Academic literature on the topic 'Asymmetrical dimers'

Protein-based drugs often require targeted drug delivery for optimal therapy. A successful strategy to increase the circulation time of the protein in the blood is to link the therapeutic protein with an albumin-binding domain. In this work, we characterized such a protein-based drug, GA-Z. Using asymmetrical flow field-flow fractionation coupled with multi-angle light scattering (AF4-MALS) we investigated the GA-Z monomer-dimer equilibrium as well as the molar binding ratio of GA-Z to HSA. Using small angle X-ray scattering, we studied the structure of GA-Z as well as the complex between GA-Z and HSA. The results show that GA-Z is predominantly dimeric in solution at pH 7 and that it binds to monomeric as well as dimeric HSA. Furthermore, GA-Z binds to HSA both as a monomer and a dimer, and thus, it can be expected to stay bound also upon dilution following injection in the blood stream. The results from SAXS and binding studies indicate that the GA-Z dimer is formed between two target domains (Z-domains). The results also indicate that the binding of GA-Z to HSA does not affect the ratio between HSA dimers and monomers, and that no higher order oligomers of the complex are seen other than those containing dimers of GA-Z and dimers of HSA.

Norovirus is one of the leading agents of gastroenteritis and is a major public health concern. In this study, the crystal structures of recombinant RNA-dependent RNA polymerase (RdRp) from murine norovirus-1 (MNV-1) and its complex with 5-fluorouracil (5FU) were determined at 2.5 Å resolution. Crystals with C2 symmetry revealed a dimer with half a dimer in the asymmetrical unit, and the protein exists predominantly as a monomer in solution, in equilibrium with a smaller population of dimers, trimers and hexamers. MNV-1 RdRp exhibited polymerization activity with a right-hand fold typical of polynucleotide polymerases. The metal ion modelled in close proximity to the active site was found to be coordinated tetrahedrally to the carboxyl groups of aspartate clusters. The orientation of 5FU observed in three molecules in the asymmetrical unit was found to be slightly different, but it was stabilized by a network of favourable interactions with the conserved active-site residues Arg185, Asp245, Asp346, Asp347 and Arg395. The information gained on the structural and functional features of MNV-1 RdRp will be helpful in understanding replication of norovirus and in designing novel therapeutic agents against this important pathogen.

Plasmonic optical tweezers with a symmetry-tunable potential well were investigated based on a heterogeneous model of nano-bowtie antennas made of different noble substances. The typical noble metals Au and Ag are considered as plasmonic supporters for excitation of hybrid plasmonic modes in bowtie dimers. It is proposed that the plasmonic optical trapping force around a quantum dot exhibits symmetry-broken characteristics and becomes increasingly asymmetrical with increasing applied laser electric field. Here, it is explained by the dominant plasmon hybridization of the heterogeneous Au–Ag dimer, in which the plasmon excitations can be inconsistently modified by tuning the applied laser electric field. In the spectrum regime, the wavelength-dependent plasmonic trapping potential exhibits a two-peak structure for the heterogeneous Au–Ag bowtie dimer compared to a single-peak trapping potential of the Au–Au bowtie dimer. In addition, we comprehensively investigated the influence of structural parameter variables on the plasmonic potential well generated from the heterogeneous noble nano-bowtie antenna with respect to the bowtie edge length, edge/tip rounding, bowtie gap, and nanosphere size. This work could be helpful in improving our understanding of wavelength and laser field tunable asymmetric nano-tweezers for flexible and non-uniform nano-trapping applications of particle-sorting, plasmon coloring, SERS imaging, and quantum dot lighting.

Using a combination of biochemistry, mass spectrometry, NMR spectroscopy and cryo-electron microscopy (cryo-EM), we have been able to show that quasi-equivalent conformer switching in the coat protein (CP) of an RNA bacteriophage (MS2) is controlled by a sequence-specific RNA–protein interaction. The RNA component of this complex is an RNA stem-loop encompassing just 19 nts from the phage genomic RNA, which is 3569 nts in length. This binding results in the conversion of a CP dimer from a symmetrical conformation to an asymmetric one. Only when both symmetrical and asymmetrical dimers are present in solution is assembly of theT = 3 phage capsid efficient. This implies that the conformers, we have characterized by NMR correspond to the two distinct quasi-equivalent conformers seen in the 3D structure of the virion. An icosahedrally-averaged single particle cryo-EM reconstruction of the wild-type phage (to ∼9 Å resolution) has revealed icosahedrally ordered density encompassing up to 90% of the single-stranded RNA genome. The RNA is seen with a novel arrangement of two concentric shells, with connections between them along the 5-fold symmetry axes. RNA in the outer shell interacts with each of the 90 CP dimers in theT = 3 capsid and although the density is icosahedrally averaged, there appears to be a different average contact at the different quasi-equivalent protein dimers: precisely the result that would be expected if protein conformer switching is RNA-mediated throughout the assembly pathway. This unprecedented RNA structure provides new constraints for models of viral assembly and we describe experiments aimed at probing these. Together, these results suggest that viral genomic RNA folding is an important factor in efficient assembly, and further suggest that RNAs that could sequester viral CPs but not fold appropriately could act as potent inhibitors of viral assembly.

