Дисертації з теми "Hydrogen deuterium exchange (HDx)"
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1
Bourguet, Maxime. "Développements méthodologiques en spectrométrie de masse structurale pour la caractérisation de complexes biologiques multiprotéiques." Thesis, Strasbourg, 2019. http://www.theses.fr/2019STRAF013.
Повний текст джерелаАнотація:
Ce travail de thèse porte sur le développement de méthodes de spectrométrie de masse (MS) structurale pour la caractérisation de systèmes protéiques complexes, souvent réfractaires aux approches biophysiques classiques. Dans ce contexte, les développements entrepris furent notamment focalisés sur la caractérisation de complexes impliqués dans la biogénèse des ribosomes et dans la régulation transcriptionnelle, fonctions cellulaires essentielles pouvant être liées à de nombreuses pathologies humaines dont certains cancers. Ainsi, les approches par MS native, pontage chimique et d’HDX-MS ont permis de renseigner sur la connectivité, les proximités spatiales ou encore la dynamique conformationnelle retrouvées au sein des complexes étudiés. Parmi ces techniques, l’HDX-MS permet une approche comparative basée sur les mesures d’incorporations en deutérium renseignant sur la dynamique conformationnelle d’une protéine sous différents états. Aussi, la combinaison d’approches de MS structurale a permis d’approfondir la caractérisation des systèmes complexes étudiés, démontrant ainsi l’intérêt d’une approche intégrative dans ce contexteThis PhD thesis focuses on developing methods in structural mass spectrometry (MS) to characterize complex protein systems, given their size and their heterogeneity, frequently inaccessible by classical biophysic approaches. In this context, methodological developments have particularly focused on the characterization of protein complexes involved in ribosomes biogenesis and transcriptional regulation. These fundamental cellular processes are related to numerous diseases such as cancers and genetic diseases. Thus native MS, crosslink, and hydrogen/deuterium exchange coupled to MS (HDX-MS) allowed gaining insights about the stoechiometry, spatial proximities and conformational dynamics of studied systems. Among these approaches, HDX-MS enables a comparative approach based on deuterium incorporation measurements giving information about the conformational dynamics of labeled proteins in various experimental conditions. Finally, the combination of structural approaches enables to deeply characterize complex protein systems, highlighting the advantages of an integrative approach in this context
2
Durazo, Armando. "Hydrogen/deuterium exchange studies on copper-zinc superoxide dismutase." Diss., Restricted to subscribing institutions, 2007. http://proquest.umi.com/pqdweb?did=1495960591&sid=1&Fmt=2&clientId=1564&RQT=309&VName=PQD.
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Mullangi, Vennela Dr. "Development and Application of Histidine Hydrogen Deuterium Exchange Mass Spectrometry." Cleveland State University / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=csu1388959354.
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4
Stofella, Michele. "Hydrogen deuterium exchange: methods to probe protein dynamics at single residue resolution." Master's thesis, Alma Mater Studiorum - Università di Bologna, 2020. http://amslaurea.unibo.it/21242/.
Повний текст джерелаАнотація:
The purpose of this work is to provide computational methods to fingerprint protein dynamics probed by hydrogen deuterium exchange mass spectroscopy (HDX-MS). Hydrogen deuterium exchange consists in the spontaneous exchange of amide hydrogens of amino acids with deuterium contained in solution. The consequent increase in mass of the protein can be monitored by mass spectroscopy. Moreover, the exchange rate (or protection factor) provides a parameter probing protein dynamics at single residue resolution. The ExPfact algorithm is a computational method implemented to extract fine-grained information out of coarse-grained HDX-MS experimental data. The method is validated through a comparison with protection factors estimated from HDX-NMR measurements probing the mouse prion protein. Also, a second application studying glycogen phosphorylase shows how structural changes between different states of the same protein can be detected at amino acidic resolution. Furthermore, fine-grained information extracted by ExPfact is coupled with a back-exchange correction to reproduce experimental spectra, suggesting that the information encoded in the centroids of the spectra is sufficient to characterize experimental data. Last but not least, an existing structural model connecting the structure of a protein to its protection factors is discusses and improved via the introduction of a dependence on the electrostatic potential of the protein.
5
Tsutsui, Yuko. "EXPLORING FUNCTIONAL AND FOLDING ENERGY LANDSCAPES BY HYDROGEN-DEUTERIUM EXCHANGE MASS SPECTROMETRY." Case Western Reserve University School of Graduate Studies / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=case1196199391.
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6
Lu, Xiaojun. "STRUCTURE OF PRION PROTEIN AMYLOID FIBRILS AS DETERMINED BY HYDROGEN/DEUTERIUM EXCHANGE." Case Western Reserve University School of Graduate Studies / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=case1205510131.
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7
Hsu, Simon. "Hydrogen/deuterium-exchange (DXMS) analysis of the carbohydrate phosphatase, starch-excess 4." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2008. http://wwwlib.umi.com/cr/ucsd/fullcit?p1459886.
Повний текст джерелаАнотація:
Thesis (M.S.)--University of California, San Diego, 2008.Title from first page of PDF file (viewed January 5, 2009). Available via ProQuest Digital Dissertations. Includes bibliographical references (p. 80-83).
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Mandell, Jeffrey G. "Protein-protein interactions studied by hydrogen-deuterium exchange and computer-aided docking /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2000. http://wwwlib.umi.com/cr/ucsd/fullcit?p9970664.
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NDONI, ENEA. "Characterization of the immune response and cross protection activity elicited by the Neisserial Heparin Binding Antigen (NHBA), a component of the 4CMenB vaccine." Doctoral thesis, Università di Siena, 2017. http://hdl.handle.net/11365/1011542.
Повний текст джерелаАнотація:
Invasive disease caused by capsular group B Neisseria meningitidis (MenB) is life threating disease causing hundred thousands of deaths every year, still remaining an unmet medical need in many countries. Although disease can be observed at all age groups, infants and adolescents are the most at risk populations showing the highest incidence in case numbers. Since the MenB capsule was not-immunogenic the development of a MenB vaccine which makes the use of other antigens becomes necessary. 4CMenB is a multicomponent vaccine against serogroup B N. meningitidis composed by three major protein antigens, factor H-binding protein (fHbp), Neisserial Heparin-Binding Antigen (NHBA) and Neisserial adhesin A (NadA), combined with outer membrane vesicles (OMVs) from the New-Zealand epidemic strain (NZ98/254). Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein expressed by all N. meningitidis strains analyzed so far and is composed of two major domains, a highly variable amino-terminal (N-term) domain which anchors the protein on the bacterial outer membrane through the lipobox motif, and a highly conserved carboxyl-terminal (C-term) domain. These domains are separated by a short and quite conserved Arginine-rich (Arg-rich) motif which has been reported to be involved in different mechanisms that mediate meningococci adhesion, infection and survival within the host’s blood stream. NHBA is susceptible to cleavage by NalP, a bacterial protease which has its cleavage site upstream of the arginine region. Moreover human proteases such as human lactoferrin (hLf) and kallikrein are able to process NHBA downstream the the Arg-rich region. Both bacterial and human proteases-mediated cleavage releases the C-term of NHBA in the supernatant, while the N-term of the protein remains anchored on the bacterial surface. NalP cleavage did not impact SBA titers elicited by anti-NHBA antibodies but little is known about the impact that host’s proteases have on bactericidal titers. Based on sequence analysis it has been reported that NHBA has two major alleles, the so called “short” and “long” variants, which differentiate by the presence or absence of a 190 bp long fragment. Despite its sequence variability, NHBA is able to induce a robust and broad immune response against meningococcal strains expressing vaccine homologous and heterologous variants. Although anti-NHBA antibodies are able to induce bacterial killing when tested in serum bactericidal activity assay (SBA), the regions involved in eliciting cross protective immune response remain still unknown. Aims of this study were to use monoclonal antibodies (mAbs) raised against the NHBA vaccine variant peptide 2 (NHBAp2) to (i) map the NHBA regions involved in eliciting the functional response, (ii) test their ability to induce cross protection against strains expressing epidemiologically relevant homologous and heterologous NHBA variants, and (iii) investigate the molecular mechanism of NHBA-mediated bactericidal activity. To this end we used a panel of anti-NHBA mAbs selected to recognize different regions of the protein. Our results showed that only anti-N-term mAbs were able to induce killing of bacterial strains expressing the homologous NHBAp2 and closely related heterologous NHBA variants. Synergy between monoclonal antibodies targeting the N-term and the C-term of NHBA resulted in a significant increase of bactericidal titers but cross protection remained restricted to closely phylogenetic NHBA variants. Anti C-term mAbs were not able to induce SBA activity when tested individually, but surprisingly they became bactericidal when tested in combination. Moreover they were able to induce full cross protection against a panel of strains expressing phylogenetically distant heterologous NHBA variants.
Our results suggest that the partial release of the NHBA C-terminal portion upon NalP and serum proteases could explain why anti-C-term mAbs are not able to induce complement mediated bactericidal killing when tested individually. However, the simultaneous binding of C-term mapping mAbs on the same NHBA molecule can induce the formation of a very stable ternary complex that probably allows a more efficient C1q engagement and C3 deposition, thus leading to the observed co-operative bactericidal activity. These results suggest that synergy between anti-NHBA antibodies is at the basis of the mechanism of NHBA-induced bactericidal activity, which could explain the robust and cross-protective immune response elicited by anti-NHBA polyclonal antibodies following immunization. Collectively, the body of experimental data suggests that both domains of NHBA are required to elicit complement mediated bactericidal activity against strains expressing the vaccine homologous and heterologous NHBA variants.
