No description available.

No description available.

Cryo-electron microscopy (cryo-EM) is a structural biology technique that determines the structure of proteins and macromolecular complexes using the transmission electron microscope under cryogenic conditions. In my Ph.D. studies, I took advantage of this technique, in the study of dynamical features of ribosomes in both eukaryotes and prokaryotes. In Chapter 2, I report my graduate research on the investigation of ribosomes from the human malaria parasite, Plasmodium falciparum, using single-particle cryo-EM. In collaboration with Dr. Jeffrey Dvorin at Harvard Medical School, we obtained five cryo-EM reconstructions of ribosomes purified from P. falciparum blood-stage schizonts, and discovered structural and dynamical features that differentiate the ribosomes of P. falciparum from those of the mammalian system. Moreover, we discovered that RACK1, a necessary ribosomal protein in eukaryotes, does not specifically co-purify with the 80S fraction in the P. falciparum schizonts stage and would mainly function in a ribosome-unbound, free state during the blood-stage. More extensive studies, using cryo-EM methodology, of translation in the parasite, will provide structural knowledge that could help in the design of effective anti-malaria drugs. In Chapter 3, I describe the cryo-EM studies of the Saccharomyces cerevisiae ribosome in response to a carbon source switch. In collaboration with Dr. Andrew Link at Vanderbilt University, we obtained reconstructions of the 80S ribosomes at selected time points after the glucose-to-glycerol carbon source shift, and observed that a fraction of ribosomes lacked densities for r-proteins, mainly eS1 (yeast rpS1) on the 40S subunit and uL16 (yeast rpL10) on the 60S subunit. We found that the binding ratio of eS1 and uL16 to ribosomes changed as a function of time, consistent with the change in translational activities as gauged by polysome profiling. On the basis of these observations, along with previous structural and genetics studies, we propose that rapid control of translation is exerted through the dissociation of r-protein eS1/rpS1 and uL16/rpL10 from the ribosome. Our studies thus open a new venue on the exploration of S. cerevisiae’s rapid adaption to carbon source shifts at the level of translation. In Chapter 4, I have documented a collaborative work on the development and application of a new technique, time-resolved cryo-EM, which can be used to study processes involving two reaction partners on a sub-second time scale. With my colleagues at the Frank and Gonzalez labs at Columbia University, we successfully applied this method to study the process of E. coli ribosomal subunits association. By mixing and reacting the two subunits for 60 ms and 140 ms, we captured the association reaction in a pre-equilibrium state, and detected different conformations of E. coli 70S ribosomes. With the current capability of this mixing-spraying method to visualize multiple states of molecules in a sub-second reaction, we expect to be able to standardize this method and apply it to more challenging biological processes, such as translation recycling and initiation processes.

abstract: The ribosome is a ribozyme and central to the biosynthesis of proteins in all organisms. It has a strong bias against non-alpha-L-amino acids, such as alpha-D-amino acids and beta-amino acids. Additionally, the ribosome is only able to incorporate one amino acid in response to one codon. It has been demonstrated that reengineering of the peptidyltransferase center (PTC) of the ribosome enabled the incorporation of both alpha-D-amino acids and beta-amino acids into full length protein. Described in Chapter 2 are five modified ribosomes having modifications in the peptidyltrasnferase center in the 23S rRNA. These modified ribosomes successfully incorporated five different beta-amino acids (2.1 - 2.5) into E. coli dihydrofolate reductase (DHFR). The second project (Chapter 3) focused on the study of the modified ribosomes facilitating the incorporation of the dipeptide glycylphenylalanine (3.25) and fluorescent dipeptidomimetic 3.26 into DHFR. These ribosomes also had modifications in the peptidyltransferase center in the 23S rRNA of the 50S ribosomal subunit. The modified DHFRs having beta-amino acids 2.3 and 2.5, dipeptide glycylphenylalanine (3.25) and dipeptidomimetic 3.26 were successfully characterized by the MALDI-MS analysis of the peptide fragments produced by "in-gel" trypsin digestion of the modified proteins. The fluorescent spectra of the dipeptidomimetic 3.26 and modified DHFR having fluorescent dipeptidomimetic 3.26 were also measured. The type I and II DNA topoisomerases have been firmly established as effective molecular targets for many antitumor drugs. A "classical" topoisomerase I or II poison acts by misaligning the free hydroxyl group of the sugar moiety of DNA and preventing the reverse transesterfication reaction to religate DNA. There have been only two classes of compounds, saintopin and topopyrones, reported as dual topoisomerase I and II poisons. Chapter 4 describes the synthesis and biological evaluation of topopyrones. Compound 4.10, employed at 20 µM, was as efficient as 0.5 uM camptothecin, a potent topoisomerase I poison, in stabilizing the covalent binary complex (~30%). When compared with a known topoisomerase II poison, etoposide (at 0.5 uM), topopyorone 4.10 produced similar levels of stabilized DNA-enzyme binary complex (~34%) at 5 uM concentration. / Dissertation/Thesis / Ph.D. Chemistry 2013

No description available.

