 2.2.3 --- Ligation of DNA fragments --- p.27 / Chapter 2.2.4 --- Screening for successful inserted plasmid clones from ligation reactions --- p.28 / Chapter 2.2.4.1 --- By PCR --- p.28 / Chapter 2.2.4.2 --- By restriction digestion --- p.28 / Chapter 2.2.5 --- DNA sequencing --- p.29 / Chapter 2.3 --- Expression and Purification of Protein --- p.29 / Chapter 2.3.1 --- "General bacterial culture, harvesting and lysis" --- p.29 / Chapter 2.3.2 --- Purification of recombinant wild type TRP and mutants --- p.30 / Chapter 2.3.3 --- Purification of recombinant wild type YRP and mutants --- p.32 / Chapter 2.4 --- Thermodynamic Studies by Circular Dichroism (CD) Spectrometry --- p.34 / Chapter 2.4.1 --- Thermodynamic studies by guanidine-induced denaturations --- p.34 / Chapter 2.4.2 --- Themodynamic studies by thermal denaturations --- p.36 / Chapter 2.4.3 --- ACp measurement of the TRP mutants --- p.37 / Chapter 2.4.3.1 --- By Gibbs-Helmholtz analysis --- p.37 / Chapter 2.4.3.2 --- By van't Hoff analysis --- p.37 / Chapter 2.5 --- Crystal Screen for the Mutant T. celer L30e --- p.38 / Chapter 2.5.1 --- T. celer L30e Pro→Ala and Pro→Gly mutants --- p.38 / Chapter 2.5.2 --- Yeast L30e K65P mutant --- p.38 / Chapter 2.6 --- Sequences of Primers --- p.39 / Chapter 2.6.1 --- Primers for TRP and its mutants --- p.39 / Chapter 2.6.2 --- Primers for YRP and its mutantsReagents and buffers --- p.40 / Chapter 2.7 --- Reagents and Buffers --- p.40 / Chapter 2.7.1 --- Reagents for competent cell preparation --- p.40 / Chapter 2.7.2 --- Nucleic acid eletrophoresis buffers --- p.41 / Chapter 2.7.3 --- Media for bacterial culture --- p.41 / Chapter 2.7.4 --- Reagents for SDS-PAGE --- p.42 / Chapter 2.7.5 --- Buffers for TRP purification --- p.44 / Chapter 2.7.6 --- Buffers for YRP purification --- p.45 / Chapter 2.7.7 --- Buffer for Circular Dichroism (CD) Spectrometry --- p.46 / Chapter Chapter Three --- Results --- p.48 / Chapter 3.1 --- "Cloning, expression and purification of the mutant proteins" --- p.48 / Chapter 3.1.1 --- "Mutagenesis, cloning and purification of the thermophilic proteins - T. celer L30e protein and its mutants" --- p.48 / Chapter 3.1.2 --- "Mutagenesis, cloning and purification of the mesophilic proteins - yeast L30e protein and its mutants" --- p.52 / Chapter 3.2 --- Stability of Pro→Ala/Gly mutants of T. celer L30e at 298K --- p.55 / Chapter 3.2.1 --- Design of alanine and glycine mutants from thermophilic homologue --- p.55 / Chapter 3.2.2 --- "Among alanine mutants, only P59A was destabilized" --- p.55 / Chapter 3.2.3 --- Ala→Gly mutations destabilized the protein --- p.59 / Chapter 3.3 --- Stability of Xaa→Pro mutants of yeast L30e at 298K --- p.61 / Chapter 3.3.1 --- Design of proline mutants from mesophilic homologue --- p.61 / Chapter 3.3.2 --- "K65P, corresponding to P59 in T. celer L30e, stabilized yeast L30e" --- p.62 / Chapter 3.3.3 --- Yeast L30e mutated with thermophilic consensus sequence did not give a more stable protein --- p.65 / Chapter 3.4 --- Temperature dependency of the stability of the mutants of T. celer L30e --- p.67 / Chapter 3.4.1 --- The trend of ΔGU was consistence through 25 to 75°C --- p.67 / Chapter 3.4.2 --- Melting temperatures of T. celer mutants determined by thermal denaturations --- p.68 / Chapter 3.5 --- pH dependency of melting temperatures --- p.75 / Chapter 3.5.1 --- ΔCP values of the P59A/G mutants determined by van't HofF's analyses increased significantly --- p.77 / Chapter 3.6 --- No structural change was observed in the crystal