10
Lau, Simon Sheen Man. "The application of capillary electrophoresis-hydrogen deuterium exchange-mass spectrometry in peptide analysis." Thesis, University of Strathclyde, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.431775.
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Brooke, Jennifer Christine. "Probing the water content of the Earth's mantle : an experimental study of hydrogen mobility under extreme conditions." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/28994.
Повний текст джерелаАнотація:
Previous research has established that the majority of nominally anhydrous minerals (NAMs) in Earth’s mantle can incorporate water in the form of structurally bound hydrogen and, correspondingly, the mantle is thought to contain a substantial volume of water. Water has been shown to play a key role in the geodynamics of the Earth’s interior and quantifying the amount, and distribution, of water in the mantle is an important step in understanding many deep-Earth processes. One of the parameters highly sensitive to the incorporation of water in the mantle is electrical conductivity, as hydrogen is highly mobile and acts as the dominant charge-carrying species. In theory, this relationship can be used in conjunction with geophysical techniques that measure mantle-scale electrical conductivity to ‘map-out’ the deep Earth’s water content – but accurate interpretation of such data requires full understanding of hydrogen mobility in NAMs under extreme conditions, which remains poorly constrained. The aim of this project is to contribute to the reconciliation of geophysical observations with laboratory measurements of electrical conductivity, by considering hydrogen-deuterium exchange in single crystals. In a novel experimental design, hydrogen in crystals synthesised under mantle conditions (such that the hydrogen defects present correspond to the conditions being studied) exchanges with deuterium from a liquid source under controlled (mantle) pressure and temperature conditions for a specified time period. This results in hydrogen-deuterium exchange profiles that can be characterised by SIMS and subsequently fitted to Fick’s law to calculate hydrogen diffusion coefficients – which in turn can be related to electrical conductivity through the Nernst-Einstein equation. Analysis of the experimental results underlines the complexity of the influence of hydrogen on electrical conductivity in NAMs, and emphasises the need for careful consideration when interpreting and applying the results of diffusion studies. Ultimately, the data obtained in this study provides a useful contribution to understanding hydrogen diffusion in mantle minerals, but the non-trivial nature of both the experimental and analytical aspects mean that the method cannot easily be applied to other mantle phases.
12
Herrmann, Kristin Ann. "Hydrogen / Deuterium Exchange and Fragmentation of Biomolecules to Probe Gas Phase Structure and Energetics." Diss., Tucson, Arizona : University of Arizona, 2005. http://etd.library.arizona.edu/etd/GetFileServlet?file=file:///data1/pdf/etd/azu%5Fetd%5F1397%5F1%5Fm.pdf&type=application/pdf.
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Masson, Glenn Robert. "New insights into the dynamics of phosphoinositide signalling through hydrogen deuterium exchange mass spectrometry." Thesis, University of Cambridge, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.709108.
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Foord, Rachel Lucy. "The effect of osmolytes on protein stability." Thesis, Imperial College London, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.244276.
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15
McCombs, Michelle. "A 59-Cobalt NMR Investigation of the Hydrogen/Deuterium Exchange Kinetics in Cobalt(III) Complexes." TopSCHOLAR®, 2003. http://digitalcommons.wku.edu/theses/554.
Повний текст джерелаАнотація:
Currently, the hydrogen/deuterium exchange kinetics in cobalt(III) complexes are being investigated. In the presence of deuterated solvents, (e.g. D2 0 and CH3CH20D) the amine hydrogens in the complexes are exchanged for deuteriums. For the hexaamminecobalt(III) ion, 19 isotopmers (H18D0 to HOD 18) are possible. For the tris(ethylenediammine)cobalt(III) ion, 13 isotopmers (HI 2D0 to HOD 12) are possible. Each hydrogen/deuteruim exchange causes a shift in the observed 59Co resonance of approximately 6 ppm. The rate constant of the hydrogen-deuterium exchange for the first H-D exchange has been determined as a function of solvent. When the chosen solvent is D20, the rate constant is 1.09 x 10° sec"1 for the hexaamminecobalt(III) ion. In methanol-d, the rate constant is 1.82 x 10"4 sec"'. Electronic effects of ligands have also been investigated. Experimental conditions (e.g., observation frequencies and solution parameters) and the representative NMR spectra are presented.
16
Hodkinson, John. "(beta)2-microglobulin from function to fibril : an investigation using hydrogen/deuterium exchange mass spectrometry." Thesis, University of Leeds, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.507734.
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Long, Yaoling. "Study of the conformation of myoglobin adsorbed on nanoparticles using hydrogen/deuterium exchange mass spectrometry." [Gainesville, Fla.] : University of Florida, 2009. http://purl.fcla.edu/fcla/etd/UFE0041357.
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Owens, Simon. "Kinetics and mechanisms of hydrogen isotope exchange over solid storage media." Thesis, University of Bath, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.687343.
Повний текст джерелаАнотація:
Hydrogen isotope separation systems using palladium (Pd) are currently being designed for both reactor designs with the aim of separating and purifying the reactor exhaust products which contain valuable unspent hydrogen isotopes. Hydrogen isotope exchange in Pd offers an efficient, ambient condition process that can produce pure isotopic species in a process far simpler and less costly than the current state of the art cryogenic distillation processes. The method is applicable whether separating hydrogen (protium), deuterium or tritium and any combination of these. If practical fusion devices are ever to be realised it is essential to produce an economical and efficient fuel cycle capable of separating and purifying hydrogen isotopes. Hydrogen isotope exchange in Pd is also of interest to the waste separation and purification industries, in particular those using hydrogen separation membranes which used Pd and Pd-alloy membranes. Understanding hydrogen isotope exchange, with particular regard to the formation of the intermediate (and often unwanted) hydrogen deuteride (HD), will aid significantly in future designs of hydrogen isotope separation systems. Novel hydrogen isotope exchange experiments involving hydrogen and deuterium at a number of temperatures (208 K, 293 K and 373 K) and pressures (1.3 bar – 8 bar) not yet explored are presented in this thesis. The experiments were carried out on a unique piece of laboratory apparatus provided to and further developed at the University of Bath. Alongside experimentation, a novel comprehensive multidimensional multi-physics model has been created to analyse the experimental data obtained using the new apparatus and elucidate the kinetics and mechanisms of the reactions occurring between hydrogen isotopic species and Pd during hydrogen isotope exchange based on Langmuir-Hinshelwood surface reaction mechanism. The surface reaction rates, kinetic rate constants and heat effects have been examined in detail, and in tandem, for the first time.
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Gingras, Isabelle. "Deactivation and regeneration processes affecting the catalyst for deuterium isotopic exchange between water vapour and hydrogen." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1995. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/mq25593.pdf.
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Arrington, Justine Victoria. "Hydrogen/Deuterium Exchange Studies of Protein and Non-Protein Amino Acids using Electrospray Ionization Mass Spectrometry." W&M ScholarWorks, 2012. https://scholarworks.wm.edu/etd/1539626931.
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Gucinski, Ashley Christine. "Gas Phase Structural Studies of Peptide Fragment Ions: Structural Insights into Mass Spectrometry Fragmentation Mechanisms." Diss., The University of Arizona, 2011. http://hdl.handle.net/10150/202766.
Повний текст джерелаАнотація:
This dissertation presents extensive structural studies of gas-phase peptide fragment ions, with a specific focus on b₂⁺ ions. Fragment ion structures can provide important insights into peptide fragmentation mechanisms. Based on the structures formed, information about the preference of competing b ion formation pathways can be obtained. b₂⁺ ion structures are of interest because of their large relative abundances in MS/MS spectra, which are difficult to predict. Prior to this work, only a few b₂⁺ ion structures were determined; these systems featured only aliphatic residues and all formed oxazolones. The work presented herein examines the influence of basic, acidic, and backbone-attached sidechains on peptide fragmentation mechanisms, as revealed by the resulting b₂⁺ fragment ion structure(s) formed. Specifically, the structures of several histidine, aspartic acid, and proline-containing b₂⁺ ions are determined by using action IRMPD spectroscopy, fragment ion HDX, and DFT calculations. The structures of a series of histidine analogue-containing b₂⁺ ions reveal that the location and availability of the pi-nitrogen is essential for diketopiperazine formation. The histidine sidechain bulk or strain interferes with the complete trans-cis isomerization required for diketopiperazine formation, so the oxazolone structure is also present. Xxx- Pro b₂⁺ ions favor oxazolone formation with aliphatic N-terminal residues. HP favors the diketopiperazine, combining the histidine effect and the proline cis conformation propensity. For Xxx-Asp b₂⁺ ions, aspartic acid significantly influences b₂⁺ ion structure only with an N-terminal histidine or lysine; both HD and KD form a mixture of oxazolone, anhydride, and diketopiperazine structures, presenting the first spectroscopic evidence for the anhydride b₂⁺ion structure. The HA and AH b₂⁺ ions feature the same structures, but HP and PH do not, showing that residue position matters. Additionally, while relative intensities and HDX rates featured some fluctuation, peptide precursor composition differences did not alter the mixture of b₂⁺ ion structures formed for a given b₂⁺ ion. To complement existing gas-phase structural methods, the utility of a new technique, QCID-HDX-IRMPD, was applied to m/z 552.28 from YAGFL-OH. Both the standard b₅⁺ fragment ion and an isobaric non-C-terminal water loss ion are present. Without separation of these isomers, MS/MS spectral interpretation would be complicated.
