Thesis (Ph. D.)--Rutgers University, 2008. / "Graduate Program in Computational Biology and Molecular Biophysics." Includes bibliographical references (p. 157-179).

Orientador: Gustavo Orlando Bonilla Rodriguez / Banca: Patrícia Caetano de Souza / Banca: Luiz Henrique Florindo / Resumo: A Glutationa (GSH) é um tripeptídeo presente nas células, que em sua forma reduzida age como antioxidante, mantendo os grupamentos tiólicos nas proteínas em estado reduzido. Ao exercer função protetora, a glutationa é oxidada para sua forma dissulfeto, expondo quatro cargas negativas. A glutationa oxidada (GSSG) poderia interagir não covalentemente com moléculas capazes de ligar ânions, como a hemoglobina. Essa condição nos levou a investigar as possíveis mudanças estruturais e funcionais das hemoglobinas bovina, humana e majoritária do peixe matrinxã (Brycon cephalus) quando interagem alostericamente com a glutationa. As amostras de sangue bovina e humana foram purificadas e submetidas à eletroforese para verificação de pureza. As propriedades de ligação com oxigênio e controle alostérico foram analisados por tonometria a 20°C, calculando a afinidade de ligação com o O2 e a cooperatividade (n50). As condições experimentais adotadas foram, stripped, cloreto e glutationa oxidada (GSSG), nessas condições, as hemoglobinas bovina e humana apresentaram efeito Bohr alcalino. O cloreto induziu o maior efeito de diminuição da afinidade em praticamente todas as condições. As hemoglobinas apresentaram processo cooperativo de ligação de oxigênio, em todas as condições e em toda faixa de pH. No caso da hemoglobina bovina adulta, a GSSG aumentou a afinidade por O2, em toda a faixa de pH testada, enquanto para a humana isso ocorreu abaixo de pH 7,0. O cloreto capaz de diminuir a afinidade de forma efetiva em todas as condições. A hemoglobina bovina apresentou uma menor afinidade de ligação ao oxigênio em relação à hemoglobina humana. A glutationa oxidada atuou como efetor alostérico heterotrópico em hemoglobinas humana, bovina e matrinxã (Hb-II), aumentando a afinidade de ligação por oxigênio da hemoglobina. Entretanto, a glutationa na forma reduzida... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Glutathione (GSH) is an intracellular tripeptide, which in its reduced form acts as an antioxidant, keeping the cysteines in the reduced state. When exerting protective function, glutathione is oxidized to form its disulfide, exposing four negative charges. The oxidized glutathione (GSSG) could interact with molecules covalently able to bind anions, such as hemoglobin. That motivated us to investigate the possible structural and functional changes of bovine, human and a fish hemoglobin from the fish 'matrinxã' (Brycon cephalus) when they interact allosterically with glutathione. Samples of human and cattle blood were purified and subject to electrophoresis to verify their purity. The functional properties of oxygen binding and allosteric control were analyzed by the tonometric method at 20°C, calculating the affinity of O2-binding and cooperativity (n50). The tested experimental conditions involved hemoglobin in the absence (stripped) and in the presence of chloride and oxidized glutathione (GSSG). Under those circumstances, the human and bovine hemoglobins displayed an alkaline Bohr effect. Chloride induced the greatest effect of reducing affinity in all conditions. For all the experimental sets oxygen binding was cooperative. For bovine adult hemoglobin GSSG increased O2-affinity for all the tested pH values, whereas it occurred for human Hb for pH values above 7.0. Bovine Hb displayed lower O2-affinity than its human counterpart. Oxidized glutahione acted as a heterotropic allosteric effector in all the tested hemoglobins, increasing O2-affinity. However, GSH decreased O2-affinity of human and bovine Hbs. We assume that there is a binding site for GSSG at the R state, increasing O2-affinity, and another one for GSH at the T state, stabilizing its conformation and therefore lowering O2-affinity. / Mestre