structure of P59A --- p.80 / Chapter Chapter Four --- Discussion --- p.84 / Chapter 4.1 --- The trend of stability from guanidine-induced denaturation agreed with that from thermal denaturations --- p.86 / Chapter 4.2 --- The magnitude of destabilization of P59A and Ala→Gly mutation was consistent with the expected destabilization due to entropy --- p.87 / Chapter 4.3 --- Entropic effect had little effect for residues in flexible region --- p.93 / Chapter 4.4 --- Stabilization forces that compensate the entropic effect --- p.96 / Chapter 4.5 --- Compensatory stabilization due to the release of amide group --- p.99 / Chapter 4.5.1 --- Intra-molecular H-bond in P88A --- p.99 / Chapter 4.5.2 --- Solvent-protein H-bond in P43A --- p.103 / Chapter 4.6 --- Consensus concept was not applicable in our model --- p.110 / Chapter 4.7 --- "Pro→Ala mutation destabilized the protein increase the protein's ACP value, however enthalpy and entropy change were difficult to be decomposed" --- p.111 / Chapter 4.8 --- Concluding Remarks --- p.112 / References --- p.113

Chu Lai On. / Thesis submitted in: July 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 152-172). / Abstracts in English and Chinese. / Acknowledgements --- p.2 / Abstract --- p.3 / Table of Content --- p.7 / Abbreviations --- p.12 / Naming system for mutant proteins --- p.13 / Abbreviations for amino acid --- p.14 / Chapter Chapter 1 --- Introduction --- p.15 / Chapter 1.1 --- Structure-function relationship of trichosanthin --- p.18 / Chapter 1.2 --- Properties of acidic ribosomal P-proteins --- p.21 / Chapter 1.3 --- Interaction among P-proteins and trichosanthin --- p.25 / Chapter 1.4 --- Properties of eukaryotic elongation factor 2 and interaction with P-proteins --- p.26 / Chapter 1.5 --- "Objectives and strategy of studying the interaction among trichosanthin, P-proteins and eukaryotic elongation 2" --- p.30 / Chapter Chapter 2 --- Materials and Methods --- p.33 / Chapter 2.1 --- General techniques --- p.33 / Chapter 2.1.1 --- Preparation and transformation of Escherichia coli competent cells --- p.33 / Chapter 2.1.2 --- Minipreparation of plasmid DNA using Wizard Plus SV Minipreps DNA purification kit from Promega --- p.34 / Chapter 2.1.3 --- Agarose gel electrophoresis of DNA --- p.36 / Chapter 2.1.4 --- Purification of DNA from agarose gel using Wizard SV Gel and PCR Clean-Up System from Promega --- p.36 / Chapter 2.1.5 --- Polymerase Chain Reaction (PCR) --- p.37 / Chapter 2.1.5.1 --- Basic Protocol --- p.37 / Chapter 2.1.5.2 --- Generation of P2 truncation mutants --- p.38 / Chapter 2.1.5.3 --- Generation of TCS mutants --- p.39 / Chapter 2.1.6 --- Restriction digestion of DNA --- p.41 / Chapter 2.1.7 --- Ligation of DNA fragments --- p.41 / Chapter 2.1.8 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.42 / Chapter 2.1.9 --- Staining of protein in polyacrylamide gel --- p.45 / Chapter 2.2 --- Expression and purification of recombinant proteins --- p.46 / Chapter 2.2.1 --- "Bacterial culture, harvesting and lysis" --- p.46 / Chapter 2.2.2 --- Purification of recombinant TCS and mutants --- p.47 / Chapter 2.2.3 --- Purification of acidic ribosomal protein P2 and mutants --- p.48 / Chapter 2.2.4 --- Purification of MBP-fusion proteins --- p.50 / Chapter 2.3 --- Purification of eEF2 from rat livers --- p.51 / Chapter 2.4 --- In vitro binding assay by NHS-activated Sepharose resin --- p.53 / Chapter 2.4.1 --- Coupling of protein sample to NHS-activated Sepharose resin --- p.53 / Chapter 2.4.2 --- In vitro binding