With thiolated oxalato bridging ligands, three strongly coupled, asymmetrical Mo₂ dimers have been studied. In these strongly mixed-valence systems, the charge is unevenly distributed and electron transfer between the two bridged Mo₂ centers occurs, as indicated by the LMCT absorption and supported by the DFT calculations.

Dual frequency liquid crystal mixtures are a technology used to achieve faster response times in electro-optical devices. At low driving frequencies the material acts as a positive dielectric, while at high frequencies the materials switches to a negative dielectric due to dielectric relaxation. This allows for voltage driven reorientation of the liquid crystal in two directions, thereby increasing the device switching speed. This project aimed to synthesise and investigate the mesomorphic properties of a series of asymmetric liquid crystal dimers targeting dual frequency mixtures. The targeted structures feature two mesogenic arms linked by a flexible methylene spacer. One section of the dimeric mesogen has lateral fluorine substituents and the other section has terminal fluorination, to generate dipoles both perpendicular and parallel to the liquid crystal director. The aim is to synthesise materials with one section of the mesogen contributing positive dielectric anisotropy at low driving frequency and the other section contributing negative dielectric anisotropy at high frequencies. The synthesis of twelve systems is presented in this thesis, these dimers vary in core length, terminal chain and parity of the spacer. The even lengthed spacers give linear shaped dimers, while the odd lengthed spacers give bent shaped materials. Synthesis proceeded through a number of conventional synthetic procedures including low temperature lithiations and Suzuki cross-couplings. Structure and purity was confirmed using NMR, MS, EA and HPLC, with the targeted materials being of high purity. The mesomorphic behaviour was investigated through a combination of optical polarising microscopy, differential scanning colorimetry and x-ray diffraction techniques. The materials exhibit nematic, smectic A, smectic C and in one case a more highly ordered nematic phase (Nx). Two of the targeted systems, one linear shaped and the other bent shaped both with asymmetric core lengths were further investigated as mixtures with each other and the stability of the Nx phase in mixtures is discussed. For the same two dimers electro-optic measurements were conducted to measure birefringence and to investigate the voltage and frequency response of the materials and it was found that the linear dimer has relatively low crossover frequency making it viable for dual frequency liquid crystal mixtures.

Marine organisms are a source of a diverse range of secondary metabolites. This thesis describes the isolation and structure elucidation of novel alkaloid and terpenoid metabolites from marine invertebrates which were collected from the Mackay - Capricorn Section of the Great Barrier Reef Marine Park (Keppel Bay and the Capricorn Bunker Group).

B-Carboline, its N,N symmetrical dimer and a series of novel asymmetrical dimers of B-carboline, were isolated from a didemnid ascidian (genus Didemnum). The asymmetrical dimers, however, were isolated in such low yield from the ascidian that their structures could not be conclusively determined. Derivatization of B-carboline allowed the preparation of these compounds in sufficient quantities to allow the elucidation of their structures by NMR spectroscopy. Two other new asymmetrical dimers of B-carboline, not observed in the ascidian, were also prepared and their structures elucidated.

Tetronic acids are commonly isolated from sponges of the genus Ircinia. A novel sesterterpene tetronic acid was isolated from the sponge Ircinia (= Psammocinia) wistarii. This novel compound (a sulfate ester) was highly unstable; rapid decomposition of the sulfate ester resulted in the formation of the known compounds ircinianin and wistarin. The isolation and structure elucidation of the novel sulfate ester is described in Chapter 3 of this thesis.

Alcyonolide 5, a novel diterpene triacetate, was isolated from two soft corals of the order Alcyonacea. This compound, is one of a series isolated from corals of the genera Alcyonium and Efflatounaria.

The bastadins are a series of (typically macrocyclic) tyrosine derivatives, commonly isolated from the sponge lanthella basta. During these investigations a new bastadin was isolated from lanthella quadrangulata.