Made available in DSpace on 2014-06-11T19:22:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-11-29Bitstream added on 2014-06-13T20:10:03Z : No. of bitstreams: 1 koga_di_me_sjrp.pdf: 1553861 bytes, checksum: f88c5801e8c2256d97ed7327d979e856 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O α-tocoferol é a principal dentre as moléculas que desempenham o papel biológico de vitamina E, sendo a que apresenta maior biodisponibilidade e atividade. Além dos papéis como vitamina, o α-tocoferol tem diversas funções no organismo incluindo a inibição de proteínas da família PLA2. Esse trabalho apresenta a análise vibracional do α-tocoferol inteiro no vácuo seguido pela otimização geométrica da molécula em ambiente protéico. O estudo do tocoferol no vácuo foi realizado dentro do formalismo do DFT usando o funcional de correlação-troca B3LYP e a base de funções 6-311G**. A análise vibracional foi realizada em um programa desenvolvido pelo autor. Em ambiente protéico, o processo foi dividido em duas etapas: a otimização geométrica do sistema inteiro no formalismo da dinâmica molecular e a otimização somente do ligante e de um envelope protéico em DFT. Por dificuldades computacionais não foi possível a análise vibracional do sistema. / The α-tocopherol is the most important molecule which has the biological role of vitamin E, having the greatest biodisponibility and activity. Besides its vitamin roles, the α- tocopherol has many functions in organisms including the inhibition of proteins of the PLA2 family. This work presents the vibrational analysis of the whole α-tocoferol in vacuum followed by the geometric optimization of the molecule in protein environment. The study of tocopherol in vacuum was done in the DFT framework using the B3LYP exchangecorrelation functional with the 6-311G** function basis and the vibrational analysis was done by a program developed by the author. The process was divided in two steps for the study in protein environment: the geometrical optimization of the whole system in the molecular dynamics framework and the optimization of the ligand and a protein envelope in DFT. Due to computational troubles it was not possible to perform the vibrational analysis of this system.

Being able to control protein function directly and in real time is attractive. In this thesis, I discuss controlling protein activity using alternating current electric fields on the order of 1 MV/m. Since protein structures result in localized and/or protein-wide charge gradients, it is expected that a sufficiently high electric field applied across a protein should result in structural distortions which can temporarily alter or halt protein function. The field is set to oscillate above the influences of the electrochemical double layer effects (1 kHz) and below the level needed for hydrodynamic rotation of proteins (10 MHz). A device is used to pass this field across a small volume of sample while allowing the solution to still be observed. Through application of high electric fields, we are able to temporarily reduce the activity of a bioluminescent luciferase reaction. Activity is inferred by measurement of the intensity and wavelength of the light emitted by the luciferase reaction. As this process is explored further, it could lead to the ability to electrically control protein function.

Yes / We describe a method for performing time-resolved X-ray crystallographic experiments based on the Hadamard transform, in which time resolution is defined by the underlying periodicity of the probe pulse sequence, and signal/noise is greatly improved over that for the fastest pump-probe experiments depending on a single pulse. This approach should be applicable on standard synchrotron beamlines and will enable high-resolution measurements of protein and small-molecule structural dynamics. It is also applicable to other time-resolved measurements where a probe can be encoded, such as pump-probe spectroscopy. / Wellcome Trust 4-year PhD program “The Molecular Basis of Biological Mechanisms” 089312/Z/09/Z. This work was also supported by the EPSRC Award “Dynamic Structural Science at the Research Complex at Harwell” EP/I01974X/1 and by BBSRC Award BB/H001905/1.

Allergic reactions are mainly mediated by the interactions between the IgE and its ligands, amongst them CD23 and CD21 in what is termed the IgE network. CD23 is involved in upregulating IgE expression by forming a trimolecular complex with CD21 and IgE on the B-cell surface, resulting in the specific activation of IgE-positive B cells. CD21 also interacts with C3d and is a bridge between the innate and the immune system. A crystal structure of the interaction has been solved (Szakonyi et al., 2001) but was controversial because it contradicted previous biochemical analyses. The aims of this thesis were to use various biophysical techniques to study the interactions between the molecules in the IgE network and its possible inhibition. Part 1: Characterisation of a phage display-derived peptide that inhibit IgE binding to CD23 A peptide was previously derived using phage display technology and tested for binding ability to CD23 using SPR and ITC. Subsequent NMR experiments were performed to identify the binding site, followed by characterization of its derivatives. Crystallisation of CD23 with the peptide and soaking with its truncated tripeptide, NWP, were also attempted. Part 2: Characterisation of CD23 and its interaction with its ligands X-ray crystallography was undertaken to solve the structure of derCD23 in complex with a phage display-derived peptide (Part1) followed by crystal soaking with a truncated tripeptide, NWP. However, a reproducible, high-resolution wild type derCD23 structure was determined at 1.9 Å. A comparison of the binding behaviour between the monomeric derCD23 and a trimeric CD23 construct was carried out in order to see the effect of oligomerisation upon IgE binding. Using the known interaction map as well as a crystal structure, the possible interacting residues between CD23 and IgE were examined. The characterisation of the CD23/CD21 interaction was continued from previous efforts in order to confirm that the binding epitope of CD23 for CD21 lies within the C-terminus of CD23. Characterisation of the interactions of CD23/IgE/FcεRI was performed to examine these multimolecular interactions and possible regulatory mechanisms in mast cell degranulation. It was shown that CD23 can form multimeric complexes with IgE-Fc that bind to FcεRI with higher apparent affinity than IgE-Fc alone, which may lead to increases in mast cell degranulation. It was also found that the IgE bound on FcεRI still binds to CD23 although with a lower binding capacity, presumably due allosteric changes. The binding of CD23 with a monoclonal antibody IDEC-152 was also characterised using SPR and NMR spectroscopy. It was proposed that IDEC-152 might interfere with the trimerisation site of CD23 thus reducing its affinity for IgE. A thermofluor assay was developed and optimised for potential screening of compounds that bind to derCD23 using a qPCR machine, which may be useful to screen compounds that bind to CD23 as part of future drug discovery project. Crystallisation of the derCD23/CD21 and IgE/triCD23/CD21 complexes was also attempted as part of ongoing crystallisation projects. Part 3: The interaction between C3d and CD21 The interaction between C3d and CD21 is believed to be a bridge between the innate and adaptive immune response, and is thought to be pivotal in the initiation of autoimmune disease. Following from previous studies on this interaction, further characterisations were performed using NMR and ITC to confirm the involved sites on CD21 (SCR1-2) in binding to C3d. Several potential salt bridges have been identified so far, allowing a high-resolution docked structure of the C3d/CD21 complex.