22
Stokasimov, Ema. "Insights into the allosteric interactions within the actin molecule." Diss., University of Iowa, 2009. https://ir.uiowa.edu/etd/890.
Повний текст джерелаАнотація:
Actin's ability to engage in a wide range of physiological functions requires that it be subject to complex spatial and temporal regulation. This regulation is achieved internally through monomer-monomer contacts and externally through interactions with actin binding proteins. The first part of my thesis focused on better understanding the role of inter-monomeric ionic interactions proposed between subdomains 2 and 3 of opposing monomers in F-actin stabilization. I studied several yeast actin mutants: A167R to disrupt a proposed ionic attraction with R39, A167E to mimic a proposed ionic attraction in muscle actin, and D275R to disrupt a proposed ionic attraction with R39. I investigated the effects of mutations in vivo, effects on filament polymerization characteristics and appearance in vitro, as well as interaction of the mutants with the filament severing protein cofilin. While both in vivo and in vitro data demonstrated the importance of the R39-D275 interaction for yeast actin and the interaction of the filament with cofilin, disruption of this interaction alone did not cause filament fragmentation. Conversely, results with A167 do demonstrate the in vivo and in vitro importance of another potential R39 ionic interaction for filament stabilization.
In the second part of my work I used amide proton hydrogen/deuterium (HD) exchange detected by mass spectrometry as a tool to gain structural insight into yeast and muscle actin and profilin isoform differences and the actin-profilin interaction. The yeast and muscle actin HD analysis showed greater exchange for yeast G-actin compared to muscle actin in the barbed end pivot region and areas in subdomains 1 and 2, and for F-actin in monomer-monomer contact areas. These results suggest greater flexibility of the yeast actin monomer and filament compared to muscle actin. For yeast-muscle hybrid G-actins, the muscle-like and yeast-like parts of the molecule generally showed exchange characteristics resembling their parent actins. There were a few exceptions to this rule, however: a peptide on top of subdomain 2 and the pivot region between subdomains 1 and 3. These exhibited muscle actin-like exchange characteristics even though the areas were yeast-like, suggesting that there is crosstalk between subdomains 1 and 2 and the large and small domains. Hybrid F-actin data showing greater exchange compared to both yeast and muscle actins are consistent with mismatched yeast-muscle actin interfaces resulting in decreased stability of the hybrid filament contacts. Actin-profilin HD exchange results demonstrated a possible differential interaction of specific profilin isoforms with specific actin isoforms. While profilin binding mostly caused a decreased exchange for yeast actin peptides, it caused an increase in exchange for muscle actin peptides. Many of the changes observed were in peptides that line or contact the nucleotide cleft, consistent with profilin's ability to alter the kinetics of nucleotide exchange.
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Zimmer, Agnes [Verfasser], and C. [Akademischer Betreuer] Ritter. "Structural Characterisation of Fungal and Bacterial Amyloids by Hydrogen/Deuterium Exchange NMR Spectroscopy / Agnes Zimmer ; Betreuer: C. Ritter." Braunschweig : Technische Universität Braunschweig, 2011. http://d-nb.info/117582495X/34.
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Lindh, Erik L. "Cellulose-water interaction: a spectroscopic study." Doctoral thesis, KTH, Tillämpad fysikalisk kemi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-199200.
Повний текст джерелаАнотація:
The human society of today has a significantly negative impact on the environment and needs to change its way of living towards a more sustainable path if to continue to live on a healthy planet. One path is believed to be an increased usage of naturally degradable and renewable raw materials and, therefore, attention has been focused on the highly abundant biopolymer cellulose. However, a large drawback with cellulose-based materials is the significant change of their mechanical properties when in contact with water. Despite more than a century of research, the extensively investigated interaction between water and cellulose still possesses many unsettled questions, and if the answer to those were known, cellulose-based materials could be more efficiently utilized. It is well understood that one interaction between cellulose and water is through hydrogen bonds, established between water and the hydroxyl groups of the cellulose. Due to the very similar properties of the hydroxyl groups in water and the hydroxyl groups of the cellulose, the specific interaction-induced effect on the hydroxyl groups at a cellulose surface is difficult to investigate. Therefore, a method based on 2H MAS NMR spectroscopy has been developed and validated in this work. Due to the verified ability of the methodology to provide site-selective information regarding the molecular dynamics of the cellulose deuteroxyl groups (i.e. deuterium-exchanged hydroxyl groups), it was shown by investigating 1H-2H exchanged cellulose samples that only two of the three accessible hydroxyl groups (on the surface of cellulose fibrils) exchange with water. This finding was also verified by FT-IR spectroscopy, and together with MD simulations we could establish that it is O(2)H and O(6)H hydroxyl groups (of the constituting glucose units) that exchange with water. From the MD simulations additional conclusion could be drawn regarding the molecular interactions required for hydrogen exchange; an exchanging hydroxyl group needs to donate its hydrogen in a hydrogen bond to water. Exchange kinetics of thin cellulose films were investigated by monitoring two different exchange processes with FT-IR spectroscopy. Specific information about the two exchanging hydroxyl/deuteroxyl groups was then extracted by deconvoluting the changing intensities of the recorded IR spectra. It was recognized that the exchange of the hydroxyl groups were well described by a two-region model, which was assessed to correspond to two fibrillary surfaces differentiated by their respective positions in the fibril aggregate. From the detailed deconvolution it was also possible to estimate the fraction of these two surfaces, which indicated that the average aggregate of cotton cellulose is built up by three to four fibrils. 2H MAS NMR spectroscopy was used to examine different states of water in cellulose samples, hydrated at different relative humidities of heavy water. The results showed that there exist two states of water adsorbed onto the cellulose, differentiated by distinct different mobilities. These two states of water are well separated and had negligible exchange on the time scale of the experiments. It was suggested that they are located at the internal and external surfaces of the fibril aggregates. By letting cellulose nanofibrils undergo an epoxidation reaction with a mono epoxide some indicative results regarding how to protect the cellulose material from the negative impact of water were presented. The protecting effect of the epoxidation were examined by mechanically testing and NMR spectroscopy. It was proposed that by changing the dominant interaction between the fibril aggregates from hydrophilic hydrogen bonds to hydrophobic π-interactions the sensitivity to moisture was much reduced. The results also indicated that the relative reduction in moisture sensitivity was largest for the samples with highest moisture content.QC 20161229
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Mikita, Natalie. "DEVELOP SPECTROSCOPIC APPROACHES TO STUDY NON-PROTEOSOMAL ATP-DEPENDENT PROTEOLYSIS." Case Western Reserve University School of Graduate Studies / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=case1401814273.
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Jin, Yining. "Water dynamics at the MHCI-peptide binding interface studied by Hydrogen-deuterium exchange and structural studies of Apo A-I mimetic peptide-lipid binding." University of Toledo Health Science Campus / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=mco1404706445.
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Sawyer, Holly Ann. "Investigation of the effect of intra-molecular interactions on the gas-phase conformation of peptides as probed by ion mobility-mass spectrometry, gas-phase hydrogen/deuterium exchange, and molecular mechanics." Texas A&M University, 2004. http://hdl.handle.net/1969.1/3093.
Повний текст джерелаАнотація:
Ion mobility-mass spectrometry (IM-MS), gas-phase hydrogen/deuterium (H/D) exchange ion molecule reactions and molecular modeling provide complimentary information and are used here for the characterization of peptide ion structure, including fine structure detail (i.e., cation-π interactions, β-turns, and charge solvation interactions). IM-MS experiments performed on tyrosine containing tripeptides show that the collision cross-sections of sodiated, potassiated and doubly sodiated species of gly-gly-tyr are smaller than that of the protonated species, while the cesiated and doubly cesiated species are larger. Conversely, all of the alkali-adducted species of try-gly-gly have collision cross-sections that are larger than that of the protonated species. The protonated and alkali metal ion adducted (Na+, K+ and Cs+) species of bradykinin and bradykinin fragments 1-5, 1-6, 1-7, 1-8, 2-7, 5-9 and 2-9 were also studied using IM-MS and the alkali metal ion adducts of these species were found to have cross-sections very close to those of the protonated species. Additionally, multiple peak features observed in the ATDs of protonated bradykinin fragments 1-5, 1-6 and 1-7 are conserved upon alkali metal ion adduction. It was observed from gas-phase H/D ion molecule reactions that alkali adducted species exchange slower and to a lesser extent than protonated species in the tyrosine- and arginine-containing peptides. Experimental and computational results are discussed in terms of peptide ion structure, specifically the intra-molecular interactions present how those interactions change upon alkali salt adduction, as well as with the sequence of the peptide.
Additionally, IM-MS data suggests the presence of a compact conformation of bradykinin fragment 1-5 (RPPGF) when starting from organic solvent conditions. As water is added stepwise to methanolic solutions, a more extended conformation is populated. When the starting solution is composed of ≈90% water, two distinct mobility profiles are observed as well as a shoulder, indicating the presence of three gas-phase conformations for RPPGF. Gas-phase H/D exchange of [M+H]+ ions prepared from aqueous solvents show a bi-exponential decay, whereas samples prepared from organic solvents show a single exponential decay. The effect of solvent on gas-phase peptide ion structure, i.e., solution-phase memory effects, is discussed and gas-phase structures are compared to know solution-phase structures.
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Rashid, Shaan. "Dual-spray Synthesis and Reactions." Thesis, Université d'Ottawa / University of Ottawa, 2017. http://hdl.handle.net/10393/35726.
