Protein homo-oligomerization is the process through which identical peptides bind together to form higher order complexes. Self-interactions in many cases are constitutive and stable, used as building blocks for biological structures, such as rings, filaments and membranes. Further, homo-oligomerization can also be a regulatory process that influences the proteins' function such as change in transcriptional activities for transcription factors. Innovative methods to measure oligomerization in live cells are needed in order to understand regulation and function of homooligomerization in the native cellular context. This thesis examines the case of the tumor suppressor p53, whose homo-tetramerization greatly influences its activity as a transcription factor. We develop methods to quantify p53's self-interaction in individual living cells and follow it in time after DNA damage. The two methods we developed have complementary qualities and different applications. We first use fluorescent correlation spectroscopy to study the molecular events occurring in the first three hours of the p53 in response to double strand breaks. We find that in the absence of stress p53 is present in a mixture of, monomers, dimers and tetramers. When damage is sensed, oligomerization is rapidly induced and nearly all p53 is found bound in tetramers. We combine our data with a mathematical framework to propose the existence of a dedicated mechanism triggering p53 oligomerization independently of protein stabilization. Next, we use bimolecular fluorescent complementation to probe for tetramerization in the longer timescales of p53's response to ultraviolet radiation. In this context we find that even though the rate of p53 accumulation increases with the dose of radiation, p53 tetramers are formed at a steady rate. We hence propose the existence of an inhibitory mechanism that prevents the oligomerization reaction from following a linear input-output relation. We identify ARC, a known cofactor of p53, as part of this inhibitory mechanism. Downregulation of ARC restore the linear relation between to total and tetrameric p53. Finally, in both experimental setups higher oligomerization lead to an increase in p53 activity, underscoring the connection between regulation of oligomerization and the transcriptional activity of p53 in cancer cells. Collectively, this work emphasizes the importance of precise measurements to investigate the regulation and function of higher order complexes and provides generally applicable methods to quantify homo-oligomerization in live single cells.

Catalytic ethylene oligomerization is a well understood industrially viable process. The large majority of scientific literature and patents concerning this process has been developed with the use of chromium catalysts. Commercial systems producing selective tri/tetramerization, non-selective oligomerization and polymerization are all based on this metal with the exception of a few systems based on other transition metals (Zr, Ti, Ni etc.). This versatility raises interesting questions about chromium’s unique behaviour. Essentially, selective or non-selective oligomerization and polymerization processes could be regarded as belonging to the same category of C-C bond forming reactions, though different mechanisms are involved. The first part of this thesis explores a variety of chromium complexes for ethylene oligomerization purposes. In order to gather further information about the unique behaviour of chromium, we have explored a variety of nitrogen and phosphorus containing ligands. We started with a simple bi-dentate anionic amidophosphine (NP) ligand and assessed the role of the ligand’s negative charge and number of donor atoms in determining the type of catalytic behaviour in relation to the metal oxidation state. This ligand proved capable of generating a series of chromium dimeric, tetrameric or polymeric and even heterobimetallic chromium-aluminate complexes in different valence states. This allowed us to isolate a “single component” self activating Cr(II) complex as well as a rare example of mixed valence Cr(I)/Cr(II) species. Additionally, each of these species acted as switchable catalyst depending on the type of co-catalyst

Catalytic ethylene oligomerization is a well understood industrially viable process. The large majority of scientific literature and patents concerning this process has been developed with the use of chromium catalysts. Commercial systems producing selective tri/tetramerization, non-selective oligomerization and polymerization are all based on this metal with the exception of a few systems based on other transition metals (Zr, Ti, Ni etc.). This versatility raises interesting questions about chromium’s unique behaviour. Essentially, selective or non-selective oligomerization and polymerization processes could be regarded as belonging to the same category of C-C bond forming reactions, though different mechanisms are involved. The first part of this thesis explores a variety of chromium complexes for ethylene oligomerization purposes. In order to gather further information about the unique behaviour of chromium, we have explored a variety of nitrogen and phosphorus containing ligands. We started with a simple bi-dentate anionic amidophosphine (NP) ligand and assessed the role of the ligand’s negative charge and number of donor atoms in determining the type of catalytic behaviour in relation to the metal oxidation state. This ligand proved capable of generating a series of chromium dimeric, tetrameric or polymeric and even heterobimetallic chromium-aluminate complexes in different valence states. This allowed us to isolate a “single component” self activating Cr(II) complex as well as a rare example of mixed valence Cr(I)/Cr(II) species. Additionally, each of these species acted as switchable catalyst depending on the type of co-catalyst

