Naturally evolving proteins must navigate a vast set of possible sequences to evolve new functions. This process depends on the genotype-phenotype map. Much effort has been directed at measuring protein genotype phenotype maps to uncover evolutionary trajectories that lead to new functions. Often, these maps are too large to comprehensively measure. Sparsely measured maps, however, are prone to missing key evolutionary trajectories. Many groups turn to computational models to infer missing phenotypes. These models treat mutations as independent perturbations to the genotype-phenotype map. A key question is how to handle non-independent effects known as epistasis. In this dissertation, we address two sources of epistasis: 1) global and 2) local epistasis. We find that incorporating global epistasis improves our predictive power, while local epistasis does not. We use our model to infer unknown phenotypes in the Plasmodium falciparum chloroquine transporter (PfCRT) genotype-phenotype map, a protein responsible for conferring drug resistance in malaria. From these predictions, we uncover key evolutionary trajectories that led high resistance. This dissertation includes previously published and unpublished co-authored material. / 2020-01-11

Thesis advisor: Eranthie Weerapana / Activity-based protein profiling (ABPP) is a chemical proteomic technique that allows for selective labeling, visualization, and enrichment of the subset of active enzymes in a complex proteome. Given the dominant role of posttranslational modifications in regulating protein function in vivo, ABPP provides a direct readout of activity that is not attained through traditional proteomic methods. The first application of chemical proteomics in C. elegans was used to identify dysregulated serine hydrolase and cysteine-mediated protein activities in the long-lived daf-2 mutant, revealing LBP-3, K02D7.1, and C23H4.2 as novel regulators of lifespan and dauer formation. The tools of ABPP were also utilized in studying protein interactions at the host-pathogen interface of V. cholerae infection, discovering four pathogen-secreted proteases that alter the biochemical composition of the host, decrease the activity of host serine hydrolases, and inhibit bacterial binding by a host-secreted lectin. Lastly, ABPP was used to study the targets of protein arginine deiminases (PADs) using a citrulline-specific activity-based probe (ABP), highlighting its utility in detecting biologically relevant PAD substrates as well as identifying mRNA processing factors as previously unknown targets of PAD. Taken together, these studies demonstrate the ability of ABPP to discover novel protein regulators of physiological and pathological processes. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