<p>The four-electron reduction of dioxygen to water is the most exothermic non-photochemical reaction available to biology. A detailed molecular description of this reaction is needed to understand oxygen-based redox processes. Horseradish peroxidase (HRP) is a haem-containing redox enzyme capable of catalysing the reduction of dioxygen to water. We developed instrumentation and experimental methodology to capture and characterise by X-ray crystallography transient reaction intermediates in this reaction. </p><p>An instrument was designed (“the vapour stream system”) to facilitate reaction initiation, monitoring and intermediate trapping. In combination with single crystal microspectrophotometry, it was used to obtain conditions for capturing a reactive dioxygen complex in HRP. X-ray studies on oxidised intermediates can be difficult for various reasons. Electrons re-distributed in the sample through the photoelectric effect during X-ray exposure can react with high-valency intermediates. In order to control such side reactions during data collection, we developed a new method based on an angle-resolved spreading of the X-ray dose over many identical crystals. Composite data sets built up from small chunks of data represent crystal structures which received different X-ray doses. As the number of electrons liberated in the crystal is dose dependent, this method allows us to observe and drive redox reactions electron-by-electron in the crystal, using X-rays.</p><p>The methods developed here were used to obtain a three-dimensional movie on the X-ray-driven reduction of dioxygen to water in HRP. Separate experiments established high resolution crystal structures for all intermediates, showing such structures with confirmed redox states for the first time. </p><p>Activity of HRP is influenced by small molecule ligands, and we also determined the structures of HRP in complex with formate, acetate and carbon monoxide.</p><p>Other studies established conditions for successfully trapping the M-intermediate in crystals of mutant bacteriorhodopsin, but the poor diffraction quality of these crystals prevented high-resolution structural studies.</p>

The four-electron reduction of dioxygen to water is the most exothermic non-photochemical reaction available to biology. A detailed molecular description of this reaction is needed to understand oxygen-based redox processes. Horseradish peroxidase (HRP) is a haem-containing redox enzyme capable of catalysing the reduction of dioxygen to water. We developed instrumentation and experimental methodology to capture and characterise by X-ray crystallography transient reaction intermediates in this reaction. An instrument was designed (“the vapour stream system”) to facilitate reaction initiation, monitoring and intermediate trapping. In combination with single crystal microspectrophotometry, it was used to obtain conditions for capturing a reactive dioxygen complex in HRP. X-ray studies on oxidised intermediates can be difficult for various reasons. Electrons re-distributed in the sample through the photoelectric effect during X-ray exposure can react with high-valency intermediates. In order to control such side reactions during data collection, we developed a new method based on an angle-resolved spreading of the X-ray dose over many identical crystals. Composite data sets built up from small chunks of data represent crystal structures which received different X-ray doses. As the number of electrons liberated in the crystal is dose dependent, this method allows us to observe and drive redox reactions electron-by-electron in the crystal, using X-rays. The methods developed here were used to obtain a three-dimensional movie on the X-ray-driven reduction of dioxygen to water in HRP. Separate experiments established high resolution crystal structures for all intermediates, showing such structures with confirmed redox states for the first time. Activity of HRP is influenced by small molecule ligands, and we also determined the structures of HRP in complex with formate, acetate and carbon monoxide. Other studies established conditions for successfully trapping the M-intermediate in crystals of mutant bacteriorhodopsin, but the poor diffraction quality of these crystals prevented high-resolution structural studies.