Catalytic ethylene oligomerization is a well understood industrially viable process. The large majority of scientific literature and patents concerning this process has been developed with the use of chromium catalysts. Commercial systems producing selective tri/tetramerization, non-selective oligomerization and polymerization are all based on this metal with the exception of a few systems based on other transition metals (Zr, Ti, Ni etc.). This versatility raises interesting questions about chromium’s unique behaviour. Essentially, selective or non-selective oligomerization and polymerization processes could be regarded as belonging to the same category of C-C bond forming reactions, though different mechanisms are involved. The first part of this thesis explores a variety of chromium complexes for ethylene oligomerization purposes. In order to gather further information about the unique behaviour of chromium, we have explored a variety of nitrogen and phosphorus containing ligands. We started with a simple bi-dentate anionic amidophosphine (NP) ligand and assessed the role of the ligand’s negative charge and number of donor atoms in determining the type of catalytic behaviour in relation to the metal oxidation state. This ligand proved capable of generating a series of chromium dimeric, tetrameric or polymeric and even heterobimetallic chromium-aluminate complexes in different valence states. This allowed us to isolate a “single component” self activating Cr(II) complex as well as a rare example of mixed valence Cr(I)/Cr(II) species. Additionally, each of these species acted as switchable catalyst depending on the type of co-catalyst

There is an increasing trend towards advancing the understanding and development of ethylene oligomerization catalysts, both in academia and industry. The metal of choice in this chemistry is invariably chromium, which has shown great versatility in selective trimerization/tetramerization, non-selective oligomerization and polymerization of ethylene. While much success has been achieved in ethylene trimerization, the same con not be said about tetramerization catalysis. Aminophosphine based ligands have demonstrated their ability towards selective 1-octene production, however, the popular PNP catalyst is able to achieve only 70% selectivity. In order to explore the possibility of developing and enhancing the selectivity of chromium based ethylene tetramerization catalyst, this thesis work was undertaken. The ligand systems we chose for our work were bidentate aminophosphine based (PN(CH2)nNP), which has yielded interesting selective oligomerization. Subtle modifications were found to result in drastic changes in selectivity, from tetramerization (PN(CH2)3NP) to trimerization (PN(CH2)2NP). We managed to successfully develop the first truly selective (over 90%) 1-octene catalyst with polymer-free behavior. Further modifications on the ligand framework, where one atom of Si was used to link the two NP units, resulted in non-selective oligomerization, in which case we determined that the oxidation-state of chromium is a key player. We explored other modifications on our selective ligands in which one of the arms on the bidentate ligand was replaced with a base-donor amine, phosphine or pyridine, and resulted in interesting selectivity changes. The final modification that we tested was a novel N(CH2)2P ligand and found it to be a highly active, non-selective oligomerization catalyst.

There is an increasing trend towards advancing the understanding and development of ethylene oligomerization catalysts, both in academia and industry. The metal of choice in this chemistry is invariably chromium, which has shown great versatility in selective trimerization/tetramerization, non-selective oligomerization and polymerization of ethylene. While much success has been achieved in ethylene trimerization, the same con not be said about tetramerization catalysis. Aminophosphine based ligands have demonstrated their ability towards selective 1-octene production, however, the popular PNP catalyst is able to achieve only 70% selectivity. In order to explore the possibility of developing and enhancing the selectivity of chromium based ethylene tetramerization catalyst, this thesis work was undertaken. The ligand systems we chose for our work were bidentate aminophosphine based (PN(CH2)nNP), which has yielded interesting selective oligomerization. Subtle modifications were found to result in drastic changes in selectivity, from tetramerization (PN(CH2)3NP) to trimerization (PN(CH2)2NP). We managed to successfully develop the first truly selective (over 90%) 1-octene catalyst with polymer-free behavior. Further modifications on the ligand framework, where one atom of Si was used to link the two NP units, resulted in non-selective oligomerization, in which case we determined that the oxidation-state of chromium is a key player. We explored other modifications on our selective ligands in which one of the arms on the bidentate ligand was replaced with a base-donor amine, phosphine or pyridine, and resulted in interesting selectivity changes. The final modification that we tested was a novel N(CH2)2P ligand and found it to be a highly active, non-selective oligomerization catalyst.