of protein sample to coupled NHS-activated resin --- p.54 / Chapter 2.5 --- Ribosome-inactivated activity assay using rabbit reticulocyte lysate in vitro translation system --- p.55 / Chapter 2.6 --- Circular dichroism (CD)spectrometry --- p.57 / Chapter 2.7 --- Isothermal titration calorimetry (ITC) experiment --- p.57 / Chapter 2.8 --- Surface plasmon resonance (SPR) experiment --- p.58 / Chapter 2.8.1 --- Immobilization of P2 onto aminosilane cuvette --- p.58 / Chapter 2.8.2 --- Interaction between eEF2 and immobilized P2 --- p.60 / Chapter 2.9 --- Preparation of Anti-P antibody --- p.61 / Chapter 2.10 --- Western blotting of protein --- p.62 / Chapter 2.11 --- Reagents and buffer --- p.64 / Chapter 2.11.1 --- Reagents for competent cell preparation --- p.64 / Chapter 2.11.2 --- Nucleic acids electrophoresis buffer --- p.65 / Chapter 2.11.3 --- Media for bacterial culture --- p.66 / Chapter 2.11.4 --- Buffers for TCS purification --- p.67 / Chapter 2.11.5 --- Buffers for eEF2 purification --- p.68 / Chapter 2.11.6 --- Reagents for SDS-PAGE --- p.68 / Chapter 2.11.7 --- Reagents and buffers for Western blot --- p.70 / Chapter 2.11.8 --- Reagents and buffers for coupling sample proteins to NHS-activated Sepharose resin --- p.72 / Chapter 2.11.9 --- Reagents and buffers for in vitro binding assay --- p.72 / Chapter 2.11.10 --- Reagents and Buffers for surface plasmon resonance --- p.72 / Chapter 2.12 --- Sequences of primers --- p.73 / Chapter Chapter 3 --- Interaction between TCS and P2 --- p.80 / Chapter 3.1 --- Introduction --- p.80 / Chapter 3.2 --- Interaction between TCS and P-proteins in rat liver lysate --- p.83 / Chapter 3.3 --- Construction of TCS mutants --- p.85 / Chapter 3.4 --- Expression and purification of TCS mutants --- p.87 / Chapter 3.5 --- Biological assay of TCS mutants --- p.91 / Chapter 3.6 --- Physical interaction of TCS mutants and P2 by surface plasmon resonance (SPR) --- p.94 / Chapter 3.7 --- Discussion --- p.100 / Chapter Chapter 4 --- Mapping the region of P2 that binds TCS and eEF2 --- p.104 / Chapter 4.1 --- Introduction --- p.104 / Chapter 4.2 --- Construction of P2 truncation mutants --- p.106 / Chapter 4.3 --- Expression and purification of P2 truncation mutants --- p.107 / Chapter 4.4 --- Mapping the region of P2 that binds TCS --- p.111 / Chapter 4.4.1 --- Interaction between TCS and P2 mutants by in vitro binding assay --- p.111 / Chapter 4.4.2 --- Interaction study of TCS and P2 mutant by isothermal titration calorimetry (ITC) --- p.116 / Chapter 4.5 --- Mapping the region of P2 that binds eEF2 --- p.120 / Chapter 4.5.1 --- Purification of eEF2 from rat liver --- p.120 / Chapter 4.5.2 --- Physical interaction of P2 and eEF2 by surface plasmon resonance (SPR) --- p.126 / Chapter 4.5.3 --- Interaction between eEF2 and P2 mutants by in vitro binding assay --- p.128 / Chapter 4.6 --- Mapping the C-terminal region of P2 by MBP-fusion proteins --- p.130 / Chapter 4.6.1 --- Construction and purification of MBP-fusion proteins --- p.131 / Chapter 4.6.2 --- "Interaction among eEF2, TCS and MBP-fusion proteins by in vitro binding assay" --- p.133 / Chapter 4.7 --- Discussion --- p.137 / Chapter Chapter 5 --- Effect of C-17 peptide on TCS biological activity --- p.143 / Chapter 5.1 --- Introduction --- p.143 / Chapter 5.2 --- Ribosome-inactivating activity of TCS with C-17 peptide --- p.145 / Chapter 5.3 --- Discussion --- p.147 / Chapter Chapter 6 --- Conclusion and suggestions for future study --- p.149 / References --- p.152 / Appendix --- p.173