京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22412号 / 工博第4673号 / 新制||工||1729(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 浜地 格, 教授 森 泰生, 教授 生越 友樹 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

This dissertation investigates non-covalent macromolecular chemistry using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) based techniques. The included studies reveal the impact of molecular structure on conformation and binding energetics. Supramolecules that might be too heavy to be dissociated in single collision-induced dissociation (CID) were dissociated using sustained off-resonance collision induced dissociation (SORI-CID) techniques. Relative binding energies and thresholds were evaluated for various macromolecular host-guest systems. Besides the non-covalent binding energies, conformation characterization was undertaken by a novel method to determine collision cross sectional areas using FTICR (CRAFTI, and multi-CRAFTI), initially developed by the Dearden lab. The systems chosen for further understanding of macromolecular interactions include calixarene-alkali metal complexes, cucurbit[5]uril-alkali halide complexes and cryptand-alkali metal complexes. The results were found to be consistent with expected behavior, and strongly correlated with predictions from computations. Size- and shape selectivity, as well as host-guest polarizability, are the main factors that govern the non-covalent macromolecular interactions that control complex conformation and dissociation. The results demonstrate the ability of FTICR to simultaneously determine binding energy, structure and conformation, which are the most important aspects for determination of comprehensive molecular characterization.

Charles University Faculty of Pharmacy in Hradec Králové Department od Biochemical Sciences Candidate: Růžena Melicharová Supervisor: PharmDr. Anna Jirkovská, Ph.D. Title of thesis: The detection of protein covalent complexes with DNA using fluorescent microscopy Anthracycline antibiotics are present one of the most potent antineoplastic drugs. The mechanism of their action is complex. They are reported to intercalate to DNA, form DNA adducts and interact with topoisomerase II (TopII) as its poisons. Catalytic cycle of TopII is interrupted when anthracyclines stabilize the covalent complex of DNA and TopII and that causes cell damage. However, using of anthracyclines is limited by several adverse effects e. g. myelotoxicity and cardiotoxicity. The mechanism of cardiotoxicity is still unclear but may be associated with poisoning of the TopIIβ isoform. Unlike the TopIIα, TopIIβ is present mostly in quiescent cells as cardiomyocytes. Furthermore, the only clinically approved cardioprotective drug dexrazoxane belongs to TopII catalytic inhibitors. Nevertheless, the details of the dexrazoxane-afforded protection are unclear. This thesis was aimed to optimize the TARDIS (trapped in agarose DNA immunostaining) assay to detect and quantify covalent cleavage complexes, compare different ways for analysis of...

Structural proteomic methods such as an ion mobility mass spectrometry, chemical cross- linking or covalent labeling have been established as powerful tools for structural studies of biomolecules in general. These methods have significantly contributed to the expansion of our knowledge about biomolecular functions, their dynamics and molecular interactions and therefore led to the understanding of important biological processes occurring in a cell. We decided to spread these methods and we developed a new radical labeling technique relaying on Fluor-alkyl radicals that does not require a laser dissociation pf hydrogen peroxide. We exploited the potential of Togni reagents to form Fluor-alkyl radicals by reducing agent under native conditions. The induction of Fluor-alkyl radicals was triggered by ascorbic acid and the labeling pulse was stopped by tryptophan. The modified proteins were analyzed by top down or bottom up approach using high resolution mass spectrometry. In case of top down approach, several fragmentation techniques (CID-ECD, ETD) were tested for protein analysis while in case of bottom up approach the analyzed proteins were digested by trypsin and separated on reverse phase column online coupled to mass spectrometer. As the model biomolecules we chose a 20 proteinogenic amino acids...

The joining of two molecular functions or the strategy of adding functions to proteins has been tremendously important for the development of proteins as tools in research and clinic. Depending on the intended application, there are a wide variety of functions that can be added to a proteins. In clinical applications drugs are a commonly conjugated to antibodies and in research adding reporter groups such as biotin, enzymes or fluorophores is a routine procedure. The chemistries and methods most often used suffer from drawbacks such as lack of stringency, which could lead to undesired effects on the protein. Many site-specific methods of labeling of antibodies require modification or insertion of handles in the antibody recombinantly, before labeling can be performed. The core of this thesis is the development of a strategy for covalent specific labeling of antibodies by exploiting the site specific binding of the Z domain to Protein A. Photoreactive Z-domains were produced by solid phase peptide synthesis, which provides the opportunity to insert a photoreactive amino acid and a reporter biotin at specific positions in the domain. The inherited binding to the Fc-part of the antibody in combination with the incorporated photoreactive amino acid, BPA, is used for site-specific interaction, and thereafter, covalent coupling to the antibody. The exposure with the appropriate wavelength of light enables the formation a covalent linkage between the Z domain and the antibody. The biotinylated photoactivable domains were subsequently used to site-specifically label a number of different types of antibodies, polyclonal rabbit IgG, monoclonal human IgG1 and monoclonal mouse IgG2a, and thereafter the antibodies was employed in a variation of applications. The photolabeling procedure of antibodies by the use of photoactivable Z domains has proven to be successful and could serve as a valuable tool in several applications. / QC 20120507