by Feng Zhang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1988. / Bibliography: leaves 104-115.

Protein synthesis in all organisms is catalyzed by a highly-conserved ribonucleoprotein macromolecular machine known as the ribosome. Prior to each round of protein synthesis in the cell, a functional ribosomal complex is assembled from its component parts at the start site of a messenger RNA (mRNA) template during the process of translation initiation. In bacteria, rapid and high-fidelity translation initiation is promoted by three canonical initiation factors: IF1, IF2, and IF3. In this thesis, I report the use of single-molecule fluorescence methods to study the role of the initiation factors and ribosome-factor interactions in regulating molecular events that occur during late stages of the translation initiation pathway. In Chapter 1, I provide a structural and biochemical framework for understanding one of the key events of the initiation pathway: docking of the large (50S) ribosomal subunit with the small subunit 30S initiation complex (30S IC). The 50S subunit joining reaction is catalyzed by GTP-bound IF2 and results in formation of a 70S initiation complex (70S IC) that contains an initiator transfer RNA (tRNA) and is primed for formation of the first peptide bond. During 50S subunit joining, IF2-GTP establishes interactions with RNA and protein components of the 50S subunit's GTPase-associated center (GAC), which play an important role in subunit recruitment as well as the subsequent activation of GTP hydrolysis by IF2. In Chapter 2, I describe the development of a single-molecule fluorescence resonance energy transfer (smFRET) signal to monitor the interactions between IF2 and the ribosome's GAC during real-time 50S subunit joining reactions. Specifically, the role of the L11 region, comprising ribosomal protein L11 and its associated ribosomal RNA (rRNA) helices, was investigated. The L11 region is a prominent structural component of the GAC that is believed to undergo large-scale conformational changes during protein synthesis; however, the nature and timescale of these conformational dynamics, and their role in regulating the biochemical activities of IF2 during initiation, are not known. I demonstrate that my smFRET-based 50S subunit joining assay is sensitive to conformational rearrangements between IF2 and L11 within the 70S IC and can thus be used as a tool for characterizing GAC dynamics and elucidating their function during initiation. Furthermore, my smFRET approach is shown to provide information on the rate of 50S subunit joining as well as the rate of IF2 dissociation from the 70S IC. Notably, IF2-dependent GTP hydrolysis was found to influence the extent of 70S IC conformational dynamics as well as the dissociation rate of IF2. The role of IF3 in regulating 50S-subunit joining dynamics is discussed in Chapter 3. IF3 plays an important role in ensuring the fidelity of translation initiation by preventing the formation of initiation complexes containing a non-initiator tRNA and/or a non-canonical mRNA start codon. Inclusion of IF3 within the 30S IC in the smFRET experiments was found to render the IF2-catalyzed 50S subunit joining reaction highly reversible. Direct observation of repetitive docking and undocking of the 50S subunit with the 30S IC indicates that IF3 may modulate translation initiation efficiency by influencing the stability of the 70S IC. The