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1
Caramia, Sara. « Purification and preliminary structural characterization by NMR spectroscopy of the "HoLaMa" DNA polymerase ». Master's thesis, Alma Mater Studiorum - Università di Bologna, 2017. http://amslaurea.unibo.it/14420/.
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HoLaMa is a Klenow sub-fragment lacking the 3’-5’ exonuclease domain, whose gene codes for residues 515-928 of Escherichia coli DNA polymerase I. The enzyme was designed in a previous study starting from different Klenow enzymes, with the aim of studying a mini-DNA polymerase with NMR spectroscopy. In the present work, we studied a new purification protocol for the production of HoLaMa in order to obtain an appropriate quantity for NMR analysis. We tested three different purification procedure and at the end, we collected 5.8 mg of HoLaMa (Volume 1.8 mL, concentration 67 μM). After the purification, we started the study of HoLaMa by NMR spectroscopy, focusing on the nature of enzyme-substrate interactions and studying the kinetics of the reaction. Our preliminary studies were designed to understand the characteristic NMR signals of HoLaMa under different conditions of temperature and buffer; finally, we also focused our analysis on the interactions between protein, DNA and nucleotides.
2
Nishizawa, Mayu. « Physicochemical Characterization of Physiological Aspects of Protein Structure ». Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/263680.
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京都大学新制・課程博士
博士(工学)
甲第23219号
工博第4863号
京都大学大学院工学研究科分子工学専攻
(主査)教授 田中 庸裕, 教授 近藤 輝幸, 准教授 菅瀬 謙治, 教授 佐藤 啓文
学位規則第4条第1項該当
Doctor of Philosophy (Engineering)
Kyoto University
DGAM
3
MERLO, SILVIA. « Characterization of biomedical relevant ligand-protein interactions using Nuclear Magnetic Resonance (NMR) Spectroscopy ». Doctoral thesis, Università degli Studi di Milano-Bicocca, 2013. http://hdl.handle.net/10281/40953.
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NMR techniques allow to obtain structural information useful for the comprehension of biological processes. The aim of this work is to investigate interactions between biological macromolecules and binding partners, including other macromolecules, small ligands and therapeutically relevant compounds. These results will be exploited for the design and development of new potential drugs and medical devices.
4
MACCHI, ELEONORA. « NMR as a tool for structural characterization of carbohydrates and glycan-protein interactions ». Doctoral thesis, Università degli Studi di Milano-Bicocca, 2015. http://hdl.handle.net/10281/69274.
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Il virus dell’influenza A è un virus a RNA formato da 8 geni, tre dei quali - emagglutinina (HA), neuramminidasi (NA) e polimerasi (PB) –, risultano particolarmente critici nell’infezione e nella trasmissione uomo-uomo. L’infezione inizia con il legame dell’HA del virus ai recettori glicanici presenti sulle cellule dell’ospite; questa interazione è altamente specifica, ed è governata dal tipo di legame tra l’acido sialico e il galattosio all’interno del recettore.
I recettori umani glicanici, siti di riconoscimento per i virus human-adapted, sono espressi principalmente nel tratto superiore dell’epitelio respiratorio umano e presentano un legame α2→6 tra l’acido sialico e il galattosio nell’estremità non riducente. I virus aviari invece, riconoscono recettori glicanici che presentano un legame α2→3 tra acido neuraminico e il galattosio.