UCP1 is a member of the mitochondrial transmembrane anion carrier protein superfamily and is required to mediate adaptive thermogenesis in brown adipose tissue (BAT). Once activated, UCP1 uncouples mitochondrial respiration from ATP synthesis, thereby wasting the protonmotive force formed across the mitochondrial inner membrane as heat. It is hypothesized that proton leaks through UCP1 could be a molecular target to combat certain forms of obesity. Although it is well established that UCP1 is regulated by allosteric mechanisms, alternative methods such as post-translational modification still remain to be explored. The aims of the present study were to confirm the phosphorylation of UCP1 and the physiological relevance of this modification. Using isoelectric focusing, we confirmed that UCP1 displayed acidic shifts consistent with phosphorylation in BAT mitochondria isolated from cold exposed versus warm acclimated mice. A mouse model that ectopically expressed UCP1 in skeletal muscle was used to explore the link between the mitochondrial redox status and UCP1 function. Our results show that the expression of UCP1 in skeletal muscle led to decreases in body and tissues weights. In contrast, glucose uptake into skeletal muscle, food intake and energy expenditure was increased with the expression of UCP1. Finally, proton leaks through UCP1 were determined to be increased in isolated mitochondria from transgenic versus wild-type mice. Taken together these results indicate a complex interplay between mitochondrial redox status, post-translational modification and UCP1 function. Elucidation of novel mechanisms regulating UCP1 offers alternatives strategies that can be explored in order to modulate BAT thermogenesis.

This investigation addressed several aspects of the covalent interaction of metabolites of the insecticide chlordane with cellular macromolecules in vitro. Microsomal preparations from the liver of mice and rats were used and covalent binding to microsomal protein and RNA or to added calf thymus DNA was studied. Pure 14C-labelled cis- and trans-chlordane isomers as well as an isomeric mixture (14C-cis-chlordane plus 14C-trans-chlordane 3:1, w:w) were used as substrates for the in vitro system. Biochemical parameters investigated included inhibition of microsomal mixed-function oxidase and epoxide hydratase plus the induction of these enzymes by pretreatment with chlordane or phenobarbital. The effect of these manipulations on covalent binding of the metabolites to the macromolecules was of interest. Isolation of the protein, RNA and DNA from the in vitro microsomal systems and determination of unextractable radioactivity indicated that the chlordane derived material bound to each of the macromolecules investigated. The only exception was that mouse liver microsomes did not activate trans-chlordane to a form which bound to DNA in measurable amounts under the conditions employed. Microsomal epoxide hydratase and aminopyrine demethylase activity were increased in both the rat and the mouse following chlordane pretreatment. The effect of this induction on the macromolecular interaction of chlordane metabolites was variable for both chlordane and phenobarbital pretreated groups. Generally, for the mouse, induction increased binding to protein and DNA but decreased binding to RNA. In the rat, induction decreased binding to each of the macromolecular species. The effect of enzyme inhibition was variable in both species under the different conditions tested except for the binding of cis-chlordane derived material to DNA in the mouse liver system. In that case inhibition of epoxide hydratase clearly reduced the concentration of material covalently interacting with the DNA to unmeasurable levels. The results indicated little possibility that the primary epoxide metabolite of chlordane, oxychlordane, is involved in the binding. The effects of epoxide hydratase inhibition, however, indicate that some secondary epoxide is involved in the cis-chlordane binding to DNA in the mouse. The possible analogy between the binding behavior of chlordane found in this study and the binding behavior of other well characterized toxic compounds is discussed.