individual 50S subunit docking events were found to result in the formation of very different classes of 70S IC, characterized by different stabilities and unique patterns of IF2-L11 interactions. I propose that these dynamics reflect an underlying conformational equilibrium of the IF3-bound 30S IC that is read out during 50S subunit joining, and that this equilibrium could be modulated in order to regulate the efficiency of translation initiation. Following initiation-factor mediated assembly of the 70S IC, the first aminoacyl-tRNA is delivered to the ribosome in ternary complex with elongation factor Tu (EF-Tu) and GTP. Accommodation of aminoacyl-tRNA into the ribosome's peptidyl transferase center leads to formation of the first peptide bond, which signals the end of initiation and entry into the elongation phase of protein synthesis. The ternary complex binding site on the ribosome overlaps with that of IF2 at the GAC; a question of key mechanistic importance in understanding how the ribosome coordinates the transition from initiation to elongation thus concerns the relative timing of ternary complex binding with respect to IF2 dissociation from the 70S IC. In Chapter 4, I present preliminary results from two- and three-color fluorescence co-localization experiments aimed at characterizing the timing of these events at the single-molecule level. The data strongly suggest the occurrence of simultaneous occupancy of the ribosome by IF2 and ternary complex, implying that the ribosome is structurally capable of recruiting ternary complex prior to IF2 release from the 70S IC. The observation that the ribosome can accommodate more than one translation factor at a time may have important implications for understanding how it efficiently coordinates factor binding and release throughout protein synthesis, and opens the door to mechanistic studies of the ribosomal L7/L12 stalk, presumed to play a prominent role in these processes.

"July 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 166-177) / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

by Lam Suet Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 109-121). / Acknowledgements --- p.i / Table of contents --- p.ii / Abstract --- p.vii / List of Abbreviations --- p.ix / List of Tables --- p.x / List of Figures --- p.xi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Ribosome-inactivating proteins (RIPs) --- p.3 / Chapter 1.1.1 --- Types of RIPs --- p.4 / Chapter 1.1.1.1 --- Type I RIPs --- p.5 / Chapter 1.1.1.2 --- Type II RIPs --- p.7 / Chapter 1.1.2 --- Physicochemical properties --- p.7 / Chapter 1.1.3 --- N-glycosidase activity of RIPs --- p.8 / Chapter 1.1.3.1 --- Specificity of N-glycosidase activity --- p.10 / Chapter 1.1.3.2 --- Inhibition of protein synthesis --- p.11 / Chapter 1.1.4 --- Other enzymatic and biological activities of RIPs --- p.11 / Chapter 1.1.4.1 --- Enzymatic activities --- p.11 / Chapter 1.1.4.2 --- Multiple depurination --- p.13 / Chapter 1.1.4.3 --- RNase activity --- p.14 / Chapter 1.1.4.4 --- DNase activity --- p.15 / Chapter 1.1.4.5 --- Biological activities --- p.16 / Chapter 1.1.5 --- Storage of RIPs in plant cells --- p.17 / Chapter 1.1.5.1 --- RIPs targeted to subcellular compartments --- p.18 / Chapter 1.1.5.2 --- Cytoplasmic RIPs --- p.20 / Chapter 1.1.6 --- Physiological roles of RIPs --- p.22 / Chapter 1.1.6.1 --- Defensive role in plants --- p.22 / Chapter 1.1.6.2 --- Metabolic role of RIPs --- p.26 / Chapter 1.1.6.3 --- RIPs as storage proteins --- p.26 / Chapter 1.1.7 --- Application of RIPs --- p.27 / Chapter 1.1.7.1 --- Therapeutic applications --- p.27 / Chapter 1.1.7.2 --- Possible use of RIPs in agriculture --- p.30 / Chapter 1.2 --- Objectives of the present study --- p.31 / Chapter 1.2.1 --- Rationale of the study --- p.31 / Chapter 1.2.2 --- Outline of the thesis --- p.32 / Chapter Chapter 2 --- Screening of hitherto unexplored plant species for RIPs --- p.33 / Chapter 2.1 --- Introduction --- p.34 / Chapter 2.2 --- Materials and methods / Chapter 2.2.1 --- Materials --- p.36 / Chapter 2.2.2 --- Preparation of crude powder --- p.36 / Chapter 2.2.3 --- Protein determination --- p.38 / Chapter 2.2.4 --- Preparation of rabbit reticulocyte lysate --- p.38 / Chapter 2.2.5 --- Protein synthesis inhibition assay --- p.39 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Preparation of crude powder --- p.41 / Chapter 2.3.2 --- Protein synthesis inhibition assay --- p.41 / Chapter 2.4 --- Discussion --- p.43 / Chapter Chapter 3 --- Purification of RIP from garden pea (Pisum sativum) --- p.45 / Chapter 3.1 --- Introduction --- p.46 / Chapter 3.2 --- Materials and methods / Chapter 3.2.1 --- Materials --- p.50 / Chapter 3.2.2 --- Purification of RIP from garden pea --- p.52 / Chapter 3.2.3 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.54 / Chapter 3.2.4 --- Precautions for working with RNA --- p.56 / Chapter 3.2.5 --- N-glycosidase assay --- p.57 / Chapter 3.2.6 --- Quantitation of RNA --- p.60 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Quantitation of RNA --- p.61 / Chapter 3.3.2 --- Affinity chromatography on Affi-gel Blue gel --- p.61 / Chapter 3.3.3 --- Iminodiacetic acid-agarose chromatography --- p.64 / Chapter 3.3.4 --- Cation exchange chromatography on Resource-S --- p.66 / Chapter 3.3.5 --- Gel filtration on Superose 12 HR 10/30 --- p.69 / Chapter 3.3.6 --- "Assessment of purity, yield and activity" --- p.72 / Chapter 3.4 --- Discussion --- p.74 / Chapter Chapter 4 --- Physicochemical and biological properties of garden pea RIP --- p.77 / Chapter 4.1 --- Introduction --- p.79 / Chapter 4.2 --- Materials and methods / Chapter 4.2.1 --- Materials --- p.81 / Chapter 4.2.2 --- Molecular weight determination --- p.82 / Chapter 4.2.3 --- Subunit composition --- p.82 / Chapter 4.2.4 --- Isoelectric focusing (IEF) --- p.83 / Chapter 4.2.5 --- Detection of glycoproteins --- p.84 / Chapter 4.2.6 --- N-terminal amino acid sequence --- p.84 / Chapter 4.2.7 --- Inhibition of cell-free protein synthesis --- p.86 / Chapter 4.2.8 --- N-glycosidase activity --- p.86 / Chapter 4.2.9 --- Deoxyribonuclease activity --- p.87 / Chapter 4.2.10 --- Activity towards tRNA --- p.88 / Chapter 4.3 --- Results / Chapter 4.3.1 --- Molecular weight determination --- p.89 / Chapter 4.3.2 --- Subunit composition --- p.91 / Chapter 4.3.3 --- Isoelectric focusing (IEF) --- p.92 / Chapter 4.3.4 --- Detection of glycoproteins --- p.94 / Chapter 4.3.5 --- N-terminal amino acid sequence --- p.96 / Chapter 4.3.6 --- Inhibition of cell-free protein synthesis --- p.97 / Chapter 4.3.7 --- N-glycosidase activity --- p.99 / Chapter 4.3.8 --- Deoxyribonuclease activity --- p.101 / Chapter 4.3.9 --- Activity towards tRNA --- p.102 / Chapter 4.4 --- Discussion --- p.103 / Chapter Chapter 5 --- General discussion and conclusion --- p.106 / References --- p.109