Studi precedenti dell’interazione tra HA e trisaccaridi hanno dimostrato che sia la conformazione dei glicani, che il diverso tipo di legame tra acido sialico e galattosio sono fattori chiave per la regolazione dell’interazione. Partendo dalle sopracitate considerazioni questo lavoro di ricerca si è occupato di studiare la dinamica e la conformazione in soluzione di due pentasaccaridi, usati come modelli per il recettore aviario (LSTa, Neu5Ac-α(2→3)-Gal-β(1→3)-GlcNAc-β(1→3)-Gal-β(1→4)-Glc) e umano (LSTc, Neu5Ac-α(2→6)-Gal-β(1→4)-GlcNAc-β(1→3)-Gal-β(1→4)-Glc), utilizzando tecniche NMR (Nuclear Magnetic Resonance) e simulazioni di dinamica molecolare (MD). I nostri studi dimostrano che in soluzione i due recettori presentano diverse conformazioni, dinamiche e topologie. Queste peculiarità uniche comportano caratteristiche molecolari diverse per il riconoscimento di HA, dimostrando quindi la specificità dell’interazione tra recettore e emagglutinina.
La relazione tra la specificità dell’HA verso i recettori e la trasmissibilità del virus è stata precedentemente dimostrata usando il prototipo del virus SC18 (H1N1) A/South Carolina/1/1918. Combinando tecniche di Risonanza Magnetica Nucleare e di dinamica molecolare, abbiamo dimostrato come, durante l’interazione, il sito di binding dell’emagglutinina imponga differenti vincoli conformazionali al recettore. Il virus pandemico SC18, che presenta un’efficacia di trasmissione negli uomini molto alta, a confronto con il singolo (NY18, Asp225 → Gly) e doppio (AV18, Asp190 → Glu e Asp225 → Gly) mutante, impone maggiori vincoli alla conformazione del recettore umano, proprietà correlata all’affinità dell’interazione recettore-HA, misurata tramite saggi biochimici.
Questa relazione tra affinità e vincoli conformazionali imposti al recettore è stata osservata anche per il virus aviario-adattato AV18, il quale impone vincoli conformazionali maggiori al recettore aviario in confronto a quelli imposti a quest’ultimo da NY18. In particolare, è interessante osservare come emagglutinine differenti impongano vincoli conformazionali diversi a seconda che leghino recettori umani o aviari. In ultimo, abbiamo esteso il nostro studio a un virus meno pandemico, H7N9, e due suoi mutanti, i quali sono in grado di legare sia il recettore umano che aviario, allo scopo di capire come avviene l’interazione tramite l’utilizzo di tecniche NMR e di dinamica molecolare.
In questo studio descriviamo le basi strutturali dell’interazione tra l’emagglutinina di nuovi virus e i recettori umani e aviari, combinando l’approccio sperimentale a tecniche computazionali. Questa metodologia potrà essere usata come strumento utile per la sorveglianza di nuovi virus pandemici.
5
Gustafsson, Robert. « Biophysical characterization of the *5 protein variant of human thiopurine methyltransferase by NMR spectroscopy ». Thesis, Linköpings universitet, Molekylär Bioteknik, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-78526.
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Human thiopurine methyltransferase (TPMT) is an enzyme involved in the metabolism of thiopurine drugs, which are widely used in leukemia and inflammatory bowel diseases such as ulcerative colitis and Crohn´s disease. Due to genetic polymorphisms, approximately 30 protein variants are present in the population, some of which have significantly lowered activity. TPMT *5 (Leu49Ser) is one of the protein variants with almost no activity. The mutation is positioned in the hydrophobic core of the protein, close to the active site. Hydrogen exchange rates measured with NMR spectroscopy for N-terminally truncated constructs of TPMT *5 and TPMT *1 (wild type) show that local stability and hydrogen bonding patterns are changed by the mutation Leu49Ser. Most residues exhibit faster exchange rates and a lower local stability in TPMT *5 in comparison with TPMT *1. Changes occur close to the active site but also throughout the entire protein. Calculated overall stability is similar for the two constructs, so the measured changes are due to local stability. Protein dynamics measured with NMR relaxation experiments show that both TPMT *5 and TPMT *1 are monomeric in solution. Millisecond dynamics exist in TPMT *1 but not in TPMT *5, even though a few residues exhibit a faster dynamic. Dynamics on nanosecond to picosecond time scale have changed but no clear trends are observable.