There is an increasing trend towards advancing the understanding and development of ethylene oligomerization catalysts, both in academia and industry. The metal of choice in this chemistry is invariably chromium, which has shown great versatility in selective trimerization/tetramerization, non-selective oligomerization and polymerization of ethylene. While much success has been achieved in ethylene trimerization, the same con not be said about tetramerization catalysis. Aminophosphine based ligands have demonstrated their ability towards selective 1-octene production, however, the popular PNP catalyst is able to achieve only 70% selectivity. In order to explore the possibility of developing and enhancing the selectivity of chromium based ethylene tetramerization catalyst, this thesis work was undertaken. The ligand systems we chose for our work were bidentate aminophosphine based (PN(CH2)nNP), which has yielded interesting selective oligomerization. Subtle modifications were found to result in drastic changes in selectivity, from tetramerization (PN(CH2)3NP) to trimerization (PN(CH2)2NP). We managed to successfully develop the first truly selective (over 90%) 1-octene catalyst with polymer-free behavior. Further modifications on the ligand framework, where one atom of Si was used to link the two NP units, resulted in non-selective oligomerization, in which case we determined that the oxidation-state of chromium is a key player. We explored other modifications on our selective ligands in which one of the arms on the bidentate ligand was replaced with a base-donor amine, phosphine or pyridine, and resulted in interesting selectivity changes. The final modification that we tested was a novel N(CH2)2P ligand and found it to be a highly active, non-selective oligomerization catalyst.

La protéine suppresseur de tumeurs p53 induit l'apoptose et l'arrêt du cycle cellulaire dans les cellules qui sont stimulées par divers stress cellulaires, dont le stress génotoxique. p53 maintient l'intégrité génomique, et ses fonctions sont les plus importantes pour la résistance à la tumorigenèse cellulaire. Des mutations de TP53, qui est le gène codant pour p53, sont fréquemment observées dans les tumeurs malignes. Comme l'apoptose n'est pas induite dans les cellules cancéreuses avec des mutations ou une délétion de TP53 en réponse à la radiothérapie ou au traitement par des agents anticancéreux, le pronostic de ces patients est très mauvais. En réponse au stress cellulaire, la p53 est stabilisée, tétramérisée et activée pour exercer des fonctions telles que l'activation de la transcription. Cette fonction est précisément régulée par des modifications post-traductionnelles telles que la phosphorylation, l'acétylation, l'ubiquitination et la méthylation de plus de 50 résidus. L'homotétramérisation de la protéine p53 par le domaine de tétramerisation (DT) à la région C-terminale est indispensable à sa fonction, et son activité transcriptionnelle est régulée par la stabilité de la structure tétramérique. Le domaine de tétramérization de la protéine p53 humaine est constitué d'un brin, d'un tour et d'une hélice et est en équilibre entre le monomère et le tétramère. La régulation par des modifications post-traductionnelles dans le domaine de la tétramérization n'est pas encore claire ; il est donc nécessaire d'élucider en détail le mécanisme de régulation par des modifications post-traductionnelles. La p53 est présente dans un large éventail d'organismes, de la lamproie, qui est un vertébré précoce, aux mammifères. Bien que le rôle de p53 soit important dans l'évolution des vertébrés, la fonction de p53 et le processus d'évolution de la structure tétramérique ne sont pas clairs. En plus de l'analyse phylogénétique de la séquence, une analyse complète de la structure et de la fonction est nécessaire. Le but de cette étude était de comprendre comment la stabilité de la structure tétramérique est modifiée par des modifications post-traductionnelles et comment la DT régule l'activité et la fonction de p53. À cette fin, j'ai exploré le mécanisme de régulation de la méthylation de l'Arg et l'évolution du domaine de tétramerisation de la p53 chez les vertébrés. Cette thèse comprend cinq chapitres. Le contexte et les objectifs de l'étude sont décrits dans une introduction générale au chapitre 1. J'ai décrit la