Chiu, Yu Hin Teddy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 118-129). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Table of Content --- p.vi / Abbreviations --- p.x / Naming system for mutant proteins --- p.xi / Abbreviation for amino acid --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- What are acidic ribosomal proteins? --- p.1 / Chapter 1.2 --- Why P-proteins are so important? --- p.13 / Chapter 1.3 --- Research objectives --- p.15 / Chapter Chapter 2 --- Materials and Methods --- p.17 / Chapter 2.1 --- List of buffers and media --- p.17 / Chapter 2.1.1 --- Preparation of buffers and media --- p.17 / Chapter 2.1.2 --- Buffers for preparing competent cells --- p.17 / Chapter 2.1.3 --- Media for bacterial culture --- p.17 / Chapter 2.1.4 --- Buffers for nucleic acid electrophoresis --- p.19 / Chapter 2.1.5 --- Buffers for protein electrophoresis --- p.19 / Chapter 2.1.6 --- Buffers for interaction studies using BIAcore 3000 --- p.21 / Chapter 2.2 --- General methods --- p.23 / Chapter 2.2.1 --- Preparation of Escherichia coli (E.coli.) competent cells --- p.23 / Chapter 2.2.2 --- Transformation of Escherichia coli (E.coli.) competent cells --- p.23 / Chapter 2.2.3 --- DNA cloning --- p.24 / Chapter 2.2.3.1 --- DNA cloning by polymerase chain reaction (PCR) --- p.24 / Chapter 2.2.3.2 --- Agarose gel electrophoresis of DNA --- p.25 / Chapter 2.2.3.3 --- Extraction and purification of DNA from agarose gels --- p.25 / Chapter 2.2.3.4 --- Restriction digestion of DNA --- p.25 / Chapter 2.2.3.5 --- Ligation of digested insert and expression vector --- p.27 / Chapter 2.2.3.6 --- Verification of insert by PCR --- p.27 / Chapter 2.2.3.7 --- Mini-preparation of plasmid DNA --- p.28 / Chapter 2.2.4 --- Polyacrylamide gel electrophoresis (PAGE) of protein --- p.29 / Chapter 2.2.4.1 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.29 / Chapter 2.2.4.2 --- Tricine SDS-polyacrylamide gel electrophoresis --- p.30 / Chapter 2.2.4.3 --- Native polyacrylamide gel electrophoresis --- p.31 / Chapter 2.2.4.4 --- Commassie brilliant blue staining of proteinin polyacrylamide gel --- p.32 / Chapter 2.2.4.5 --- Zinc Imidazole staining of protein in polyacrylamide gel --- p.33 / Chapter 2.2.5 --- Protein concentration determination --- p.33 / Chapter 2.2.6 --- Expression of recombinant proteins --- p.33 / Chapter 2.2.6.1 --- Expression of recombinant proteins using LB --- p.33 / Chapter 2.2.6.2 --- Expression of recombinant proteins using minimal medium --- p.34 / Chapter 2.2.6.3 --- Harvest and lysis of bacterial cell culture --- p.34 / Chapter 2.3 --- Protein purification --- p.36 / Chapter 2.3.1 --- Purification of ribosomal protein P1 and its deletion mutants --- p.36 / Chapter 2.3.1.1 --- Purification of P1 --- p.36 / Chapter 2.3.1.2 --- Purification of P1ΔC25 --- p.36 / Chapter 2.3.1.3 --- Purification of HisMBP-P1ΔC40 and HisMBP-P1ΔC47 --- p.37 / Chapter 2.3.2 --- Purification of ribosomal protein P2 and its deletion mutants --- p.38 / Chapter 2.3.2.1 --- Purification of P2 --- p.38 / Chapter 2.3.2.2 --- Purification of P2ΔC46 and P2ΔC55 --- p.39 / Chapter 2.4 --- "Preparation and purification of protein complexes formed by P1, P2 and their truncation mutants" --- p.40 / Chapter 2.4.1 --- Preparation of complexes by Co-refolding in urea buffer --- p.40 / Chapter 2.4.1.1 --- Preparation of P1 or P1ΔC25 involved complexes --- p.40 / Chapter 2.4.1.2 --- Preparation of P1ΔC40/ P2ΔC46 