by Wong Kam Bo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 149-153). / Acknowledgments --- p.i / Abstract --- p.ii / Contents --- p.iii / Abbreviations --- p.vii / Short names for mutants --- p.viii / One letter symbol for amino acids --- p.x / Chapter Chapter 1 --- Introduction / Chapter 1.1. --- Chemical and Physical Properties of Trichosanthin --- p.1 / Chapter 1.2. --- Activities of Trichosanthin at the cellular level --- p.2 / Chapter 1.3. --- Activities of Trichosanthin at the molecular level --- p.3 / Chapter 1.4. --- Objective and Strategy of Protein engineering of Trichosanthin --- p.8 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1. --- General Techniques --- p.15 / Chapter 2.1.1. --- Ethanol Precipitation of DNA and RNA --- p.15 / Chapter 2.1.2. --- Spectrophotometric quantification of DNA and RNA --- p.15 / Chapter 2.1.3. --- Minipreparation of Plasmid DNA --- p.15 / Chapter 2.1.4. --- Preparation of Plasmid DNA using Qiagen-pack 100 Cartridge --- p.16 / Chapter 2.1.5. --- Preparation of Plasmid DNA using Magic´ёØ Minipreps DNA Purification kit from Promega --- p.17 / Chapter 2.1.6. --- Preparation and Transformation of Escherichia coli Competent Cell --- p.18 / Chapter 2.1.7. --- Agarose Gel Electrophoresis of DNA --- p.19 / Chapter 2.1.8. --- Purification of DNA from Agarose Gel using GeneClean® (BIO 101 Inc.) kit --- p.20 / Chapter 2.1.9. --- Polymerase Chain Reaction (PGR) --- p.21 / Chapter 2.1.10. --- Restriction Digestion of DNA --- p.23 / Chapter 2.1.11. --- Ligation of DNA fragments --- p.23 / Chapter 2.1.12. --- Autoradiography --- p.24 / Chapter 2.1.13. --- SDS-Polyacrylamide Gel Electrophoresis (SDS- PAGE) --- p.24 / Chapter 2.1.14. --- Staining of Protein in polyacrylamide gel --- p.25 / Chapter 2.1.15. --- Western Blot detection of TCS --- p.25 / Chapter 2.1.16. --- Liquid Scintillation Counting --- p.27 / Chapter 2.1.17. --- Minimization of Ribonuclease (RNAase) activity in experiments involving RNA --- p.27 / Chapter 2.2. --- Site-Directed Mutagenesis of Trichosanthin --- p.28 / Chapter 2.2.1. --- "Construction of E160D,El60A and SEAAR deletion mutants" --- p.28 / Chapter 2.2.2. --- Construction of E189A mutant and El60A E189A double mutant --- p.31 / Chapter 2.2.3. --- Construction of E189D mutant and El60A E189D double mutant --- p.36 / Chapter 2.2.4. --- Construction of Q156A mutant --- p.38 / Chapter 2.2.5. --- Construction of Q156A El60A mutant (Fig. 2.7) --- p.41 / Chapter 2.2.6. --- Construction of Q156A El89A mutant (Fig. 2.8) --- p.43 / Chapter 2.3. --- DNA sequencing --- p.45 / Chapter 2.3.1. --- DNA Sequencing Reaction --- p.45 / Chapter 2.3.2. --- DNA Sequencing Electrophoresis --- p.46 / Chapter 2.3.3. --- Resolving GC band compression --- p.48 / Chapter 2.4. --- Overexpression of mutated TCS in Escherichia coli --- p.48 / Chapter 2.5. --- Purification of mutated TCS --- p.49 / Chapter 2.6. --- Ribosome inactivating activity Assay using Rabbit Reticulocyte Lysate In Vitro Translation system --- p.50 / Chapter 2.7. --- N-glycosidase activity Assay --- p.51 / Chapter 2.7.1. --- Inactivation of ribosome in rabbit reticulocyte lysate --- p.51 / Chapter 2.7.2. --- RNA extraction --- p.51 / Chapter 2.7.3. --- Aniline Degradation --- p.52 / Chapter 2.7.4. --- Electrophoresis of RNA in Agarose Gel containing Formamide --- p.52 / Chapter 2.8. --- Reagents and buffers --- p.53 / Chapter 2.8.1. --- Nucleic Acid Electrophoresis Buffers --- p.53 / Chapter 2.8.2. --- Reagents for preparation of plasmid DNA --- p.54 / Chapter 2.8.3. --- Media for bacterial culture --- p.54 / Chapter 2.8.4. --- Reagents for SDS-PAGE --- p.55 / Chapter 2.8.5. --- Reagents for western blot --- p.56 / Chapter 2.8.6. --- Reagents for DNA sequencing --- p.57 / Chapter Chapter 3 --- Construction of TCS mutants / Chapter 3.1. --- Introduction --- p.59 / Chapter 3.2. --- Results --- p.59 / Chapter 3.2.1. --- "Construction of E160D,El60A and ASEAAR" --- p.59 / Chapter 3.2.2. --- Construction of E189A and E160AE189A mutants --- p.66 / Chapter 3.2.3. --- Construction of E189D and E160AE189D mutants --- p.80 / Chapter 3.2 --- A Construction of Q156A mutant --- p.82 / Chapter 3.2.5. --- Construction of Q156AE160A and Q156AE189A --- p.86 / Chapter 3.3. --- Discussion --- p.90 / Chapter Chapter 4 --- Expression and Purification of mutated TCS proteins / Chapter 4.1. --- Introduction --- p.94 / Chapter 4.2. --- Results --- p.95 / Chapter 4.2.1. --- Expression and purification of E160D and El60A mutants --- p.95 / Chapter 4.2.2. --- Expression and purifcation of E189D and E160AE189D mutants --- p.99 / Chapter 4.2.3. --- Expression and purifcation of E189A and E160AE189A mutants --- p.104 / Chapter 4.2.4. --- Expression and purifcation of Q156A and Q156AE160A mutants --- p.109 / Chapter 4.2.5. --- Expression and purifcation of Q156AE189A mutant --- p.114 / Chapter 4.2.6. --- Analysis of protein purity by SDS-PAGE and Western immunoblotting --- p.114 / Chapter 4.3. --- Discussion --- p.119 / Chapter Chapter 5 --- Biological Assay of mutated proteins / Chapter 5.1. --- Introduction --- p.125 / Chapter 5.2. --- Results --- p.125 / Chapter 5.2.1. --- Ribosome inactivating activity assay --- p.125 / Chapter 5.2.2. --- N-glycosidase activities of El60AE189A mutant --- p.131 / Chapter 5.3. --- Discussion --- p.133 / Chapter 5.3.1. --- Role of glutamate-160 --- p.133 / Chapter 5.3.2. --- A putative mechanism for N-glycosidase activity of TCS --- p.137 / Chapter 5.3.3. --- Role of glutamate-189 and glutamine-156 --- p.143 / Chapter 5.3.4. --- Prospective and future studies --- p.145 / Chapter 5.4. --- Concluding remarks --- p.147 / Appendix / Chapter A.l --- Size of molecule weight markers --- p.148 / Chapter A.2 --- Reference --- p.149