The norcembrenolides, a series of norditerpenes, are commonly isolated from the soft corals of the genus Sinularia. The soft coral Sinularia numerosa was found to contain two of these compounds. One was the known compound norcembrenolide (the first representative of this class), the second was a stereoisomer of norcembrenolide. The chemical literature reports several compounds of this type, however, there appears to be a degree of confusion surrounding the structures of these stereoisomers. This thesis resolves the confusion surrounding the stereochemistry of these isomers, and assigns a structure to the minor metabolite of S. numerosa.

Approximately 200 marine invertebrates were collected during the course of these investigations. Many known compounds were isolated from the marine invertebrates that were collected from the Mackay - Capricorn Section of the Great Barrier Reef Marine Park. This afforded an opportunity to survey the natural products which may be found in this region, and highlights some of the difficulties associated with finding novel compounds within this region.

Tese de mestrado, Neurociências, Universidade de Lisboa, Faculdade de Medicina, 2019
Os astrócitos são um tipo de células nervosas essenciais para um bom funcionamento do Sistema Nervoso Central (SNC). Normalmente, são responsáveis pela regulação da corrente sanguínea, manutenção da barreira hematoencefálica, fornecimento de energia através dos metabólicos, participação na função e plasticidade sináptica e manutenção do equilíbrio de iões extracelulares, fluidos e transmissores. Estas células são também os primeiros elementos a responderem após um insulto do SNC. Os astrócitos perante traumas apresentam uma variedade de mecanismos de defesa e um deles e a via do JAK/STAT3. A ativação desta via ocorre durante a astrogliose reativa, um processo pelo qual os astrócitos sofrem grandes alterações, principalmente na expressão dos seus genes, na morfologia e proliferação. Em casos mais graves, a ativação de STAT3 permite a formação da cicatrização glial, uma barreia física que protege as células saudáveis das danificadas e da própria inflamação. STAT3 (signal transducer and activator of transcription 3) no geral participa em inúmeras funções biológicas em forma de homo-dímero e hetero-dímero (com STAT1, outro membro da família das proteínas STAT). Esta proteína é um fator de transcrição citoplasmático que tem como principal função a regulação da expressão de genes específicos envolvidos no crescimento, sobrevivência e diferenciação celular, assim como no desenvolvimento e na inflamação, entre outros processos biológicos. No entanto, tem sido demonstrado que a desregulação da ativação de STAT3 pode contribuir para o desenvolvimento de várias doenças. Por exemplo, STAT3 aberrante ou constitutivamente ativo está associado a uma grande variedade de cancros e malignidades hematológicas. A clássica via de sinalização de STAT3 e a JAK/STAT3. Esta começa quando citocinas ou fatores de crescimento se ligam a recetores permitindo que proteínas como JAK fosforilem o domínio citoplasmático do mesmo. Dímeros de STAT3 são recrutados para o recetor e fosforilados na tirosina na posição 705 também por JAK, ativando-os. Estes dímeros ativos entram para o núcleo e regulam a expressão de genes alvo. Inicialmente pensava-se que a formação de dímeros ocorria apenas depois da ativação de STAT3, ou seja, depois da fosforilação da tirosina 705. No entanto, nos últimos anos tem sido demonstrado que STAT3 encontra-se maioritariamente no citoplasma em forma de dímero não-fosforilado (U-STAT3) em células não estimuladas. Para além disso, dímeros de U-STAT3 conseguem entrar no núcleo e na mitocôndria para regular a atividade transcricional e aumentar a respiração celular, respetivamente. Esta descoberta levantou bastantes questões na comunidade científica pois STAT3 está a revelar ser uma proteína muito mais complexa do que se pensava. STAT3 apresenta seis domínios, cada um com funções específicas, e é alvo de inúmeras alterações como mutações e modificações pós-traducionais em certos resíduos. Estas alterações são capazes de influenciar o comportamento de STAT3 e, por conseguinte, a sua função. Por esta razão, neste projeto foram estudados vários resíduos. Cinco deles estão relacionados com modificações pós-traducionais: K49, K140, K685, Y705 e S727. K49 pode ser acetilado ou dimetilado, podendo regular positivamente a transcrição de genes e a ligação do STAT3 ao ADN. A dimetilação de K140 atenua a expressão de um regulador negativo de STAT3. Também ocorre acetilação do resíduo K685 que participa na dimerização e permite amplificar o efeito da fosforilação de Y705. A fosforilação de Y705 é, por sua vez, a modificação pós-traducional mais estudada, pois ela é responsável por ativar STAT3 para a sua função transcricional. Outra fosforilação acontece no resíduo S727 que tanto aumenta o efeito da fosforilação de Y705 ao ajudar na regulação da expressão de genes, como está envolvida na respiração mitocondrial. Para além destes resíduos, ainda foi estudado um resíduo relacionado com adenomas hepatocelulares inflamatórios quando está mutado (L78); outro relacionado com a estabilidade estrutural dos dímeros (R609); e o terminal carboxílico que inclui os dois últimos domínios de STAT3, SH2 e TAD. Para avaliar a contribuição relativa destes resíduos na dimerização e na distribuição intracelular, foram geradas combinações simétricas (com a mesma mutação nos dois monómeros) e assimétricas (com mutações diferentes em cada monómero). Ao longo do projeto foi