Повний текст джерелаАнотація:
By using two electrospray emitters containing different solutions (“dual-spray”) we have recently conducted in-source hydrogen/deuterium exchange (HDX) reactions and synthesized organometallic species. For dual-spray HDX reactions, peptide and protein solutions were electrosprayed through one emitter and the deuterating agent D2O through the secondary electrospray emitter. Clear shifts in isotope distributions indicated hydrogen-deuterium exchange occurring within the ion source. By ion mobility, simultaneous deuterium exchange for two isobaric species, the oxytocin monomer and dimer, was observed. Lysozyme has a linear relation between the charge state and the average number of exchanges, indicating that lysozyme becomes increasingly unfolded as the charge state increases. Based on deuterium uptake data and the lack of a temperature dependence, the dual-spray HDX reaction is thought to occur mostly in the gas phase. Tris(2,2’-bipyridine)ruthenium(II) and similar complexes containing the 1,10-phenanthroline ligand were formed by spraying a ligand solution and the ruthenium trichloride solution through two independent ESI emitters. This was confirmed by comparing ion mobility drift time, mass spectra, and CID fragmentation with the reference standard compounds. Tris(2,2’-bipyridine)iron(II), tris(1,10-phenantroline)iron(II) and mixed ligand complexes of iron(II) formed by dual-spray showed two additional hydrogens bonded to the complex. By CID, these unique gas phase complexes showed similar initial ligand loss to the reference standards however the secondary ligand loss showed dissimilar dissociation channels and energetics. Using DFT calculations, geometry optimizations for the [Fe(phen)3 + 2H]2+ complex and its fragment ions were done. After the initial ligand loss, the additional hydrogens are believed to transfer to the central iron atom. The relative energy of the dissociation channels showed good agreement with experimental breakdown curves.
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Marini, Joseph Thomas. "Development and implementation of a FT-ICR mass spectrometer for the investigation of ion conformations of peptide sequence isomers containing basic amino acid residues by gas-phase hydrogen/deuterium exchange." Diss., Texas A&M University, 2003. http://hdl.handle.net/1969.1/115.
Повний текст джерелаАнотація:
The gas-phase hydrogen/deuterium (H/D) exchange of protonated di- and tripeptides containing a basic amino acid residue has been studied with a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Bimolecular reactions are monitored as a function of time providing exchange efficiencies and temporal distributions for the peptide ions. Results from these experiments indicated that position of the basic residue within the peptide (i.e. N-terminal, internal, or C-terminal) influences gas-phase H/D exchange, suggesting unique peptide ion conformations. The FT-ICR mass spectrometer employed for these gas-phase H/D exchange studies was modified from its original design. Instrument modifications include development of an internal matrix assisted laser desorption ionization (MALDI) source for peptide protonation. In addition, a two-section cell was utilized, allowing control of ion motion and factors affecting gas-phase ion molecule reactions. Systems investigated in these gas-phase H/D exchange studies are peptides containing the same amino acid residues but different sequences. These sequence isomers display dissimilar reaction efficiencies and temporal distributions for deuterium incorporation depending on the primary structure of the peptide ion. Specifically, [M+H]+ peptide ions containing a N-terminal basic residue demonstrate unique H/D exchange behavior when compared to their internal and C-terminal counterparts. These differences are attributed to dissimilar intramolecular bridging interactions involved with inductive stabilization of the charge site. Gas-phase H/D exchange of peptide sequence isomers was also probed with various deuterium reagents. Findings suggest that different reagents also influence H/D exchange reaction rate efficiencies and temporal distributions. These dissimilarities are ascribed to relative gas-phase basicity and proposed mechanistic exchange differences for the deuterium reagents.
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Topbas, Celalettin. "Apolipoprotein A-I Self-Association and the Formation of High Density Lipoprotein." Cleveland State University / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=csu1440347923.
Повний текст джерела
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Hagman, Charlotte. "Method Development in Quantitative and Structural Proteomics using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry." Doctoral thesis, Uppsala University, Department of Engineering Sciences, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-4761.
Повний текст джерелаАнотація:
In this thesis, methods for studying different aspects of proteomics were developed with Fourier Transform Ion Cyclotron Resonance, (FTICR), mass spectrometry. The FTICR technique provides ultra-high mass resolving power, mass accuracy at sub ppm level and sensitivity in the attomole region.
Methods for quantifying biomarkers in body fluids such as cerebrospinal fluid, (CSF), and plasma were developed. Two sets of global markers with different properties were used for quantitative analysis; S-Methyl Thioacetimidate, (SMTA), and S-Methyl Thiopropionimidate, (SMTP), and [H4]- and [D4]-1-Nicotinoyloxy succinimide ester. Reduced ion suppression and higher sensitivity was obtained by coupling a High Performance Liquid Chromatography, (HPLC), system to the FTICR mass spectrometer.
In body fluids, proteins and peptides are present in a broad dynamic concentration range. Therefore, depleting abundant proteins prior to analysis results in decreased ion suppression and increased sensitivity. Two commercial depletion kits were evaluated with the SMTA- and SMTP-markers.
For both types of global markers, the experimental error for quantitative analysis of abundant proteins was less than 30%. This provides a lower limit for the protein up- and down regulations in complex solutions that can be monitored with HPLC-FTICR mass spectrometry.
Together with the identity and quantity of selected proteins the structure, dynamics and interactions with other molecules are of great importance. The later can be elucidated with Hydrogen/Deuterium Exchange, (HDX), mass spectrometry. Structural information at high resolution can be obtained with Collision-Induced Dissociation, (CID), HDX mass spectrometry. In this thesis, exchange rates of amide hydrogens in peptides were in excellent agreement with NMR results.
In some cases, the CID-fragments have different gas-phase exchange properties and as a consequence the solution phase exchange process can not be monitored. By applying Electron Capture Dissociation, (ECD), at ultra-high vacuum, the exchange process at a specific residue could be monitored.
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Terral, Guillaume. "Apports de l'échange hydrogène/deutérium couplé à la spectrométrie de masse en protéomique structurale pour la caractérisation de complexes multi-protéiques." Thesis, Strasbourg, 2016. http://www.theses.fr/2016STRAF019/document.
Повний текст джерелаАнотація:
Ce travail de thèse porte sur développement de méthodes en spectrométrie de masse structurale pour l’analyse de protéines recombinantes et de leurs complexes associés. L’objectif central s’est porté sur des développements méthodologiques en échange hydrogène/deutérium couplé à la spectrométrie de masse (HDX-MS). Les techniques biophysiques de caractérisation structurale à haute résolution comme la cristallographie ou la RMN se heurtent régulièrement à des problèmes de productions de cristaux, de taille de complexes analysables ou encore de quantité de matériel nécessaire importante. Le développement de méthodes spécifiques HDX-MS a permis de réaliser une caractérisation structurale de systèmes protéiques variés, et réfractaires aux approches haute résolution. La combinaison de cette approche à différents outils de MS structurale est aussi illustrée, et montre tout son intérêt pour l’obtention d’informations à résolution augmentéeThis thesis work focuses on development of structural mass spectrometry methods for the analysis of recombinant proteins and their associated complex. The central objective has focused on the development of hydrogen/deuterium exchange coupled to mass spectrometry approaches (HDX-MS). The high resolution biophysical techniques for structural characterization such as crystallography or NMR regularly face problems of crystal productions, size analyzable complex or quantity of material required. The development of specific HDX-MS methods allowed the characterization of various, and refractory protein systems to high resolution approaches. The combination of this approach with complementary structural MS tools is also illustrated, and shows its interest to obtain increased resolution information
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Hofmann, Lukas. "Structural Endeavors in the Retinoid (Visual) Cycle." Case Western Reserve University School of Graduate Studies / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=case1497045464455384.
Повний текст джерела
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Javed, Waqas. "Etude des états conformationnels d'un transporteur ABC bactérien de drogues multiples, BmrA Functionality of membrane proteins overexpressed and purified from E. coli is highly dependent upon the strain Assemblies of lauryl maltose neopentyl glycol (LMNG) and LMNG-solubilized membrane proteins." Thesis, Université Grenoble Alpes, 2020. https://thares.univ-grenoble-alpes.fr/2020GRALV046.pdf.