Yung, Yuk Lin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 65-73). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Sou, Weng Hong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 96-106). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

"The class IX myosin is a member of the myosin superfamily and found in variety of tissues. Myosin IX is quite unique among the myosin superfamily in that the tail region contains a GTPase activating protein (GAP) domain for the small GTP-binding protein, Rho. Recently it was reported that myosin IX shows processive movement that travels on an actin filament for a long distance. This was an intriguing discovery, because myosin IX is a “single-headed” myosin unlike other processive myosins which have “double-headed” structure. It has been thought that “processive” motors walk on their track with their two heads, thus traveling for a long distance. Therefore, it is reasonable to expect that the processive movement of single headed myosin IX is based on the unique feature of myosin IX motor function. In this study, I investigated the mechanism of processive movement of single-headed myosins by analyzing the mechanism of ATPase cycle of myosin IX that is closely correlated with the cross-bridge cycle (the mechanical cycle of actomyosin). In the first part, I performed the transient enzyme kinetic analysis of myosin IX using the motor domain construct to avoid the complexity raised by the presence of the tail domain. It was revealed that the kinetical characteristics of myosin IX ATPase is quite different from other processive myosins. It was particularly notable that the affinity of the weak actin binding state of Myosin IX was extremely high comparing with known myosins. It is thought that the high affinity for actin throughout the ATPase cycle is a major component to explain the processive movement of myosin IX. In the second part of this study, I cloned full length human myosin IX construct to further investigate the regulation of motor activity of myosin IX. It was revealed that the basal ATPase activity but not the actin dependent ATPase activity of myosin IX is inhibited by its tail region. Furthermore full-length myosin IX is regulated by calcium, presumably due to the calcium binding to the CaM light chain. These result suggest that the tail domain serves as a regulatory component of myosin IX."

Le mécanisme d’épissage dans les organites est décrit comme étant l’ancêtre du spliceosome nucléaire. Cependant même si les protéines composant ce dernier sont bien connues, seulement quelques facteurs d’épissage ont été identifiés et caractérisés dans les chloroplastes et les mitochondries. Beaucoup de protéines ayant la faculté de se lier à l’ARN ont acquis des fonctions dans l’épissage, en effet un certain nombre de protéines sans véritable lien ont un rôle essentiel, avec différents degrés de spécificité dans l’épissage de la plupart des introns chloroplastiques chez les plantes. La plus grande famille de protéines se liant à l’ARN est la famille des protéines à domaines « pentatricopetide repeat » (PPR). Ces protéines sont impliquées dans la plupart des processus post-transcriptionnels dans les organites. En 2006, parmi les centaines de protéines PPR décrites chez les plantes, seulement une PPR avait été décrite comme nécessaire à l’épissage d’un intron. Ainsi, PPR4 est absolument et spécifiquement nécessaire pour l’épissage en trans de l’intron 1 de rps12 dans les plastes (Schmitz-Linneweber et al., 2006), suggérant que d’autres protéines PPR pourraient être impliquées dans l’épissage des ARN des organites. Le sujet de cette thèse porte sur la caractérisation d’autres protéines PPR impliquées dans ce processus. En utilisant des approches de génétique inverse et des outils mis en place dans le cadre de la thèse afin de détecter des défauts d’épissage par PCR quantitative, sept nouvelles PPRs impliquées dans l’épissage d’un certain nombre d’introns dans les plastes et les mitochondries ont pu être caractérisées. Dans l’optique de rechercher si des protéines PPR, impliquées dans l’épissage mais aussi dans l’édition des ARN, interagissent avec d’autres protéines, des approches de TAP-TAG ont été réalisées et sont également présentées dans ce manuscrit. L’identification de partenaires protéiques pour 3 PPRs impliquées, nous a ainsi permis de redessiner nos modèles et d’émettre de nouvelles hypothèses. Enfin, une dernière partie est consacrée à la découverte d’isoformes d’épissage pour des gènes PPR sans introns. Phénomène qui permettrait de réguler l’expression des gènes PPR, et/ou d’augmenter la diversité des protéines PPR. / The RNA splicing mechanism in organelles is described to be ancestral to that of the nuclear spliceosome. However, whereas this last complex is well known, only very few splicing factors have been identified and characterized in chloroplasts and mitochondria. Many RNA binding proteins have acquired roles in RNA splicing, and indeed a variety of often unrelated RNA binding proteins have essential functions in splicing of many plastid introns in plants, with varying degrees of specificity. The largest family of RNA binding proteins in plant organelles is the pentatricopeptide repeat (PPR) family. PPR proteins are involved in diverse post-transcriptional processes in organelles. In 2006, among hundreds of higher plant proteins of this family, only one was described as being required for a splicing event - PPR4 was shown to be absolutely and specifically required for the trans-splicing of the rps12 intron 1 in plastids (Schmitz-Linneweber et al., 2006). The main purpose of this PhD thesis was to characterize other PPR proteins involved in this process. By using a reverse genetics approach and by developing tools for the detection of splicing defects, seven new PPR proteins involved in RNA splicing of a subset of chloroplast or mitochondria introns have been characterized. In parallel, in order to characterize proteins involved in PPR-containing complexes, a TAP-TAG approach has been carried out on a few PPR proteins involved in splicing or editing of organellar RNA. The identification of partner proteins of 3 PPR proteins allows us to draw new mechanistic models and new hypotheses. Finally, the final part of the manuscript describes the discovery of splicing isoforms of PPR-encoding mRNAs. Alternative splicing may be involved in regulation of PPR gene expression and/or in increasing the diversity of the PPR protein family.