fonction suppresseur de tumeur de p53, sa structure tétramérique et son évolution chez les vertébrés. Au chapitre 2, j'ai décrit le contrôle de la fonction par méthylation de la DT p53. La fonction de la p53 est régulée par des modifications post-traductionnelles, y compris la méthylation de trois résidus Arg dans la DT. Il a été rapporté que cette méthylation par la protéine Arg méthyltransférase 5 (PRMT5) favorise l'arrêt du cycle cellulaire, mais réprime l'apoptose. Pour clarifier le mécanisme de régulation par la méthylation, j'ai effectué une analyse de la stabilité de la structure du fragment p53 méthylé et un essai de méthylation in vitro avec la PRMT5. La méthylation des résidus Arg a déstabilisé la structure oligomérique, en particulier, Arg337 a largement contribué à la déstabilisation. J'ai identifié les sites de méthylation de PRMT5 en utilisant la CLN-SM/SM et j'ai révélé une cascade de méthylation qui a commencé par la monométhylation à l'Arg335. Ces résultats suggèrent un nouveau mécanisme de régulation via la modulation de la stabilité structurelle de la DT p53. L'affinité entre l'élément de réponse du gène cible et la protéine p53 semble être élevée avec les gènes d'arrêt du cycle cellulaire et faible avec les gènes pro-apoptotiques. Lorsque p53 se lie à un élément de réponse et contrôle la transcription, l'ADN se plie. On pense que même la protéine p53 méthylée et déstabilisée peut supporter la flexion des gènes d'arrêt du cycle cellulaire parce que l'affinité est élevée, mais ne peut supporter la flexion des gènes pro-apoptotiques. Au chapitre 3, j'ai décrit l'évolution de la stabilité structurelle de la DT p53 chez les mammifères. La séquence de la DT p53 des mammifères varie de 3 à 10 résidus au sein des espèces. J'ai synthétisé le fragment de 35 résidus de la DT p53 de l'humaine, de la musaraigne arboricole, du cobaye, du hamster chinois, du mouton et de l'opossum et j'ai analysé la thermostabilité de la structure oligomérique par spectrométrie DC. La musaraigne arboricole ressemble à un écureuil mais a été classée dans un ordre indépendant ; il y a eu des substitutions de seulement quatre résidus par rapport à la variante humaine, et sa structure oligomérique s'est avérée plus stable que celle de l'humain. En analysant les mutants, il a été déterminé que la substitution à l'origine de la stabilisation était du Met354 (Gln dans la version humaine). La modélisation de l'homologie de la structure tétramérique suggère que la chaîne latérale Met située dans l'hélice C-terminale a stabilisé la structure de l'hélice de l'extrémité par la chaîne latérale Met d'une autre chaîne protéique via de nouvelles interactions hydrophobes. Récemment, la musaraigne arboricole a attiré beaucoup d'attention, car les musaraignes arboricoles sauvages boivent régulièrement et sont sensibles à l'infection par l'hépatite B, dont on pensait qu'elle n'infectait que les chimpanzés et les humains. Elle est donc maintenant utilisée comme un modèle de primate. L'aldéhyde cause des dommages à l'ADN, et il a été suggéré que la musaraigne arboricole peut maintenir l'intégrité génomique grâce à son p53 stable. Au chapitre 4, j'ai décrit une analyse plus approfondie de l'évolution de la structure, de la stabilité et de la fonction de la DT p53 chez les vertébrés. En plus de l'analyse phylogénique de l'arbre, cette étude a analysé la structure, la stabilité et la fonction. L'analyse phylogénique a montré que la DT p53 des vertébrés avait une distance d'évolution plus longue que les DT de p63 et p73, qui sont des membres de la famille p53 qui forment un tétramère de la même manière. De plus, il a été révélé que le DT p53 est plus diversifié que le domaine de liaison à l'ADN (DBD). J'ai choisi 19 espèces dont des poissons sans mâchoires, des poissons cartilagineux, des poissons à nageoires de rayons, des poissons à nageoires de lobes, des Amphibia, des Reptilia, des oiseaux et des Mammifères. Bien qu'il y ait eu des substitutions de nombreux résidus, y compris chez la lamproie, le fragment DT a formé un tétramère chez toutes les espèces. J'ai effectué une analyse de la stabilité de la structure et une analyse de l'activité transcriptionnelle de la chimère p53. La lamproie avait une stabilité structurelle et une activité faibles, et les poissons comme le poisson-zèbre avaient une activité élevée en raison