Повний текст джерелаАнотація:
La résistance aux antibiotiques est une réalité à laquelle nous devons faire face. La résistance bactérienne aux antibiotiques peut être conférée par plusieurs mécanismes, dont la surexpression de pompes à efflux, certaines appartenant à la superfamille des transporteurs ABC (“ATP-binding cassette”). Les transporteurs ABC sont des protéines omniprésentes qui utilisent l'hydrolyse de l'ATP pour pomper une large gamme de substrats. Ils sont également responsables du développement des phénotypes de résistance à de multiple drogues dans les cellules cancéreuses et les microorganismes pathogènes.L'exportateur bactérien ABC BmrA (“Bacillus multidrug resistance ATP”), est homologue à ABCB1, un transporteur humain impliqué dans les phénotypes de résistance dans les cellules cancéreuses. Avec une connaissance approfondie de sa surexpression et de sa purification, BmrA est un archétype utile pour obtenir des informations sur le fonctionnement des transporteurs ABC de multiples drogues. Notre objectif est de déchiffrer les changements conformationnels associés au transport des médicaments.Nous avons montré que BmrA existe dans au moins deux conformations différentes, dans des micelles de détergent ou reconstitué dans des nanodisques. En l'absence de ligand (forme apo), différentes partie de BmrA fixe rapidement du deutérium comme le montre l'échange hydrogène deutérium couplé à la spectrométrie de masse (HDX-MS). La forme piégée par l'ADP induite par le vanadate montre une grande protection globale contre l'incorporation de deutérium. De plus, il a été observé que BmrA dans les nanodisques présente un profil de deutération différent en présence de médicament, indicatif d'une nouvelle conformation intermédiaire. De plus, en utilisant deux mutants affectés dans différentes étapes du cycle catalytique, il a été montré comment BmrA change de conformations au cours du cycle d'export des médicaments. Les résultats obtenus à partir de la diffusion de neutrons aux petits angles (SANS), brossent un tableau similaire et renforcent les résultats obtenus sur le cycle catalytique de BmrA.Ces résultats conduisent à une meilleure compréhension des changements de conformation de BmrA qui s’opèrent pour permettre le phénotype de résistance aux médicamentsAntibiotic resistance is not the story of the future but a reality today. Bacterial resistance to antibiotics can be conferred by several mechanisms, including the overexpression of dedicated efflux pumps, some of them belonging to the ABC (“ATP-binding cassette”) transporters superfamily. ABC transporters are ubiquitous proteins that use ATP hydrolysis to pump a wide range of substrates. They are also responsible for the development of MDR (“MultiDrug Resistance”) phenotypes in cancer cells and pathogenic microorganisms.The bacterial ABC exporter BmrA (“Bacillus multidrug resistance ATP”), is structurally and functionally close to ABCB1, a human transporter involved in MDR phenotypes in cancer cells. Together with extensive knowledge in its overexpression and purification, BmrA is a useful archetypical transporter to gain information on the functioning of multidrug ABC transporters. Our goal is to decipher the conformational changes associated with drug transport.We showed that BmrA exists in at least two different conformations, in detergent micelles or when reconstituted in nanodiscs. In the absence of ligand (apo form), BmrA gets quickly exchanged with deuterium as shown by Hydrogen Deuterium Exchange Coupled to Mass Spectrometry (HDX-MS). The vanadate-induced ADP trapped form shows a large overall protection against deuterium incorporation. Moreover, it was observed that BmrA in nanodiscs shows a different deuteration profile in the presence of drug, indicative of a new intermediate conformation. In addition, using two different catalytic mutants of BmrA, that are trapped in two opposite conformations of the catalytic cycle, it was shown how BmrA changes conformations during the drug export cycle. The results obtained from Small Angle Neutron Scattering (SANS), on WT BmrA and the mutants, paint a similar picture and strengthen the results obtained on the catalytic cycle of BmrA.These results could potentially lead to a better understanding of the structural basis of MDR
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Villoria, del Álamo Beatriz. "Síntesis de catalizadores sólidos orgánicos e híbridos orgánicos-inorgánicos y su aplicación." Doctoral thesis, Universitat Politècnica de València, 2021. http://hdl.handle.net/10251/163789.
Повний текст джерелаАнотація:
[ES] En este trabajo de tesis doctoral, la investigación se ha centrado en el desarrollo de diferentes procesos catalíticos heterogéneos empleando materiales híbridos orgánico-inorgánicos porosos (MOFs y sílices funcionalizadas) y materiales orgánicos aromáti-cos (PAFs), que se han estudiado en diversas reacciones orgánicas. Tras la preparación de los MOFs en estudio, se han caracterizado sus propiedades estructurales y se han determinado sus centros activos en los clústeres metálicos (circonio, hafnio o cerio). La reactividad de estos MOFs y de los materiales híbridos sílice-aminas se ha estudia-do teniendo en cuenta sus centros catalíticos; estas reacciones se han optimizado lle-vando a cabo un estudio de los mecanismos de reacción. Finalmente, se han preparado sólidos homoquirales de tipo PAF que presentan el sistema binaftilo, cuya reactividad también ha sido probada. Más específicamente, en el capítulo 3 se ha estudiado la esterificación de amidas, que permite convertirlas en ésteres, grupos funcionales más versátiles. Esta transfor-mación se ha abordado desde la catálisis heterogénea via MOFs basados en circonio, hafnio y cerio de las series MOF-808, UiO-66 y MOF-801. El catalizador más eficien-te para la esterificación de amidas ha sido el MOF-808-Zr. Mediante análisis TGA y la adsorción de una molécula sonda básica (CO) estudiada utilizando espectroscopia FT-IR, se han determinado los centros ácidos de Lewis y Brönsted presentes en ellos. De los MOFs preparados en este trabajo, el MOF 808-Zr posee una menor conectividad de los clústeres metálicos y un mayor tamaño de poro mayor que el UiO-66 y el MOF-801; además, tiene el balance adecuado de centros ácidos y básicos de Brönsted y Lewis para activar los sustratos de la reacción. El alcance de la alcoholisis con n-butanol se ha extendido a un gran número de sustratos (amidas primarias, secundarias y terciarias; aromáticas y alifáticas). La reacción también se ha estudiado en condicio-nes no solvolíticas con alcoholes más complejos. El catalizador es estable durante la reacción y puede ser reutilizado fácilmente. El mecanismo de reacción en la esterifica-ción de benzamida con n butanol catalizada por MOF-808-Zr se ha investigado me-diante el análisis cinético empleando el modelo de LHHW y el estudio in situ de las interacciones moleculares por FT-IR. En el capítulo 4, se ha investigado la deuteración por intercambio isotópico deute-rio/hidrógeno catalizada por aminas soportadas en sílice comerciales empleando D2O como fuente de deuterio. Este procedimiento es aplicable a una gran gama de sustra-tos, como compuestos carbonílicos, sales de organofosfonio, nitrocompuestos e, inclu-so, hormonas esteroideas. La estabilidad del catalizador, SiO2-(CH2)3-NH2, se mantie-ne hasta en 10 usos de reacción sin pérdidas significativas de la actividad. Por último, en el capítulo 5, se afronta la síntesis y aplicación de PAFs homoquira-les donde se ha integrado el esqueleto del BINOL (1,1′-binaftil-2,2′-diol) y del BIN-BAM (1,1' binaftil-2,2'-disulfonimida) generando tres nuevos PAFs activos en catáli-sis asimétrica: PAF-3,3'-(S)-BINOL, PAF-6,6'-(R)-BINOL y PAF 3,3'-(S)-BINBAM. En concreto, el PAF-6,6'-(R)-BINOL ha demostrado su actividad catalítica en la reacción de alquilación de aldehídos aromáticos con dietil-zinc y el catalizador PAF-3,3'-(S)-BINBAM es activo en la reacción aldólica de Mukaiyama y la reducción del doble enlace de compuestos carbonílicos a,b-insaturados.[CA] En aquesta tesi doctoral, la investigació s'ha centrat en el desenvolupament de dife-rents processos catalítics heterogenis emprant materials híbrids orgànic-inorgànics porosos (MOFs i sílices funcionalitzades) i materials orgànics aromàtics (PAFs), que s'han estudiat en diverses reaccions orgàniques. Després de la preparació dels MOFs en estudi, s'han caracteritzat les seues propietats estructurals i s'han determinat els seus centres actius en els clústers metàl·lics (zirconi, hafni o ceri). La reactivitat d'aquests MOFs i dels materials híbrids sílice-amines s'ha estudiat tenint en compte els seus cen-tres catalítics; aquestes reaccions s'han optimitzat duent a termini un estudi dels meca-nismes de reacció. Finalment, s'han preparat sòlids homoquirals de tipus PAF que presenten el sistema binaftilo, la reactivitat del qual també ha sigut provada. Més específicament, en el capítol 3 s'ha estudiat l'esterificació d' amides, que per-met convertir-les en èsters, grups funcionals més versàtils. Aquesta transformació s'ha abordat des de la catàlisi heterogènia via *MOFs basats en zirconi, hafni i ceri de les sèries MOF-808, UiO-66 i MOF-801. El catalitzador més eficient per a l'esterificació d'amides ha sigut el MOF-808-Zr. Mitjançant anàlisi TGA i l'adsorció d'una molècula sonda bàsica (CO) estudiada utilitzant espectroscopia FT-IR, s'han determinat els cen-tres àcids de Lewis i Brönsted presents en ells. Dels MOFs preparats en aquest treball, el MOF 808-Zr posseeix una menor connectivitat dels clústers metàl·lics i una major grandària de porus que el UiO-66 i el MOF-801; a més, té el balanç adequat de centres àcids i bàsics de Brönsted i Lewis per a activar els substrats de la reacció. L'abast de l'alcoholisi amb n-butanol s'ha estés a un gran nombre de substrats (amides primàries, secundàries i terciàries; aromàtiques i alifàtiques). La reacció també s'ha estudiat en condicions no solvolítiques amb alcohols més complexos. El catalitzador és estable durant la reacció i pot ser reutilitzat fàcilment. El mecanisme de reacció en l'esterifica-ció de benzamida amb n-butanol catalitzada per MOF-808-Zr s'ha investigat mitja-nçant l'anàlisi cinètica emprant el model de LHHW i l'estudi in situ de les interaccions moleculars per FT-IR. En el capítol 4, s'ha investigat la deuteració per intercanvi isotòpic deuteri/hidrògen catalitzada per amines suportades en sílices comercials emprant D2O com a font de deuteri. Aquest procediment és aplicable a una gran gamma de substrats, com a com-postos carbonílics, sals d'organofosfoni, nitrocompostos i, inclosa, hormones esteroi-dals. L'estabilitat del catalitzador, SiO2-(CH2)3-NH2, es manté fins a 10 usos de reac-ció sense pèrdues significatives de l'activitat. Finalment, en el capítol 5, s'afronta la síntesi i aplicació de PAFs homoquirals on s'ha integrat l'esquelet del BINOL (1,1′-binaftil-2,2′-diol) i del BINBAM (1,1'-binaftil-2,2'-disulfonimida) generant tres nous PAFs actius en catàlisi asimètrica: PAF-3,3'-(S)-BINOL, PAF-6,6'-(R)-BINOL i PAF 3,3'-(S)-BINBAM. En concret, el PAF-6,6'-(R)-BINOL ha demostrat la seua activitat catalítica en la reacció d'alquilació d'aldehids aromàtics amb dietil-zinc i el catalitzador PAF-3,3'-(S)-BINBAM és actiu en la reacció aldólica de Mukaiyama i la reducció del doble enllaç de compostos carbonílics a,b-insaturats.