No description available.

Accurate chromosome segregation requires a spindle apparatus composed of microtubules that arise from the spindle to attach to the kinetochore, a protein complex assembled at the centromere of each chromosome. Failure to segregate chromosomes accurately may lead to lethal early developmental defects and tumorigenesis. To achieve proper kinetochore binding to microtubules, mammalian cells have evolved elaborate mechanisms to correct attachment errors and stabilize correct ones. Current models suggest that tension between kinetochore pairs (inter-kinetochore stretch) and tension at the kinetochore (intra-kinetochore stretch) produces a spatial separation of Aurora B kinase from kinetochore-associated and microtubule-binding substrates, subsequently reducing their phosphorylations and increasing their microtubule affinity. However, the tension-based models do not explain how the initial microtubule binding at unattached kinetochores occurs, where there is no tension and kinetochore-associated substrates are highly phosphorylated and, hence unable to bind to microtubules. Therefore, there must be a mechanism that explains how the phosphorylation of kinetochore substrates by Aurora B is reduced in the absence of tension. In the first part of this thesis, I examine the structural features of the coiled-coil domain of the kinetochore-associated kinesin motor protein, CENP-E. Using Single-Molecule High-Resolution Colocalization (SHREC) microscopy analysis of kinetochore-associated CENP-E, I show that CENP-E undergoes structural rearrangements prior to and after tension generation at the kinetochore. Chemical inhibition of the motor motility or genetic perturbations of the coiled-coil domain of CENP-E increases Aurora B-mediated Ndc80 phosphorylation in a tension-independent manner. Importantly, metaphase chromosome misalignment caused by inhibition of CENP-E can be rescued by chemical inhibition of Aurora B kinase. Therefore, CENP-E regulates the initial kinetochore binding to microtubules and the stabilization of kinetochore-microtubule attachments. Formin-dependent actin assembly is known to play a role in multiple processes, including cytokinesis, filopodia formation, cell polarity, and cell adhesion. Thus, formin malfunction is directly linked to various pathologies, including defects in cell migration and tumor suppression. Although the role of formins in actin polymerization has been well described, the mechanistic processes that regulate the actin assembly function of formins remain poorly understood, especially the interplay among the various sub-families of formins and how they are spatiotemporally regulated. In the second part of this thesis, I show that Aurora B-mediated phosphorylation of the formin, mDia3 regulates actin assembly. Previous studies identified two Aurora B phosphorylation sites in the FH2 domain of mDia3. To this end, phosphomimetic and non-phosphorylatable mutants of a constitutively active form of mDia3 were designed to test whether phosphorylation by Aurora B regulates actin assembly. Using an in vitro actin polymerization kinetic assay and expression of fluorescently-tagged constitutively active mDia3 in cells, I show that phosphorylation of mDia3 by Aurora B induces the actin assembly function of mDia3. Furthermore, using a phospho-specific antibody, I show that mDia3 is phosphorylated by Aurora B. Live-cell analysis shows that perturbations of these phosphorylation sites affect cell migration and cell spreading. Therefore, I illustrate a novel regulatory mechanism for the actin assembly function of mDia3 that is dependent on Aurora B kinase activity.

M.Sc. (Biochemistry) / Please refer to full text to view abstract