usado o sistema de complementação bimolecular de fluorescência (BiFC) em células não estimuladas. Venus foi a proteína fluorescente usada neste sistema. Ambas metades não fluorescentes desta proteína (Venus 1 e Venus 2) foram fundidas com o terminal amínico de STAT3 em dois plasmídeos independentes. Quando duas moléculas de STAT3 dimerizam, as metades de Venus são capazes de interagir diretamente uma com a outra tornando-se numa proteína funcional e emitir fluorescência. O sinal que é emitido é diretamente proporcional à quantidade de dímeros formados. Na primeira parte deste projeto foi focada a dimerização. Através da técnica de citometria de fluxo, conseguiu-se perceber que esta não foi afetada pelos mutantes simples e duplos (Y705F e S727A) resistentes a fosforilação. No entanto, foi parcialmente diminuída pelo mutante que não apresentava os domínios SH2 e TAD, por um inibidor de STAT3 e pelas combinações assimétricas e simétrica do mutante L78R. Isto significa que a dimerização ocorre independentemente da fosforilação, e que o resíduo L78 e o domínio SH2 apresentam um papel crucial na formação e estabilização da mesma, respetivamente. Na segunda parte, foi avaliada a distribuição intracelular de STAT3 através da técnica de microscopia de fluorescência. Os resultados obtidos revelaram que os resíduos estudados afetam de maneira diferente a distribuição intracelular de STAT3. K49, Y705 e S727 são os que mais afetam a localização de STAT3. Os mutantes K49R e S727A aumentam a translocação de STAT3 entre o núcleo e o citoplasma apenas quando são combinados assimetricamente. Em contraste, o mutante Y705F aumenta este movimento apenas quando está acoplado com ele próprio. Enquanto que o mutante L78R aumenta a localização nuclear e a formação de agregados com praticamente todas as combinações (simétrica e assimétricas), a combinação simétrica de R609Q aumenta apenas a localização mitocondrial. Por fim, os pares contendo as mutações K140R e K685R apresentam uma tendência de alterar a localização citoplasmática dos dímeros de STAT3 para o núcleo, mas só com um par é que foi significativo. No entanto, vendo noutra perspetiva, as combinações com duas mutações K-R foram as que mais alteraram a translocação entre citoplasma e núcleo. As combinações com apenas uma mutação K-R ou com um mutante resistente à fosforilação conseguiram aumentar a quantidade de STAT3 no núcleo. Em suma, a ocorrência de diferentes modificações pós-traducionais nos dois monómeros do mesmo dímero influenciam a distribuição intracelular de STAT3, assim como o resíduo L78. Os resultados obtidos sugerem que as modificações pós-traducionais que ocorrem nas lisinas são as que mais regulam a circulação de STAT3 entre o citoplasma e o núcleo mesmo combinadas com outras modificações. O STAT3 que vai para a mitocôndria parece também ser regulado por estas mesmas modificações. Para além disso, estes resultados também indicam que é bastante importante que o resíduo L78 se mantenha intacto durante o funcionamento de STAT3. Esta mutação pode causar grandes alterações no comportamento de STAT3 tanto combinado com ele próprio como com outros mutantes. Estas combinações conseguiram diminuir a formação de dímeros e alterar a distribuição intracelular. Concluindo, há a possibilidade de haver um novo nível da regulação da atividade de STAT3 e, consequentemente, novos alvos terapêuticos. Para além disso, estes resultados revelaram ser importantes para outros complexos de proteínas que também sejam regulados por modificações pós-traducionais. Sendo STAT3 uma proteína envolvida no desenvolvimento, imunidade e em várias doenças, o comportamento destes resíduos estudados e das modificações pós-traducionais podem ter implicações relevantes para o diagnóstico, tratamento e o estudo de uma grande variedade de patologias humanas.
STAT3 is a transcription factor involved in many biological functions, such as cell proliferation, differentiation and survival, development and immunity, among others. STAT3 is functional when it dimerizes with itself or with STAT1. These dimers can be phosphorylated and become active, but inactive STAT3 dimers can influence its activity as well. Dysregulation of STAT3 activation initiates, contributes and sustains a variety of human diseases, including cancer. The aim of this project was to study specific residues/domains that had been described to influence STAT3 activity in the literature: K49, L78, K140, R609, K685, Y705, S727 and the SH2 domain. A Venus-STAT3 BiFC system was used in order to study the dimerization and intracellular distribution of STAT3 dimers in unstimulated cells. Asymmetric post-translational modifications change the intracellular distribution of STAT3 homodimers more strikingly than symmetric ones. The symmetric combinations carrying the L78R, R609Q and Y705F mutations were the only ones to affect intracellular distribution. Meanwhile, combinations carrying one or more K-R substitutions affected the nucleocytoplasmic shuttling. The L78 residue is also important for dimerization, mostly when combined with K49 and R609, as well as C-terminal of STAT3. This could mean a new level of regulation of STAT3 activity, and therefore a new possible therapeutic target. These results could be highly relevant for other protein complexes regulated by posttranslational modifications beyond STAT3. Given the essential roles of STAT3 in development, immunity, tissue stress and cancer, our findings could have important implications for the diagnosis, treatment and understanding of a wide spectrum of human pathologies.