and P1ΔC47/ P2ΔC46 --- p.41 / Chapter 2.4.2 --- Preparation of complexes by direct mixing --- p.42 / Chapter 2.5 --- Laser light scattering for the determination of molecular weight of protein and their complexes --- p.43 / Chapter 2.5.1 --- Chromatography mode light scattering experiment (SEC/LS) --- p.43 / Chapter 2.6 --- Interaction study of P1 and P2 using BIAcore 3000 surface plasmon resonance (SPR) biosensor --- p.45 / Chapter 2.6.1 --- Immobilization of P2 onto CM5 sensor chips --- p.45 / Chapter 2.6.2 --- Kinetic measurements of P1 and P2 interaction --- p.46 / Chapter Chapter 3 --- Determination of domain boundaries for dimerization of P1/P2 --- p.46 / Chapter 3.1 --- Introduction --- p.48 / Chapter 3.2 --- Preparation of P1，P2 and their truncation mutants --- p.50 / Chapter 3.2.1 --- Construction of P1 and P2 N-terminal domains (NTDs) --- p.50 / Chapter 3.2.2 --- P1 and its truncation mutants were purified in denaturing condition --- p.53 / Chapter 3.2.3 --- "P2, P2AC46 and P2AC55 were purified" --- p.56 / Chapter 3.3 --- Formation of complexes from P1，P2 and their truncation mutants --- p.59 / Chapter 3.3.1 --- "P1, P2 and their truncation mutants interact to yield protein complexes" --- p.49 / Chapter 3.3.2 --- P1AC47/P2AC46 is the smallest N-terminal domain complex --- p.63 / Chapter 3.4 --- Perturbation of P2 NTD upon binding with P1 --- p.65 / Chapter 3.4.1 --- "1H, 15N 一 HSQC spectrum of P2AC46 changed significantly upon binding with P1" --- p.65 / Chapter 3.4.2 --- P1/P2AC46 prepared by co-refolding and direct mixing give the same HSQC spectra --- p.66 / Chapter 3.5 --- Discussion --- p.69 / Chapter Chapter 4 --- Stochiometry of P1/P2 Complex is revealed by Light scattering --- p.72 / Chapter 4.1 --- Introduction --- p.72 / Chapter 4.2 --- P1 and P2 interact in 1:1 molar ratio --- p.77 / Chapter 4.2.1 --- Purified P2 exists as homo-dimer in solution --- p.77 / Chapter 4.2.2 --- The stochiometry of P1/P2 complex is 1:1 --- p.78 / Chapter 4.3 --- Stochiometries of P1 and P2 truncation mutant complexes varied from the full-length counterparts --- p.81 / Chapter 4.3.1 --- P2AC46 and P2AC55 exist as homo-dimer in solution --- p.81 / Chapter 4.3.2 --- "P1/P2AC46, P1AC25/P2 and P1AC40/P2AC46 retain the hetero-dimeric stochiometry of 1:1" --- p.82 / Chapter 4.3.3 --- P2AC55 involved complexes show a different stochiometry --- p.83 / Chapter 4.4 --- Discussion --- p.87 / Chapter Chapter 5 --- Binding kinetics of P1/P2 complex studied by surface plasmon resonance --- p.92 / Chapter 5.1 --- Introduction --- p.92 / Chapter 5.2 --- Kinetic parameters of P1 and P2 interaction is revealed by surface plasmon resonance --- p.95 / Chapter 5.2.1 --- P2 was coupled to CM5 sensor chip surface for kinetic studies --- p.95 / Chapter 5.2.2 --- Reduction of basal response after the 1st binding of P1 --- p.96 / Chapter 5.2.3 --- P1 induced a great change in response unit than P2 upon binding with immobilized P2 --- p.99 / Chapter 5.2.4 --- Kinetic parameters of P1 and P2 interaction was studied by introducing P1 to the sensor chip surface --- p.101 / Chapter 5.2.5 --- Dissociation constant derived from 1:1 Langmuir binding isotherm --- p.102 / Chapter 5.2.6 --- Dissociation constant derived from responses at equilibrium (Req) --- p.103 / Chapter 5.3 --- Discussion --- p.106 / Chapter Chapter 6 --- Conclusion and discussion of the study --- p.112 / References --- p.118 / Appendix --- p.130