d'un effet de stabilisation de la deuxième hélice dans la région C-terminale. Cependant, l'espèce de Coelacanthe atteignant l'Homme dans le but d'avancer vers la terre a gagné une stabilité qui n'était élevée que dans la première hélice. Comme une mutation du DBD affecte directement la liaison avec l'ADN et que l'évolution du DT régule indirectement l'activité de p53, on pense que la stabilité de la structure tétramérique a évolué davantage que le DBD. Au chapitre 5, j'ai décrit la conclusion globale de cette étude. Pour comprendre le mécanisme de régulation in vivo, il est très important de clarifier le mécanisme de modulation de la stabilité de la structure tétramérique de p53. Dans cette étude, il a été déterminé que la stabilité structurelle est régulée par la méthylation de la DT p53. De plus, je propose que le modèle de contrôle du choix du gène cible utilise les différences d'affinité avec l'élément de réponse et l'ajustement de la stabilité de la structure tétramérique. En outre, outre l'analyse phylogénétique de la protéine, qui est exprimée chez le vertébré entier, j'ai effectué pour la première fois une analyse de la stabilité structurelle et fonctionnelle. Les résultats suggèrent que les vertébrés se sont adaptés à l'environnement grâce à la stabilité de la DT p53, et que la p53 a joué un rôle extrêmement important dans le processus d'évolution. / Tumor suppressor protein p53 induces apoptosis and cell cycle arrest in cells that are stimulated by a variety of cellular stresses including genotoxic stress. p53 maintains genomic integrity, and its functions are the most important for resistance to cellular tumorigenesis. Mutations of TP53, which is the p53-encoding gene, are frequently found in malignant tumors. Because apoptosis is not induced in cancer cells with mutations or deletion in TP53 in response to radiation therapy or treatment with anticancer agents, the prognosis of these patients is very poor. In response to cellular stress p53 is stabilized, tetramerized, and activated to exert functions such as transcription activation. This function is precisely regulated by posttranslational modifications such as phosphorylation, acetylation, ubiquitination, and methylation of more than 50 residues. Homotetramerization of the p53 protein through the tetramerization domain (TD) at the C-terminal region is indispensable for its function, and its transcriptional activity is regulated by the stability of the tetrameric structure. The TD of human p53 consists of a β-strand, turn, and α-helix and is in equilibrium between the monomer and tetramer. The regulation by posttranslational modifications in the tetramerization domain is still unclear; thus, detailed elucidation of the regulatory mechanism through posttranslational modifications is required. p53 is found in a wide range of organisms from Lamprey, which is an early vertebrate, to mammals. Though the role of p53 is important in the evolution of vertebrates, the function of p53 and the evolution process of the tetrameric structure are unclear. As well as phylogenetic analysis of the sequence, a comprehensive analysis of the structure and the function is required. The aim of this study was to understand how the stability of the tetrameric structure is changed by posttranslational modifications and how the TD regulates p53’s activity and function. To this end, I explored the Arg methylation regulatory mechanism and the evolution of the p53 tetramerization domain in vertebrates. This thesis consists of five chapters. The background of the study and the study goals are described as a general introduction in Chapter 1. I outlined the tumor suppressor function of p53, its tetrameric structure, and its evolution in vertebrates. In Chapter 2, I described the function control by methylation of the p53 TD. p53’s function is regulated by posttranslational modifications, including the methylation of three Arg residues in the TD. It has been reported that this methylation by protein Arg methyltransferase 5 (PRMT5) promotes cell cycle arrest, but represses apoptosis. To clarify the regulatory mechanism through methylation, I carried out structure stability analysis of the methylated p53 fragment and an in vitro methylation assay with PRMT5. The methylation of the Arg residues destabilized the oligomeric structure, in particular, Arg337 