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Hake, Michael James. « Spectroscopic Characterization of the Interaction of Nck Domains with the Epidermal Growth Factor Receptor Juxtamembrane Domain ». Case Western Reserve University School of Graduate Studies / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=case1207340174.
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Silvennoinen, L. (Laura). « ERp57—Characterization of its domains and determination of solution structures of the catalytic domains ». Doctoral thesis, University of Oulu, 2006. http://urn.fi/urn:isbn:9514280547.
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Abstract
The correct three dimensional structures of proteins are essential for their ability to function properly. Proteins start to fold as soon as they are synthesized in the ribosomes from activated amino acids. Many secreted, cell-surface, secretory pathway and endoplasmic reticulum (ER) lumenal proteins have in their amino acid sequence cysteine residues which form intra- and intermolecular disulfide bridges that stabilize the overall fold of the proteins and protein complexes. The formation of correct disulfide bonds is a complex process which takes place within the ER.
Protein disulfide isomerase (PDI) is the key enzyme in the formation and rearrangement of correct disulfide bonds in the ER. It is an archetypal and the best studied member of the PDI family, i.e. a group of ER proteins that resemble thioredoxin (TRX), a protein reductase, in their structure. PDI has a four domain a-b-b'-a' structure the a and a' domains having the catalytic activity and amino acid sequence similarity to TRX. In addition to its function as a thiol-disulfide oxidoreductase, PDI acts as the β subunit in two protein complexes: collagen prolyl 4-hydroxylase (C-P4H) and microsomal triglyceride transfer protein (MTP).
The closest homologue of PDI is the multifunctional enzyme and chaperone ERp57 that functions in concert with two lectins, calnexin (CNX) and calreticulin (CRT) specifically in the folding of proteins that have sugar moieties linked to them. ERp57 is 56% similar to PDI in its amino acid sequence and has also the four-domain architecture. Despite the high similarity in their structures ERp57 cannot substitute for PDI as the β subunit of C-P4H. The minimum requirement for the C-P4H tetramer assembly is fulfilled by domains b' and a' of PDI, while domains a and b enhance this function and can be substituted in part by those of ERp57.
Until very recently the structural information of any of the PDI family members, which contains the TRX active site was limited to solution structures of human PDI domains a and b. In this research the domain boundaries of the full length ERp57 were defined and the individual domains characterized. Furthermore the solution structures of the catalytically active domains a and a' of ERp57 were studied by nuclear magnetic resonance (NMR).
8
Ahlner, Alexandra. « Improved Methods for Characterization of Protein Dynamics by NMR spectroscopy and Studies of the EphB2 Kinase Domain ». Doctoral thesis, Linköpings universitet, Kemi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-117076.
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Proteins are essential for all known forms of life and in many lethal diseases protein failure is the cause of the disease. To understand proteins and the processes they are involved in, it is valuable to know their structures as well as their dynamics and interactions. The structures may not be directly inspected because proteins are too small to be visible in a light microscope, which is why indirect methods such as nuclear magnetic resonance (NMR) spectroscopy have to be utilized. This method provides atomic information about the protein and, in contrast to other methods with similar resolution, the measurements are performed in solution resulting in more physiological conditions, enabling analysis of dynamics. Important dynamical processes are the ones on the millisecond timeframe, which may contribute to interactions of proteins and their catalysis of chemical reactions, both of significant value for the function of the proteins. To better understand proteins, not only do we need to study them, but also develop the methods we are using. This thesis presents four papers about improved NMR techniques as well as a fifth where the kinase domain of ephrinB receptor 2 (EphB2) has been studied regarding the importance of millisecond dynamics and interactions for the activation process. The first paper presents the software COMPASS, which combines statistics and the calculation power of a computer with the flexibility and experience of the user to facilitate and speed up the process of assigning NMR signals to the atoms in the protein. The computer program PINT has been developed for easier and faster evaluation of NMR experiments, such as those that evaluate protein dynamics. It is especially helpful for NMR signals that are difficult to distinguish, so called overlapped peaks, and the soft- ware also converts the detected signals to the indirectly measured physical quantities, such as relaxation rate constants, principal for dynamics. Next are two new versions of the Carr-Purcell-Maiboom-Gill (CPMG) dispersion pulse sequences, designed to measure millisecond dynamics in a way so that the signals are more separated than in standard experiments, to reduce problems with overlaps. To speed up the collection time of the data set, a subset is collected and the entire data set is then reconstructed, by multi-dimensional decomposition co-processing. Described in the thesis is also a way to produce suitably labeled proteins, to detect millisecond dynamics at Cα positions in proteins, using the CPMG dispersion relaxation experiment at lower protein concentrations. Lastly, the kinase domain of EphB2 is shown to be more dynamic on the millisecond time scale as well as more prone to interact with itself in the active form than in the inactive one. This is important for the receptor function of the protein, when and how it mediates signals. To conclude, this work has extended the possibilities to study protein dynamics by NMR spectroscopy and contributed to increased understanding of the activation process of EphB2 and its signaling mechanism.