[EN] In this Doctoral Thesis, the research has been focused on the development of different heterogeneous catalytic processes using hybrid porous organic-inorganic materials (MOFs and functionalized silicas) and organic aromatic materials (PAFs), which have been studied in various organic reactions. After the preparation of the MOFs under study, their structural properties have been characterised and their active centres in the metal clusters (zirconium, hafnium or cerium) have been determined. The reactivity of these MOFs and the hybrid silica-mine materials has been studied considering their catalytic centres; these reactions have been optimised by carrying out a study of the reaction mechanisms. Finally, homochiral PAF-type solids have been prepared with the binafil system, whose reactivity has also been tested. More specifically, the esterification of amides has been studied in Chapter 3. This reaction allows to convert the amides into esters, which are more versatile functional groups. This transformation has been approached from the heterogeneous catalysis via MOFs based on zirconium, hafnium and cerium of the MOF-808, UiO-66 and MOF-801 series. The most efficient catalyst for amide esterification has been MOF-808-Zr. Using TGA analysis and the adsorption of a basic probe molecule (CO) studied using FT-IR spectroscopy, the acid centres of Lewis and Brönsted present in them have been determined. Among the MOFs prepared in this work, MOF 808-Zr has a lower metal cluster connectivity and a larger pore size than UiO-66 and MOF-801; it also has the appropriate balance of acid and basic Brönsted and Lewis centres to activate the reaction substrates. The scope of n-butanol alcoholysis has been extended to a large number of substrates (primary, secondary and tertiary amides; aromatic and aliphatic). The reaction has also been studied in non-solvolitic conditions with more complex alco-hols. The catalyst is stable during the reaction and can be easily reused. The reaction mechanism in the esterification of benzamide with n-butanol catalysed by MOF-808-Zr has been investigated through kinetic analysis using the LHHW model and the in situ study of molecular interactions by FT-IR. In Chapter 4, the deuteration by isotopic deuterium/hydrogen exchange catalysed by commercial silica-supported amines using D2O as a source of deuterium has been investigated. This procedure is applicable to a wide range of substrates, such as carbonylic compounds, organophosphonium salts, nitro compounds and, even, steroid hormones. The stability of the catalyst, SiO2-(CH2)3-NH2, is maintained for up to 10 reaction uses without significant loss of activity. Finally, in Chapter 5, the synthesis and application of homochiral PAFs, in which the structure of BINOL (1,1′-binaftil-2,2′-diol) and BIN-BAM (1,1' binaftil-2,2'-disulfonimide) has been integrated, is discussed. Three new PAFs active in asymmetric catalysis has been generated: PAF-3,3'-(S)-BINOL, PAF-6,6'-(R)-BINOL and PAF 3,3'-(S)-BINBAM. In particular, PAF-6,6'-(R)-BINOL has demonstrated its catalytic activity in the alkylation reaction of aromatic aldehydes with diethyl zinc and the catalyst PAF-3,3'-(S)-BINBAM is active in the Mukaiyama aldolic reaction and the reduction of the double bond of carbonylic a,b-unsaturated compounds.
Villoria Del Álamo, B. (2021). Síntesis de catalizadores sólidos orgánicos e híbridos orgánicos-inorgánicos y su aplicación [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/163789
TESIS
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Serpa, Jason John. "Structure of prion β-oligomers as determined by structural proteomics". Thesis, 2017. https://dspace.library.uvic.ca//handle/1828/8548.
Повний текст джерелаАнотація:
The conversion of the native monomeric cellular prion protein (PrPC) into an aggregated pathological β-oligomeric (PrPβ) and an infectious form (PrPSc) is the central element in the development of prion diseases. The structure of the aggregates and the molecular mechanisms of the conformational change involved in this conversion are still unknown.
My research hypothesis was that a specific structural rearrangement of normal PrPC monomers leads to the formation of new inter-subunit interaction interfaces in the prion aggregates, leading to aggregation. My approach was to use constraints obtained by structural proteomic methods to create a 3D model of urea-acid induced recombinant prion oligomers (PrPβ). My hypothesis was that this model would explain the mechanism of the conformational change involved in the conversion, the early formation of the β-structure nucleation site, and would describe the mode of assembly of the subunits within the oligomer.
I applied a combination of limited proteolysis, surface modification, chemical crosslinking and hydrogen/deuterium exchange (HDX) with mass spectrometry for the differential characterization of the native and the urea-acid converted prion β-oligomer structures to get an insight into the mechanism of the conversion and aggregation. Using HDX, I detected a region of the protein in which backbone amides become more protected from exchange in PrPβ compared to PrPC. In order to obtain the inter-residue distance constraints to guide the assembly of the oligomer model, I then applied zero-length and short-range crosslinking to an equimolar mixture of 14N/15N-metabolically labeled β-oligomer thereby enabling the classification of the crosslinks as either intra-protein or inter-protein. Working with the Dokholyan group at the University of North Carolina at Chapel Hill, I was able to assemble a structure of the β-oligomer based on the combination of constraints obtained from all methods. By comparing the structures before and after the conversion, I was able to deduce the conformational change, that occurs during the conversion as the rearrangement and disassembly of the beta sheet 1– helix 1 – beta sheet 2 (β1-H1-β2) region from the helix 2 – helix 3 (H2-H3) core, forming new β-sheet nucleation site and resulting in the exposure of hydrophobic residues patches leading to formation of inter-protein contacts within aggregates.Graduate
2018-06-14
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Yang, Sheng-Wei, and 楊勝惟. "Hydrogen / Deuterium Exchange Study of Cdc42 Activation Mechanism." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/37802924059104156942.
Повний текст джерелаАнотація:
碩士東海大學
化學系
101
Hydrogen/deuterium exchange (HDX) coupled with electrospray ionization mass spectrometry (ESI-MS) has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. Cdc42 (cell division cycle protein) is a GTPase which is a member of the Rho family. Like other members of the Rho family, Cdc42 possesses an inactive GDP-bound state and an active GTP-bound state. GTP-bound Cdc42 can bind to and activate downstream effector proteins that are responsible for mediating a diversity of cellular functions, including actin cytoskeletal remodeling, cell polarity, intracellular trafficking, growth, and transcription. In this study, we analyzed the conformational change of Cdc42 upon binding to GTP by hydrogen/deuterium exchange (HDX) coupled with ESI-MS. We also compared HDX of the inactive Cdc42 with the GTP bound form Cdc42. When GTP bound on the P-Loop of Cdc42 and formed hydrogen bonds, the active form was shown to bind GTP in the active site, with significant change in P-Loop. And the binding site around was also affected, it showed a significantly reduced hydrogen-deuterium exchange, including Switch I and Switch II (sequences 27-40 and 54-65 sequence). Besides P-Loop、Switch I and Switch II, there are other sequences showing significantly decreased hydrogen -deuterium exchange, such as residue 54-65, 90-107, 125-145, 156-168, and 171-191. The result of our experiments indicat that GTP binding to P-Loop leads to a conformational change of Cdc42. This conformational change further stabilizes the structure to activate Cdc42. The comparison between the results of the hydrogen- deuterium exchange experiments and surface electrostatic potential showed that GTP、Mg2+ binding decreased HDX in P-Loop and the charge is shifted from negative to positive. On the other hand, GTP binding site changes the charge from negative charge to neutral. Moreover, when the result of hydrogen-deuterium exchange compared with B-factor, P-Loop is 40-50 Å2 that correspond to low hydrogen/deuterium exchange site. In contrast, outside of the binding region was about 60 Å2 and less stable.
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(10900263), Rishabh Tukra. "MECHANISMS AND APPLICATIONS OF SOLID-STATE HYDROGEN DEUTERIUM EXCHANGE." Thesis, 2021.
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To prolong their long-term stability, protein molecules are commonly dispensed as lyophilized powders to be reconstituted before use. Evaluating the stability of these biomolecules in the solid state is routinely done by using various analytical techniques such as glass transition temperature, residual moisture content and other spectroscopic techniques. However, these techniques often show poor correlation with long term storage stability studies. As a result, time intensive long term storage stability studies are still the golden standard for evaluating protein formulations in the solid state. Over the past few years, our lab has developed solid-state hydrogen deuterium exchange- mass spectrometry (ssHDX-MS) as an analytical tool that probes the backbone of a protein molecule in the solid state. ssHDX-MS gives a snapshot of protein-matrix interactions in the solid state and has a quick turnaround of a few weeks as opposed to a few months for accelerated stability testing. Additionally, various studies in the past have demonstrated that ssHDX-MS can be used for a wide range of biomolecules and shows strong correlation to long term stability studies routinely employed.