had a large contribution to the destabilization. I identified PRMT5 methylation sites using nLC-MS/MS and revealed a methylation cascade that started with mono-methylation at Arg335. These findings suggest a novel regulatory mechanism via structural stability modulation of the p53 TD. The affinity between the response element of the target gene and the p53 protein appeared to be high with pro-cell cycle arrest genes and low with pro-apoptotic genes. When p53 binds to a response element and controls transcription, the DNA bends. It is thought that even methylated and destabilized p53 can endure bending of pro-cell cycle arrest genes because the affinity is high, but cannot bear bending of the pro-apoptotic genes. In Chapter 3, I described the evolution of the structural stability of the p53 TD in mammals. The sequence of the mammalian p53 TD varies in 3–10 residues within species. I synthesized the 35-residue fragment of the p53 TD of human, tree shrew, guinea pig, Chinese hamster, sheep, and opossum and analyzed the thermostability of the oligomeric structure using CD spectrometry. The tree shrew resembles a squirrel but classified independent order; there were substitutions of only four residues in comparison with the human variant, and its oligomeric structure has been shown to be more stable than the human one. By analyzing the mutants, it was determined that the substitution causing the stabilization was of Met354 (Gln in the human version). The homology modeling of the tetrameric structure suggests that the Met side chain located in the C-terminal α-helix stabilized the helix structure of the end through the Met side chain of other protein chain via new hydrophobic interactions. Recently, the tree shrew has attracted a lot of attention, as wild tree shrews drink routinely and are susceptible to hepatitis B infection, which has been thought to infect only chimpanzees and humans. Thus, it is now used as a primate-like model. Aldehyde causes DNA damage, and it has been suggested that the tree shrew can maintain genomic integrity because of its stable p53. In Chapter 4, I described more extensive analysis of the evolution of the structure, stability, and function of the p53 TD in vertebrates. As well as phylogenic tree analysis, this study analyzed structure, stability, and function. The phylogenic analysis showed that the p53 TD of vertebrates had a longer evolution distance than the TDs of p63 and p73, which are p53 family members that formed a tetramer in the same way. In addition, it was revealed that the p53 TD is more diversified than the DNA-binding domain (DBD). I chose 19 species including jawless fish, cartilaginous fish, ray-finned fish, lobe-finned fish, Amphibia, Reptilia, birds, and Mammalia. Though there were substitutions of many residues including in Lamprey, the TD fragment formed a tetramer in all species. I carried out structure stability analysis and transcriptional activity analysis of chimeric p53. Lamprey had low structural stability and activity, and fish such as Zebrafish had high activity due to a stabilization effect of the second helix in the C-terminal region. However, the Coelacanth species reaching Human aiming at an advance to the land gained stability that was high only in the first helix. Because a mutation in the DBD directly affects binding with DNA and the evolution of the TD regulates the activity of p53 indirectly, it is thought that the stability of the tetrameric structure evolved more than the DBD. In Chapter 5, I described the all-inclusive conclusion of this study. To understand the regulatory mechanism in vivo, it is very important to clarify the stability modulation mechanism of the p53 tetrameric structure. In this study, it was determined that structural stability is regulated by the methylation of the p53 TD. Furthermore, I propose that the target gene choice control model uses the differences in the affinity with the response element and adjustment of the tetrameric structure stability. Furthermore, as well as phylogenetic analysis of the protein, which is expressed in the whole vertebrate, I performed structural stability and functional analysis for the first time. The results suggest that vertebrates adapted to the environment via the stability of the p53 TD, and p53 played an extremely important role in the evolution process.