9
Ghosh, Madhumita. « Structural and biochemical characterization of proteins involved in cancer ». [S.l. : s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=974284823.
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Pondaven, Simon Pierre. « Conformational Flexibility and Amyloid Core Characterization of Human Immunoglobulin Light Chain Domains by Multidimensional NMR Spectroscopy ». The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1354113457.
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Singh, Sameer Kumar Verfasser], Dieter [Akademischer Betreuer] [Willbold et Bernd [Akademischer Betreuer] König. « Characterization of HIV accessory protein interactions using NMR and microscale thermophoresis / Sameer Kumar Singh. Gutachter : Dieter Willbold ; Bernd König ». Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2013. http://d-nb.info/1035274116/34.
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Singh, Sameer Kumar [Verfasser], Dieter [Akademischer Betreuer] Willbold et Bernd [Akademischer Betreuer] König. « Characterization of HIV accessory protein interactions using NMR and microscale thermophoresis / Sameer Kumar Singh. Gutachter : Dieter Willbold ; Bernd König ». Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2013. http://d-nb.info/1035274116/34.
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Hudson, Frederick Michael Lewis. « NMR characterization guides the design of beta hairpins and sheets while providing insights into folding cooperativity and dynamics / ». Thesis, Connect to this title online ; UW restricted, 2006. http://hdl.handle.net/1773/8639.
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D'Silva, Loyola. « Monitorng the effects of antagonists on protein-protein interactions with NMR spectroscopy and structural characterization of the major intermediate in the oxidative folding of the leech carboxypeptidase inhibitor ». [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=978966600.
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Voreck, Anja Marion [Verfasser]. « Structural Characterization of a Transmembrane Protein by Solid-state NMR : A Biophysical and Functional Study of the ABC Transporter ArtMP-J / Anja Marion Voreck ». Berlin : Freie Universität Berlin, 2015. http://d-nb.info/1065670214/34.
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Yao, Xuejun [Verfasser], Markus [Akademischer Betreuer] Zweckstetter et Kai [Akademischer Betreuer] Tittmann. « Solution NMR-based characterization of the structure of the outer mitochondrial membrane protein Tom40 and a novel method for NMR resonance assignment of large intrinsically disordered proteins / Xuejun Yao. Gutachter : Markus Zweckstetter ; Kai Tittmann. Betreuer : Markus Zweckstetter ». Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2014. http://d-nb.info/1050873122/34.
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Feldmann, Erik A. « Biophysical characterization of heterocyst differentiation regulators, HetR and PatS, from the cyanobacterium, Anabaena sp. strain PCC 7120 and structural biology of bacterial proteins from the Northeast Structural Genomics Consortium ». Miami University / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=miami1342801532.