The main aim of this dissertation is to provide an initial understanding of the mechanism behind ssHDX-MS in structured protein formulations. Specifically, this dissertation is an attempt at studying the effects of various experimental variables on the ssHDX-MS of myoglobin formulations as well as demonstrating the utility of this analytical technique. Firstly, the effects of varying temperature and relative humidity on ssHDX-MS of myoglobin formulations is studied with the help of statistical modeling. Secondly, the effects of pressure on ssHDX-MS of myoglobin formulations are evaluated at an intact and peptide digest levels. Finally, ssHDX-MS is used as a characterization tool to evaluate the effects of two different lyophilization methods on the structure and stability of myoglobin formulations. The results of studies described in this dissertation show ssHDX-MS to be sensitive to changes in experimental parameters, namely temperature, relative humidity, pressure, and excipients. Additionally, ssHDX-MS results were in good agreement with other routinely employed analytical and stability testing techniques when used to compare the effects of two lyophilization methods on myoglobin formulations.
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(9095855), Rajashekar Kammari. "SOLID-STATE HYDROGEN-DEUTERIUM EXCHANGE MASS SPECTROMETRY OF LYOPHILIZED PEPTIDES." Thesis, 2020.
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Proteins are susceptible to physical and chemical degradation in solution, which can lead to the loss of therapeutic activity and increase the potential for immunogenic responses when administered. Many degradation reactions are mediated by water, and therefore the proteins are often formulated as solids in which degradation rates are slowed significantly. Lyophilization is the most common method for producing solid protein formulations, which removes the water by sublimation and desorption under vacuum from the frozen protein solutions. Lyophilization requires excipients to protect the protein from the inherent stresses involved in the process. Degradation can still occur during lyophilization and storage, and needs to be characterized in order to develop a successful formulation with desired storage stability. The analytical techniques to characterize solid-state proteins are limited, however, and many do not provide site-specific information and lack the ability to predict stability beforehand.
Recently, solid-state hydrogen-deuterium exchange mass spectrometry (ssHDX-MS) has been developed to characterize proteins in solid powders with peptide level resolution. The technique was found to be sensitive to formulation and process changes. The ssHDX-MS metrics are highly correlated to the long-term storage stability, suggesting that the method can serve as a formulation screening tool. This dissertation aims to evaluate the factors affecting ssHDX kinetics and to develop a mechanistic understanding of the exchange process in solid samples, which in turn will support the solid-state protein development and enable it to be conducted in a more a cost and time-effective way. First, the contribution of peptide-matrix interactions to deuterium incorporation kinetics in the absence of higher-order structure was assessed using lyophilized poly-D, L-alanine peptides. Deuterium incorporation depended on excipient type and D2O(g) activity in the solid samples. A reversible pseudo-first-order kinetic model was proposed and validated using the experimental data. Second, the reversibility of the hydrogen-deuterium exchange reaction in the solid-state was evaluated to support the ssHDX mechanistic model further. The reaction was found to be reversible irrespective of initial conditions and independent of the excipient type. Pre-hydration of the peptide samples prior to deuterium labeling did not affect deuterium incorporation in amorphous samples compared to the controls not subjected to pre-hydration. Third, the contribution of peptide secondary structure to deuterium uptake kinetics was quantified using structured PDLA analogs. The deuterium incorporation in structured peptides was less than that of the PDLA peptides suggesting that both peptide structure and peptide-matrix interactions contribute to ssHDX-MS. Finally, a quantitative data analysis method was presented that allows the interpretation of ssHDX-MS data of a protein relative to controls. Altogether, the findings present a comprehensive mechanistic understanding of the ssHDX-MS of proteins that is relevant to the industry.
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Chen, Maolian. "Characterization of A [Beta] aggregates using hydrogen/deuterium exchange mass spectrometry." 2006. http://etd.utk.edu/2006/ChenMaolian.pdf.
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Yeh, Ting-Kai, and 葉庭愷. "Dynamics of the H+-translocating Pyrophosphatase Revealed by Hydrogen-Deuterium Exchange." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/95767489815625538408.
Повний текст джерелаАнотація:
碩士國立清華大學
生物資訊與結構生物研究所
103
H+-translocating pyrophosphatases (H+-PPase; EC 3.6.1.1) exists in various endomembranes of plants, bacteria, and some prokaryotes. It transports H+ into lumens at the cost of hydrolyzing the ''product of anabolic reactions'', inorganic pyrophosphate (PPi). Although the crystal structure of mung bean H+-PPase has been solved recently, the motions of H+-PPase is still unclear. Here, we applied hydrogen/deuterium exchange (HDX) coupled to mass spectrometry (MS) to monitor the dynamic of H+-PPase between the resting (apo form) and initiated states (bound with substrate analogue). When proteins were placed in a D2O solution, the backbone hydrogens, which exchange with deuterium, would be identified by MS. Accordingly, we discerned through the deuterium uptake to insight into the structural dynamic and conformational changes. HDX on the VrH+-PPase shows a significant protection againat exchange upon binding with substrate analogue, especially in the highly conserved PPi binging motif and reveals a rigid structure to form a narrow transport pathway. Additionally, Loop1, Loop5and Loop11, in the cytosolic site, exhibit a solvent inaccessible region and form a lid to cover the substrate binding pocket; N terminal and vacuolar lumen Loop12 may prevent proton back flow into the cytosol. These results highlight the more detail understanding the mechanism of VrH+-PPase and presumably for biological and agricultural interests.
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Hoerner, Joshua K. "Characterizing protein conformation and dynamics using hydrogen -deuterium exchange electrospray ionization mass spectrometry." 2007. https://scholarworks.umass.edu/dissertations/AAI3254902.
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Monitoring hydrogen-deuterium exchange of proteins can yield a wealth of information about not only the native state, but also not native states of proteins. These non-native states have important biological roles including protein folding intermediates as well as ligand binding/delivery and protein-protein interactions. Structural and dynamic properties of a partially folded conformation (A-state) of ubiquitin are studied using amide hydrogen exchange in solution (HDX) and mass spectrometric detection. A clear distinction between the native state of the protein and the A-state can be made when HDX is carried out in a semicorrelated regime. Furthermore, combination of HDX and protein ion fragmentation in the gas phase by means of collision-induced dissociation (CAD)] is used to evaluate the conformational stability of various protein segments specifically in the molten globular state. Chain flexibility appears to be distributed very unevenly in this non-native conformation. This study also demonstrates the power of mass spectrometry as a tool in providing conformer-specific information about the structure and dynamics of both native and non-native protein states coexisting in solution under equilibrium. This dissertation has been broken down into several subsections. First, we evaluate electrospray ionization amide hydrogen exchange collision assisted dissociation mass spectrometry's (ESI HDX CAD MS) methodology to better understand the determinants of hydrogen scrambling in the gas phase, which can be used to probe non-native states of proteins. Secondly we examined the structure of a molten globule of ubiquitin using HDX CAD MS under mildly denaturing conditions and compare this with the proposed NMR and crystal structures of the A-state and native state, respectively. Lastly, we have conducted studies include ESI MS studies of non-covalent interactions between cellular retinoic acid binding protein II (CRABP II) and the ligand-binding domain (LBD) of retinoic acid receptor (RAR) as it pertains to delivery of retinoic acid (RA). The backbone dynamics of CRABP II will be investigated in order better understand the ligand binding and delivery properties of CRABP II and how they differ from CRABP I.
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Huang, Yi-Cyuan, та 黃翊銓. "Hydrogen/deuterium exchange study of VPPase dynamic conformational changes, and the expression of iPLA2γ". Thesis, 2018. http://ndltd.ncl.edu.tw/handle/4fx2uk.
Повний текст джерелаАнотація:
碩士東海大學
化學系
106
Amide hydrogen/deuterium exchange coupled with mass spectrometry (DXMS) had been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. The Vigna radiata H+-translocating pyrophosphatases (VPPase) transported H+ into lumens at the cost of hydrolyzing PPi, the product of anabolic reactions. VPPase transported H+ to keep acidic environments in the vacuolar lumen. It was necessary for physiological and metabolic reactions. VPPase could help plants to grow in drought and high salinity environment. Although the crystal structure of VPPase had been solved recently, the H+ translocation mechanism of VPPase was still unclear. Therefore, we applied DXMS to study VPPase conformational changes upon VPPase H+ transportion. When VPPase bound with substrate (PPi), the structure was tighter and more stable. Then the cover of cytoplasmic end opened to release the hydrolyzate. The vacuole end would be more close to prepare for proton transport. At last, proton would be transported into vacuole quickly, and the structure recovered.Calcium-independent phospholipase A2γ (iPLA2γ) played a key role in cardiolipin remodeling. The mechanism of iPLA2γ remodeling was still unknown. There was not any research about iPLA2γ structure. We used protein expression systems to express iPLA2γ from E. coli, yeast, and insect cell. E. coli system didn’t express iPLA2γ at all. Yeast system probably expressed two fragments of iPLA2γ (63, and 30 kD), which may be the degradation products of proteases. Perhaps 63 kD was N-Terminal and 30 kD C-Terminal iPLA2γ. The mRNA of iPLA2γ increased about 50 fold after baculovirus infection, but insect cell system didn’t express iPLA2γ protein.
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Xiao, Hui. "Probing protein small ligand binding dynamics by hydrogen/deuterium exchange and electrospray ionization mass spectrometry." 2005. https://scholarworks.umass.edu/dissertations/AAI3163719.