Linear alpha olefins (LAOs) are key petrochemical precursors for the synthesis of larger polymers, detergents, plasticizers, and lubricants. Most catalytic ethylene oligomerization processes generate a wide distribution of LAO carbon chain lengths. A major ongoing industrial challenge is to develop homogeneous catalysts that result in selective and tunable ethylene oligomerization to 1-hexene and 1-octene alkenes. Quantum mechanical calculations coupled with rapidly advancing technology have enabled the ability to calculate small molecule systems with high accuracy. Employing computational models to advance from empirical to quantitative prediction of product selectivities has become an active area of exploration. In this work, we demonstrate the development and use of a density-functional theory (DFT) transition-state model that provides highly accurate quantitative prediction of phosphinoamidine (P,N) Cr catalysts for controllable selective ethylene trimerization and tetramerization. This model identified a new family of highly selective catalysts that through computational-based ligand design results in a predictable shift from 1-hexene selectivity to 1-octene. Subsequent experimental ligand synthesis and catalyst testing verified the quantitative computational predictions. DFT calculations also provide key insights to factors controlling catalytic activity and present important design criteria for the development of active Cr-based ethylene oligomerization systems. Non-selective ethylene transformations, referred to as full range processes, provide access to a range of LAOs (C4-C20) that are used to produce polyethylene, surfactants, and other commercial products. During full-range oligomerizations, undesired byproducts degrade the purity of LAOs mostly consisting of branched oligomers. Computational mechanistic investigations reveal the origin of linear versus branched selectivity in Fe-catalyzed ethylene oligomerization reactions.

<p> We developed some universal platforms to overexpress the minimal functional entities of ion channels. The modular property of ion channels have been demonstrated from many aspects, such as crystal structures, chimeric channel experiments and discovery of similar modules in distantly related protein families. Thus it should be feasible to express each module independent of other channel modules. The pore-forming module of ion channels has multiple important properties as selectivity, conductivity and drug-binding. If it can be overexpressed, it will provide valuable information about channel selectivity to different ions and structural bases for drug binding as well as important application in drug screening and rational drug design. </p> <p> To test this, we first used the model channel KcsA to identify the minimal requirements for a pore-forming domain to functionally exist independently. Chapter 2 of this thesis explains in detail how the wild type C-terminal cytoplasmic domain of KcsA functions. We found that this domain has dual function as pH-sensor and tetramerization domain, and it is essential for the expression of the pore-forming domain of KcsA. Once we knew the physiological role of the cytoplasmic domain, the scenario was set to answer the question of how to make it better for the application of structural and functional studies. </p> <p> In chapter 3 and chapter 4, we replaced the wild type C-terminal domain with non-native tetramerization domains. We identified the direct correlation between protein expression level and overall thermostability of pore-forming domains. The C-terminal tetramerization domains stabilize channels in a cooperative way and play a critical way in in vivo channel assembly. The selection of the linker between pore-forming domain and tetramerization domain, the splicing motif, and the handedness of C-terminal tetrameric coiled coils all affect channel expression level and stability. </p> <p> We applied our finding in KcsA to a wide range of ion channels in chapter 5, including voltage-gated potassium channels, Ca2+-gated potassium channels, inwardrectifying potassium channels, cyclic nucleotide-gated potassium channels and voltagegated sodium channels. We managed to express similar minimal structural modules from these more structurally complicated channels with the assistance of different cytoplasmic tetramerization domains. Several minimal channels expressed well and showed similar biophysical and functional property as the wild type channels. </p> <p> These studies demonstrate that the pore-forming modules of ion channels can be expressed independently while retaining the proper structure and drug-binding properties as their wild type predecessors when using our universal expression platform. The potential application in structural studies and drug-screening is promising. </p> / Thesis / Doctor of Philosophy (PhD)