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Korste, Annika [Verfasser], Raphael [Akademischer Betreuer] Stoll et Peter [Akademischer Betreuer] Bayer. « Structural characterization of the antiapoptotic protein Bcl-x\(_L}\) and the NDH-1 complex subunit CupS by X-ray crystallography and NMR spectroscopy / Annika Korste. Gutachter : Raphael Stoll ; Peter Bayer ». Bochum : Ruhr-Universität Bochum, 2016. http://d-nb.info/1095884832/34.
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Rath, Parthasarathi. « Structural and functional characterization of PorA and PorH : the two major porins from Corynebacterium glutamicum ». Toulouse 3, 2012. http://thesesups.ups-tlse.fr/1856/.
Texte intégralRésumé :
PorA (5 kDa) et PorH (7kDa) sont les deux protéines membranaires majeures de la membrane externe de Corynebacterium glutamicum qui appartient au groupe supragénérique des bactéries Gram-positives contenant plusieurs agents pathogènes i. E. Mycobacterium tuberculosis, M. Leprae et C. Diphtheriae. Les deux protéines forment des canaux ioniques hétéromériques et présentent la particularité d'avoir une modification post-traductionnelle : estérification par l'acide mycolique. Les deux protéines ont été produites dans deux systèmes d'expression : chez C. Glutamicum et dans un système acellulaire utilisant des extraits de E. Coli. La présence ou l'absence de modification post-traductionnelle, sur les protéines produites in vivo et in vitro a été caractérisée par spectrométrie de masse MALDI-TOF. Les spectres de dichroïsme cellulaire et de RMN de PorA et PorH uniformément marquées et solubilisées en micelles de LDAO sont caractéristiques de protéines mono-disperses, partiellement structurées, et de qualité compatible avec une détermination de structure par RMN. Le test fonctionnel des protéines, par mesure de conductivité ionique après reconstitution dans une membrane lipidique (technique dite de BLM pour " black lipid membrane ") a montré que a) la modification post-traductionnelle de PorA par un acide mycolique est essentielle (contrairement à celle de PorH) b) la présence simultanée de PorA et de PorH est requise pour la formation d'un canal ionique voltage dépendant typique d'une porine. Afin de mieux comprendre l'importance de l'acide mycolique pour l'activité canal ionique, le complexe protéique PorA-PorH a été reconstitué dans son environnement naturel. Les principaux lipides de la membrane externe du C. Glutamicum [Tréhalose dimycolate (TDM), tréhalose monomycolate (TMM) et cardiolipide (CL)] ont été extraits et purifiés par chromatographie d'adsorption et échange d'ions, sous forme protonée et sous forme perdeutériée. Après formation de protéoliposomes les propriétés membranaires de TDM seul ou en mélange avec CL ont été étudiées RMN du deutérium, diffusion dynamique de la lumière et microscopie électronique. L'insertion de PorA et PorH dans des vésicules de TDM a permis de mettre en évidence la reconstitution de l'hétéro-oligomère (contrairement aux micelles de LDAO). Ceci ouvre la voie à la détermination de structure 3D du complexe PorA-PorH fonctionnel par RMN solide et/ou liquidePorA (5 kDa) and PorH (7 kDa) are two major membrane proteins from the outer membrane of Corynebacterium glutamicum which belongs to the suprageneric group of Gram-positive bacteria containing number of human pathogens such as Mycobacterium tuberculosis, M. Leprae and C. Diphtheriae. Both PorA and PorH have been shown to form heteromeric ion channels and to be post-translationally modified by mycolic acids (a-alkyl, beta-hydroxy fatty acids). Both proteins were produced in their natural host with mycolic acid modification, as well as in E. Coli based continuous exchange cell-free expression system and thus devoid of mycolic acid modification. The presence or absence of mycolic acid modification on in vivo and in vitro expressed proteins was confirmed by MALDI-TOF mass spectrometry. CD and NMR spectra of 15N/13C uniformly labeled PorA and PorH solubilized in LDAO micelles indicated mono-dispersed and partially folded proteins, compatible with structure determination by NMR. However, functional