京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18693号 / 工博第3971号 / 新制||工||1611(附属図書館) / 31626 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 北川 進, 教授 梅田 眞郷, 教授 濵地 格 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19182号 / 工博第4059号 / 新制||工||1626(附属図書館) / 32174 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 北川 進, 教授 杉野目 道紀, 教授 濵地 格 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

No description available.

The overall goal of my work was to develop and apply new chemical methods for the study of epigenetic DNA and protein modifications. In Chapter 3 the development of Suzuki-Miyaura cross coupling (SMcc) for the post-synthetic modification of DNA is described. DNA modification by SMcc is efficient (4-6h) and proceeds under mild conditions (37°C, pH 8.5). The incorporation of various groups useful for biological investigations is demonstrated using this methodology. Using a photocrosslinker, introduced into the DNA by SMcc capture experiments are performed to identify potential binding partners of modified DNA. In Chapter 4 a dehydroalanine (Dha) based chemical protein modification method is described that enables the introduction of posttranslational modification (PTM) mimics into histones. The PTM mimics introduced by this method are tested using western- and dot-blot and binding and enzymatic assays, confirming they function as mimics of the natural modifications. Chapter 5 describes the use of a generated PTM mimics to elucidate the function of O-linked β-Nacetylglucosamine (GlcNAc) of histones in transcriptional regulation. It is shown that GlcNAcylation of Thr-101 on histone H2A can destabilize nucleosome by modulating the H2A/B dimer – H3/H4 tetramer interface. N- and C-terminal histone tails play an important role in transcriptional regulation. In Chapter 6, nuclear magnetic resonance is used to investigate the structure of the histone H3 N-terminal tail in a nucleosome. The H3 tail, while intrinsically disordered, gains some α-helical character and adopts a compact conformation in a nucleosome context. This H3 tail structure is shown to be modulated by Ser-10 phosphorylation. The effect of a new covalent DNA modification, 5- hydroxymethylcytosine (5hmC), on transcription factor binding is investigated in Chapter 7. 5hmC influences HIF1α/β, USF and MAX binding to their native recognition sequence, implying involvement of this modification in epigenetic regulation.

Site-selective protein modification has become an important tool to study protein functions in chemical biology. In the preliminary work, allyl sulfides were found to be reactive substrates in aqueous cross-metathesis (CM) enabling the first examples of protein modification via this approach. In order to access the enhanced CM reactivity of allyl sulfide on proteins, facile chemical methods to install S-allyl cysteine on protein surface were developed. In particular, a cysteine-specific allylating reagent – allyl selenocyanate was used on protein substrate for the first time. The substrate scope of allyl sulfide-tagged proteins and factors that affect the outcome of CM was also investigated. A range of metathesis substrates containing different olefin tether of various lengths were screened; allyl ethers were found to be most suitable as CM partners. By reducing the steric hindrance around the allyl sulfide on protein surface through a chemical spacer, the rate and conversion of metathesis reaction on proteins was greatly enhanced. Moreover, allyl selenides were found to be more reactive than allyl sulfides in CM and enabled reactions with substrates that were previously impossible for the corresponding sulfur-analogue. Through this work, substrate selection guidelines for successful metathesis reaction on proteins were established. Rapid Se-relayed CM was further investigated through biomimetic chemical access to Se-allyl selenocysteine (Seac) via dehydroalanine. On-protein reaction kinetics revealed rate constants of Seac-mediated CM to be comparable or superior to off-protein rates of many current bioconjugations. This CM strategy was applied to histone proteins to install a mimic of acetylated lysine (K9Ac, an epigenetic marker). The resulting synthetic H3 was successfully recognized by antibody that binds natural H3-K9Ac. A Cope-type selenoxide elimination subsequently allowed the removal of such modification to regenerate dehydroalanine. Finally, preliminary research efforts towards metabolic incorporation of allyl sulfide-containing amino acid into proteins, and CM on cell surfaces were discussed.

The covalent chemistry of individual reactants bound within a protein nanopore can be monitored by observing the ionic current flow through the pore, which acts as a nanoreactor responding to bond-making and bond-breaking events. However, chemistry investigated in this way has been largely confined to the reactions of thiolates, presented by the side chains of cysteine residues. The introduction of unnatural amino acids would provide a large variety of reactive side chains with which additional single-molecule chemistry could be investigated. An efficient method to incorporate unnatural amino acid is semisynthesis, which allows site-specific modification with a chemically-defined functional group. However, relatively little work has been done on engineered membrane proteins. This deficiency stems from attributes inherent to proteins that interact with lipid bilayer, notably the poor solubility in aqueous buffer. In the present work, four different derivatives α-hemolysin (αHL) monomer were obtained either by two- or three-way native chemical ligation. The semisynthetic αHL monomers were successfully refolded to heptameric pores and used as nanoreactors to study single-molecule chemistry. The semisynthetic pores show similar biophysical properties to native αHL pores obtained from an in vitro transcription and translation technique. Interestingly, when αHL pores with one semisynthetic subunit containing a terminal alkyne group were used to study Cu(I)-catalyzed azide-alkyne cycloaddition, a long-lived intermediate in the reaction was directly observed.