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Cellular Retinoic acid binding protein I (CRABP I), a member of intracellular lipid binding proteins (iLBPs), binds physiologically to a mostly hydrophobic ligand, all-trans Retinoic Acid (RA). The binding site is inside an internal cavity, inaccessible in a static apo-protein conformation. Binding of RA to CRABP I does not result in significant changes of the protein tertiary structure, suggesting significance of dynamics in the ligand recognition and binding processes. One of the proposed scenarios for the protein-ligand binding process invokes the notion of a flexible portal region adjacent to the binding site, while another model suggests that the requisite dynamic events are induced by dimerization of the apo-protein in solution. In this work RA binding to CRABP I is studied in dilute solutions (low micro-molar range), where no dimer and/or oligomer formation occurs. Modulation of backbone dynamics within various segments of the protein by its ligand is assessed using a combination of hydrogen exchange, electrospray ionization mass spectrometry and collision-induced dissociation of protein ions in the gas phase. Consistent with the portal model of ligand entry, several protein segments are flexible in the absence of the ligand. At the same time, the two segments containing arginine residues forming a salt bridge with RA form the least flexible region in the apo-form of the protein. Although the presence of RA in solution reduces flexibility of all protein segments, the largest effect is observed within four strands that form one of the two β-sheets enveloping a cavity, which houses the ligand-binding site. These results are consistent with a model in which ligand binding occurs through a partially unstructured state of the protein with unobstructed access to the ligand-binding site. This intermediate (whose core is formed by the two stable arginine-containing strands) corresponds to a relatively low-energy local minimum on the apo-protein energy surface and is frequently sampled under native conditions. Intermediate states were visualized under various mildly denaturing conditions and characterized by HDX/CAD ESI MS. The similar amide backbone protection pattern suggests that the intermediate states bear significant resemblance with the activated intermediate state.
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Chou, S. C., and 周興中. "Statistical thermodynamics analysis of isotope-exchange reaction of hydrogen and deuterium on the transition metal surface." Thesis, 1993. http://ndltd.ncl.edu.tw/handle/94838382341395764849.
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Blakney, Gregory Terrell. "Investigations of biological interactions by hydrogen deuterium exchange Fourier transform ion cyclotron mass spectrometry novel methods, automated analysis and data reduction /." Thesis, 2003. http://wwwlib.umi.com/cr/utexas/fullcit?p3110730.
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Chan, Pin Chuan, and 陳品全. "Structure dynamics of exit regions in proton channel of VrH+-PPase as explored by hydrogen-deuterium exchange." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/j3thfv.
Повний текст джерелаАнотація:
碩士國立清華大學
生物資訊與結構生物研究所
105
The Vigna radiata H+-translocating pyrophosphatases (VrH+-PPase; EC 3.6.1.1) exists in various endomembranes of plants, bacteria, archaea, and some prokaryotes. It transports H+ into lumens at the cost of hydrolyzing PPi, the product of anabolic reactions. Although the crystal structure of H+-PPase has been solved recently, the H+ translocation mechanism of H+-PPase is still unclear. Therefore, we applied hydrogen/deuterium exchange (HDX) coupled to mass spectrometry (MS) to investigate the dynamic of H+-PPase between the resting (apo form), initiated (bound with substrate analogue) and transient states (bound with Pi). When proteins replaced hydrogen in a D2O solution, the backbone hydrogens, which exchange with deuterium, would be identified by MS. Accordingly, we determined the structural dynamic and conformational changes via the deuterium uptake. In the highly conserved substrate binding and exit regions, HDX on H+-PPase showed a compact conformation against deuterium exchange upon binding with substrate analogue and product. In addition, the exit region of proton channel exhibited a rapid-changed deuteration in the short time in the presence of phosphate. These results revealed more details about the mechanism of proton translocating by H+-PPase during PPi hydrolyzing, which are useful for biological and agricultural applications.
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Bahrainwala, Tasneem M. "Use of hydrogen/deuterium exchange-mass spectrometry in the study of cell wall degrading enzymes and their inhibitors." 2005. http://purl.galileo.usg.edu/uga%5Fetd/bahrainwala%5Ftasneem%5Fm%5F200508%5Fphd.
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Thesis (Ph. D.)--University of Georgia, 2005.Directed by Ron Orlando. Includes articles submitted to Rapid communications in mass spectrometry and Analytical biochemistry. Includes bibliographical references.
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WU, TING-YUAN, and 吳庭遠. "Analysis of the interactions of Tafazzin and Beta-2 Glycoprotein I with lipid membrane by hydrogen/deuterium exchange mass spectrometry." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/uqu7b2.
Повний текст джерелаАнотація:
碩士東海大學
化學系
106
Hydrogen/deuterium exchange (HDX) coupled with mass spectrometry (ESI-MS) has been widely used to study the mechanisms of protein dynamics, domain structure, protein-ligand interactions and protein conformational changes. Beta 2 Glycoprotein I (β2GPI) is a membrane protein and it was discovered to be the major antigen for the antiphospholipid antibodies(aPL Abs) in the antiphospholipid syndrome. The β2GPI complex binds with antibody will interact with some receptors, such as annexin A2, TLR family, glycoprotein Ibα, LRP8 to induce inflammation and prothrombotic. Many studies have suggested that β2GPI is not recognized by aPL Abs in the blood circulation. When negatively charged protein surface become exposed, the domain V of β2GPI will bind to the surface and change conformation. Then the aPL Abs are able to recognize the epitope in domain I of β2GPI. Here, we prepared 1,2-dioleoyl-sn-glycero-3-phospho-L-serine(18:1DOPS) and cardiolipin (CL) vesicles to simulate anion surface membrane. The interactions of the β2GPI with the anion membrane vesicles were analyzed by hydrogen/deuterium exchange mass spectrometry (HDXMS). The exchange level of sequence 21-27 significantly increased after β2GPI interacted with DOPS for 10 min. This results indicated that the interaction between domain I and domain V decreased, which caused the sequence 21-27 protruding out of the circular shape of the protein structure. β2GPI still maintained the circular conformation while interacting with DOPS. The exchange levels of the highly accessible sequences 1-20, 53-77, 175-188, 259-268 and 294-306 slightly decreased due to the nonspecific adsorption between β2GPI and DOPS by electrostatic force and hydrogen bonds. After β2GPI interacted with CL 10 mins. The exchange amount of sequence 21-27 significantly increased. This result was same with DOPS, suggesting the perturbation of sequence 21-27 is a preliminary behavior caused by the phospholipid binding. After β2GPI interacted with CL for 30 min, the exchange levels in several sequences significantly increased, including 1-20, 21-27, 41-51, 70-86, 153-162, 191-198, 196-205, 273-279, 297-306 and 310-316.The increasing of 1-20, 21-27, 41-51, 297-306 and 310-316 indicated that domain I did not interact with domain V and these sequences have been exposed. The increasing deuteration levels in 70-86, 153-162, 191-198, 196-205 and 273-279 indicated β2GPI conformation changed from ring conformation to chain conformation, leading to the exposure of the inner region. Overall, β2GPI could not change the ring conformation while initial contact with lipid membrane, but sequence 21-27 will be exposed. β2GPI continued to drastically change its conformation from ring to chain conformation while staying on the lipid membrane surface.
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LO, YI-TING, and 羅伊婷. "Analysis of the Transfer RNA Binding Effects on Cytochrome c and the Interactions of Tafazzin with Lipid Membrane by Hydrogen/Deuterium Exchange Mass Spectrometry." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/70915267660525113142.
Повний текст джерелаАнотація:
碩士東海大學
化學系
105
Hydrogen/deuterium exchange (HDX) coupled with mass spectrometry (ESI-MS) has been widely used to study the mechanisms of protein dynamics, domain structure, protein-ligand interactions and protein conformational changes.The redox protein cytochrome c plays an important role as an electron carrier in mitochondrial respiration. Release of cytochrome c from mitochondria triggers an intrinsic apoptosis pathway. The cytosolic transfer RNAs(tRNA) bind to cytochrome c preventing cytochrome c interaction with Apaf-1, blocking the formation of the apoptosome complex. To understand the interactions between tRNA and cytochrome c, we use HDXMS to analyze tRNA interaction with cytochrome c.Trnaphe and natural total tRNA are isolated from brewer’s yeast.tRNA binds to cyt.c to decrease the deuteration level, indicating the tRNA has caused cytochrome c to form a more compact conformation. To clarify the cause of binding, we use unstructured single strand oligonucleotidesto form complexes with cyt c in the HDXMS experiment. We compared with 12-mer dA and dT, although adenine and thymine are not important factors for the interactions, but cytochrome c in two regions 1-10 and 65-82 still showed to decreases upon unstructured dA or dT binding. Insummary, we conclude that N-terminal 20-32 is the selective region interacts with tRNA and N-terminus 1-10 interacts with oligonucleotides electrostatically. Tafazzin is an acyl transferase responsible for the remodeling of mitochondrial cardiolipin (CL). Natural mutation caused aberrant tafazzin in the Barth syndrome patients lost the remodeling function, leading to the abnormity of CL and monolyso-CL (MLCL) content. The activities of the tafazzin enzyme are tightly related to its interactions with lipid membrane. The 20% CL/ 80% PC phospholipid vesicle was prepared to simulate the mitochondrial membrane. We observed the interactions of the purified tafazzin with the CL vesicles by hydrogen/deuterium exchange mass spectrometry (HDXMS). Tafazzin protein contains a His69/Asp74 active site. The active sites and the surrounding loops show significant decreases of deuteration, which are potentially the membrane insertion regions. Based on the mechanism of the model of phospholipases, we suggest the surrounding loops positioned actives sites for the extraction of phospholipids upon lipid vesicle interaction.