博士
國立清華大學
化學系
98
In this thesis, asymmetric syntheses of methyl lactate and its antipode has been discussed. Application of these chiral synthons culminated in the asymmetric syntheses of both the antipodes of polymethylated rosmarinic acid, salvianolic acid A, salvianolic acid C, and salvianolic acid D. In addition, a modular, flexible, and convergent route to the heptamethyl lithospermic acid has been described. The heptamethyl lithospermic acid was accomplished in 5 steps from the known compounds in 28% overall yield. The trans-dihydrobenzofuran core of heptamethyl lithospermic acid was achieved by stereoselective reduction of benzo[b]furan with magnesium in methanol. Furthermore, an alternative method for the synthesis of trans-cinnamic acid derivative has been devised and can be applicable for enantioselective synthesis of lithospermic acid. The biological activity of polymethylated caffeic acids against HIV-1 virus replication was evaluated. Among them, polymethylated (+)-rosmarinic acid and (±)-salvianolic acid C were found to inhibit the replication of HIV-1RTMF (AZT-resistant) virus with IC50 values of 34 µM and 50 µM respectively. Also, this thesis presents the total synthesis of polyphenolic acids which exploits tert-butyldimethylsilyl (SiMe2t-Bu) as an ideal protecting group. In order to prove that, total synthesis of (±)-rosmarinic acid was completed in 7 steps and 42% overall yield from caffeic acid. Furthermore, efficient and flexible routes were established for the syntheses of orthogonally protected skeleton of oresbiusin, salvianolic acid C, and salvianolic acid D.

Matrix studies featuring the FTIR instrumental advantages of resolution and sensitivity for a finite amount of time are described. Ammonia clusters have a complicated spectrum in the N–H stretching region. One antisymmetric and two symmetric stretching modes in the dimer verify an asymmetric cyclic dimer structure.1 Chlorine isotopic splittings have been resolved for the four H/D substituted (HC1)3 species, which confirm the cluster stoichiometry and triangular structure.2 The ammonia/hydrogen cyanide system reveals 1:1 and 1:2 complexes, which are identified from their FTIR spectra and characterized by comparison to similar complexes.3 The ozone/hydrogen fluoride system gives the O3–HF complex, and mixed oxygen isotopic spectra show inequivalent terminal oxygen atoms.4 The symmetrical PH3–O3 complex photolyzes to give an asymmetric HOPO2 species based on mixed isotopic spectra.5

In the majority of studies of the Bezold–Brucke phenomenon, the observers have matched spectral stimuli of disparate luminance in hue by a wavelength adjustment. We used an asymmetric matching technique to match stimuli of disparate luminance in hue and saturation. We used a PIXAR imaging system to generate 28 equiluminant (110 effective td) colors on a Sony monitor. Two rectangular stimuli (1.3° × 2°) were presented, one to each eye, in a haploscope. One stimulus was reduced in luminance by placing a 1-log-unit filter before one eye. The observer adjusted the hue and saturation of the brighter stimulus until the two patches looked chromatically identical, although of different brightness (as if a mesh curtain obscured the dimmer side). One match per color was repeated on three separate days. The entire experiment was performed with a dark surround and with an equiluminant surround. With a dark surround, the matched colors were set redder or greener, consistent with the Bezold–Brucke phenomenon for spectral colors. Additionally the saturation was reduced for reds and greens. With an equiluminant surround, only desaturation was required.