Epigenetic regulation is a complex process involving the interplay of multiple different cellular factors. Work described in this thesis concerned the characterisation of proteins involved in the binding to, and demethylation of, histone 3 (H3) tails modified by N-methylation. Initial work focussed on the biophysical characterisation of the tandem plant homeodomains (PHD) of the chromatin remodeller CHD4. NMR spectroscopy was used to investigate the solution structure of the tandem PHDs. Studies on a more native-like construct including the C terminal tandem chromodomains are also presented. Binding studies of the PHDs with H3 peptides reveal that the individual PHD fingers can independently bind a histone peptide. The remainder of the work involved characterisation of JmjC histone demethylases (KDMs), enzymes that catalyse removal of Nε-methyl groups from histone lysyl-residues. Initially, two members of the KDM7 subfamily, PHF8 and KIAA1718, were studied; a high throughput screening assay for them was developed, which enabled identification of a selective inhibitor of the KDM2/7 subfamilies of KDMs, the plant growth regulator Daminozide. A disease relevant mutation in PHF8 was studied and shown to cause mis-localisation of the enzyme to the cytoplasm, providing a potential explanation for the clinically observed phenotype. Subsequent chapters describe unprecedented activities for the JmjC KDMs. 2OG oxygenases catalyse a wide range of oxidative reactions, predominantly mediated by initial substrate hydroxylation. The activity of PHF8 with lysine analogous was tested; the results demonstrated that PHF8, and other KDMs, can oxidatively remove Nε-alkyl groups other than methyl groups, such as ethyl and isopropyl groups. The substrate scope of the JmjC KDMs thus has the potential to be wider than previously thought. Observation of β-hydroxylation of the Nε-isopropyl group of a histone peptide including Nε methylisopropyllysine by JMJD2A/E supports the presumed mechanism of histone lysine demethylation as proceeding via initial hydroxylation. This work led to the discovery that JmjC KDMs can catalyse arginine demethylation. This novel arginine demethylase activity by JmjC KDMs was characterised and the work extended to encompass potential arginine demethylase activity in cells. Biochemical characterisation of UTY, a homologue of the H3 K27 demethylases JMJD3 and UTX, which is reported to be inactive, was carried out; UTY was shown to catalyse demethylation at H3 trimethylated at K27 on peptidic substrates, albeit it at substantially lower rates than the other family members. To investigate the reason for this reduced activity, two variants were made, S1142G and P1214I; the latter variant was shown to be considerably more active than wildtype UTY, likely due to an increased peptide-binding interaction. Preliminary experiments in cells did not conclusively demonstrate histone demethylation, but a luciferase assay suggested that UTY may have catalytic activity in cells. Overall the findings in the thesis suggest that the process of cellular epigenetic regulation is likely even more complex than previously thought, with the potential that JmjC KDMs carry out multiple, context dependent functions.

The physical properties of viral capsids are major determinants of vaccine efficacy for several picornaviruses which impact on human and animal health. Current picornavirus vaccines are frequently produced from inactivated virus. Inactivation often reduces the stability of the virus capsid, causing a problem for Foot and Mouth Disease Virus (FMDV) where certain serotypes fall apart into pentameric assemblies below pH 6.5 or at temperatures slightly above 37°C, destroying their effectiveness in eliciting a protective immune response. As a result, vaccines require a cold chain for storage and animals need to be frequently immunised. FMDV is a member of the Aphthovirus genus of the Picornaviridae. Globally there are seven FMDV serotypes: O, A, Asia1, C and SAT-1, -2 and -3, contributing to a dynamic pool of antigenic variation. As part of collaboration between the Division of Structural Biology, Oxford University, The Pirbright Institute, Reading University and ARC, Ondespoort, South Africa we sought to rationally engineer thermo-stable FMDV capsids either as infectious copy virus or recombinant empty capsids with improved thermo-stability for improved vaccines. In this project, in silico molecular dynamics (MD) simulations, molecular modelling, free energy calculations, X-ray crystallography, electron microscopy and various biochemical/biophysical techniques were used to design and help characterise the capsids. For the most unstable FMDV serotypes (O and SAT2), panels of stabilising mutants were characterised by MD. Promising candidates were then engineered and shown to confer increased thermo- and pH-stability. Thus, in silico predictions translate into marked stabilisation of both infectious and recombinant empty viral capsids. A novel in situ method was used to determine crystal structures for quality assessment and to verify that no unanticipated structural changes have occurred as a consequence of the modifications made. The structures of the wildtype and two of the stabilised mutants were solved and the antigenic surfaces shown to be unchanged. Animal trials showed stabilised particles can generate a similar or improved neutralising antibody response compared to the traditional vaccines and may therefore lead to a new generation of stable and safe vaccines.