This thesis focuses on developing a new photocoupling surface, base on polyallyamine (PAAm), to increase the efficiency of the photocoupling agent perfluorophenyl azide (PFPA) in the immobilization of nanoparticles, carbohydrates and graphene. Extensive studies have been carried out in our lab on the covalent immobilization of polymers and graphene using PFPA-functionalized surfaces. Here we show that PAAm-based PFPA surface can be used to efficiently immobilize not only graphene and polymers but also nanomaterials and small molecules. This was accomplished by first silanizing silicon wafers with PFPA-silane followed by attaching a thin film of PAAm by UV radiation. Treating the PAAm surface with N-hydroxysuccinimide-derivatized PFPA (PFPA-NHS) yielded the PAAm-PFPA surface. The functionalized surfaces were characterized by ellipsometry (layer thickness), contact angle (surface tension), and ATR-FTIR. The PAAm surface was further characterized by determining the density of amino groups on the surface. The PAAm-PFPA surfaces were subsequently used to covalently immobilize polymers, nanomaterials, carbohydrates and graphene by a simple procedure of coating the molecules or materials on the PAAm-PFPA surface followed by UV irradiation. The resulting surfaces were characterized using ellipsometry, AFM, optical microscopy. The attached carbohydrates were further evaluated using lectins, i.e., carbohydrate-binding proteins.

The growing renewable energy consumption has stimulated the rapid development of diverse energy storage systems (ESSs) in our electronic society. As a successful representative, lithium-ion batteries (LIBs) play a vital role in meeting today's energy storage demand. However, LIBs are plagued by intrinsic unsafety and detrimental environmental contamination. In this respect, rechargeable aqueous zinc-ion batteries (ZIBs) and supercapacitors (SCs) as potential alternatives have attracted considerable attention due to their characteristics such as innate safety, environmental friendliness, cost-effectiveness, competitive gravimetric energy density, and loose fabrication process. Inspired by these merits, massive efforts have been devoted to designing and exploring high-performance aqueous Zn-based energy storage devices. The key for advanced Zn-based energy storage devices is to exploit high-performance cathode materials. Covalent organic frameworks (COFs) are an emerging class of organic polymer with periodic skeletons showing attractive properties in structural tunability, well-defined porosity, functional versatility, and high chemical stability. The distinguishing features of COFs make them promising electrode materials for electrochemical energy storage applications. However, the electrochemical storage capability and charge storage mechanism of COF materials have been rarely investigated, and their potential applications have not been evaluated yet so far. In this thesis, COFs are proposed as cathode materials for rechargeable aqueous Zn-ion energy storage. Initially, a new phenanthroline COF (PA-COF) material was synthesized and used as an electrode for Zn-ion supercapatteries (ZISs) for the first time. The as-synthesized PA-COF shows abundant nucleophilic sites and suitable pore structure, demonstrating the efficient storage capability of Zn2+ and H+. Further, hexaazatriphenylene-based COF (HA-COF) material with and without precisely grafted quinone functional groups has been proposed to understand structure-activity relationships. In this chapter, the influence of quinone groups on the electrochemical performance of HA-COF has been systematically studied, disclosing an enhancement coordination capability of Zn ions against protons in the quinone-functionalized HA-COF. Lastly, we synthesized a radical benzobisthiazole COF (BBT-COF) and deeply investigated the electrochemical performance. As expected, this COF electrode shows an ultrastable cycling performance and demonstrates a radical reaction pathway.

Gas-phase ion/ion covalent modification was coupled to ion mobility/mass spectrometry analysis to directly correlate the structure of gaseous ubiquitin to its solution structures with selective covalent structural probes. Collision cross section (CCS) distributions were measured prior to ion/ion reactions to ensure the ubiquitin ions were not unfolded when they were introduced to the gas phase. Ubiquitin ions were electrosprayed from aqueous and methanolic solutions yielding a range of different charge states that were analyzed by ion mobility and time-of-flight mass spectrometry. Aqueous solutions stabilizing the native state of ubiquitin generated folded ubiquitin structures with CCS values consistent with the native state. Denaturing solutions favored several families of unfolded conformations for most of the charge states evaluated. Gas-phase covalent labeling via ion/ion reactions was followed by collision induced dissociation of the intact, labeled protein to determine which residues were labeled. Ubiquitin 5+ and 6+ electrosprayed from aqueous solutions were covalently modified preferentially at the lysine 29 and arginine 54 residues, indicating that elements of secondary structure as well as tertiary structure were maintained in the gas phase. On the other hand, most ubiquitin ions produced in denaturing conditions were labeled at various other lysine residues, likely due to the availability of additional sites following methanol and low pH-induced unfolding. These data support the conservation of ubiquitin structural elements in the gas phase. The research presented here provides the basis for residue-specific characterization of biomolecules in the gas phase