Hydrogenases are a family of enzyme that catalyses the bidirectional interconversion of H⁺ and H₂. There are two major classes of hydrogenases: the [NiFe(Se)]- and [FeFe]-hydrogenases. Both of these benefit from characteristics which would be advantageous to their use in technological devices for H₂ evolution and the generation of energy. These features are explored in detail in this thesis, with a particular emphasis placed on defining the conditions that limit the activity of hydrogenases when reducing H⁺ to produce H₂. Electrochemistry can be used as a direct measure of enzymatic activity; thus, Protein Film Electrochemistry, in which the protein is adsorbed directly onto the electrode, has been employed to probe catalysis by hydrogenases. Various characteristics of hydrogenases were probed. The catalytic bias for H₂ production was interrogated and the inhibition of H₂ evolution by H₂ itself (a major drawback to the use of some hydrogenases in technological devices to produce H₂) was quantified for a number of different hydrogenase. Aerobic inactivation of hydrogenases is also a substantial technological limitation; thus, inactivation of both H₂ production and H₂ oxidation by O₂ was studied in detail. This was compared to inhibition of hydrogenases by CO so as to elucidate the mechanism of binding of diatomic molecules and determine the factors limiting inactivation. This allows for a preliminary proposal for the genetic redesigning of hydrogenases for biotechnological purposes to be made. Finally, preliminary investigation of the binding of formaldehyde, potentially at a site integral to proton transfer, opens the field for further research into proton transfer pathways, the structural implications thereof and their importance in catalysis.

The centriole is a complex cylindrical assembly found in the cells of ciliated eukaryotes. It serves two important roles in the cell: templating the growth of cilia, and forming the basis of the centrosome, which is the major microtubule organising centre in the cell. Cilia and centrosomes are involved in many cellular processes, from signalling to cell division and differentiation. As such, defects in centriole assembly can have downstream consequences on these processes and are linked to a variety of human diseases including cancer and microcephaly. The complex superstructure of the centriole has fascinated biologists for decades. It comprises a nine-fold, radially symmetric array of microtubule triplet blades attached to a central cartwheel structure. During the last two decades, proteomic analyses have identified many proteins that are associated with the centriole. However, genetic studies have shown that only a surprisingly small number of these proteins are essential for the biogenesis of the centriole. In Drosophila melanogaster, three such essential proteins, SAS-6, Ana2 and SAS-4 are required in the early stages of centriole biogenesis. In this thesis I have investigated the assembly steps involving these key players from a structural perspective. I have identified and recombinantly expressed functional domains of these proteins in order to characterise them in vitro. Using X-ray crystallography and other biophysical techniques, I have been able to define mechanisms for several steps involved in the assembly of these proteins. In collaboration with colleagues in the laboratory I have been able to investigate the biological significance of these essential assembly steps in vivo. This information has provided novel insights into the molecular, and even atomic, detail of the initial steps of centriole assembly, including an explanation of a natural point mutation involved in human microcephaly.

Foi levado a efeito estudo sobre formas múltiplas de aldolase de músculo de coelho. A enzima foi purificada a pH 7,5 por eluição com substrato a partir de coluna de fosfocelulose. A enzima foi ainda cristalizada por diálise dessas preparações contra solução saturada de sulfato de amônio. Formas múltiplas de aldolase foram obtidas por fracionamento a diferentes pI por eletrofocalização em gradiente de Ampholine na faixa de pH entre 7,0 a 10,0. Nessas condições foram separados cinco híbridos resultantes da associação ao acaso das sub-unidades α e β, os quais foram analisados em estado de dissociação a partir de proteínas carboximetiladas e separadas por eletroforese em gel de poliacrilamida na presença de uréia 8M. Foi também estudado o aparecimento de sub-unidade α e β em músculo de coelhos de idades que variavam de 1 a 240 dias, verificando-se que em coelhos de 1 dia existia apenas a proteína α4, surgindo sub-unidades β já em animais de 10 dias. / Not available.

Staphylococcal leukocidin pores are formed by the obligatory interaction of two distinct polypeptides, one of class F and one of class S, making them unique in the family of β-barrel pore-forming toxins (β-PFTs). By contrast, other β-PFTs form homooligomeric pores. For example, the staphylococcal α- hemolysin is a homoheptamer. Limited and controversial data exist on the assembly and molecular architecture of the leukocidin pore. In this work, biochemical and biophysical methods were used to characterize the leukocidin pore produced by the LukF (HlgB) and LukS (HlgC) components encoded by Staphylococcus aureus. I demonstrate that LukF and LukS assemble to form an SDS-stable pore on rabbit erythrocyte membranes. In addition, the pore-forming properties of recombinant leukocidin were investigated with planar lipid bilayers. Although leukocidins and staphylococcal α-hemolysin share partial sequence identity and related folds, LukF and LukS produce a pore with a unitary conductance of 2.5 nS (1 M KCl, 5 mM HEPES, pH 7.4), which is over three times greater than that of α-hemolysin measured under the same conditions. The subunit composition and stoichiometry of a leukocidin pore were determined by two independent methods, gel shift electrophoresis and sitespecific chemical modification during single channel recording. Four LukF and four LukS subunits were shown to co-assemble into an octameric transmembrane structure. The existence of an additional subunit in part explains properties of the leukocidin pore, such as its high conductance. Additionally, this is the first time that either technique has been applied successfully to assess the composition of a heteromeric membrane protein. It is also relevant to understanding the mechanism of assembly of β-PFT pores, and suggests new possibilities for engineering these proteins. In additional studies, the HlyII pore encoded by Bacillus cereus was found to form a homoheptameric transmembrane pore with properties conforming in general with those of other members of the class of β-PFTs. HlyII possesses additional properties which make it an attractive candidate for applications in biotechnology, such as an oligomer with a high thermal stability in the presence of SDS and the ability of the pore to remain open at high transmembrane potentials.