assays (via black lipid membrane ion-channel conductance measurements) confirmed that a complex associating both proteins is required for function and that the mycolic acid modification on PorA (but not PorH), is an absolute requirement for the formation of a voltage dependent ion-channel. To understand further the importance of covalent or non-covalent interaction of their natural lipid environment on the complex formation, the major C. Glutamicum outer membrane lipids [Trehalose dimycolate (TDM), Trehalose monomycolate (TMM) and Cardiolipin (CL)] were purified using adsorption and ion exchange chromatography, both in protonated and perdeuterated form. Prior to proteoliposome reconstitution, the membrane forming properties of TDM alone or in mixture with CL were studied by 2H-NMR, Dynamic Light Scattering and Electron Microscopy. Furthermore, after in vitro reconstitution of PorA and PorH in TDM vesicles (and not in LDAO micelles or DMPC vesicles), evidence for the formation of the hetero oligomeric complex was observed. The 3D structure determination, by liquid and/or solid state NMR, of a functional PorA-PorH complex in its natural lipid environment is now feasible
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De, Cicco Maristella Verfasser], Sonja A. [Akademischer Betreuer] [Gutachter] Dames et Aymelt [Gutachter] [Itzen. « NMR characterization of the membrane-localized interaction network between the kinase TOR, the GTPase Rheb and the FKBP12-like protein FKBP38. / Maristella De Cicco ; Gutachter : Aymelt Itzen, Sonja A. Dames ; Betreuer : Sonja A. Dames ». München : Universitätsbibliothek der TU München, 2017. http://d-nb.info/1147566178/34.
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Fogeron, Marie-Laure. « Development of a wheat germ cell-free expression system for the production, the purification and the structural and functional characterization of eukaryotic membrane proteins : application to the preparation of hepatitis C viral proteins ». Thesis, Lyon 1, 2015. http://www.theses.fr/2015LYO10081/document.
Texte intégralRésumé :
Alors que 30% du génome code pour des protéines membranaires, moins de 3% des structures protéiques dans la Protein Data Bank correspondent à ces protéines. En raison de leur nature hydrophobe, les protéines membranaires sont en effet très difficiles à produire dans des systèmes d'expression classique en cellules, notamment en bactéries. L'étude structurale des protéines membranaires du virus de l'hépatite C (VHC) sous forme entière et native a donc été pendant longtemps entravée. Le VHC est un virus à ARN positif dont le complexe de réplication est basé sur un réarrangement spécifique des membranes induit par l'action concertée de plusieurs protéines non structurales du virus dont NS2, NS4B et NS5A. La structure tridimensionnelle et le rôle de ces protéines dans la réplication virale sont encore mal connus. Pour surmonter les limitations qui empêchent leurs études structurales et fonctionnelles, un système d'expression acellulaire à base d'extrait de germe de blé a été développé avec succès, permettant la production des protéines NS2, NS4B et NS5A entières directement sous une forme solubilisée en présence de détergent. Ces protéines membranaires sont produites et purifiées par chromatographie d'affinité dans des quantités de l'ordre du milligramme. Des analyses par filtration sur gel indiquent que les échantillons obtenus sont homogènes. De plus, des analyses structurales par dichroïsme circulaire montrent que les protéines produites dans ce système sont bien repliées. Leur reconstitution dans des lipides est en cours d'optimisation. Le but ultime est en effet de déterminer leur structure par RMN du solide dans un environnement lipidique mimant l'environnement natifWhile 30% of the genome encodes for membrane proteins, less than 3% of protein structures in the Protein Data Bank correspond to such proteins. Due to their hydrophobic nature, membrane proteins are indeed notoriously difficult to express in classical cell-based protein expression systems. The structural study of the membrane proteins of hepatitis C virus (HCV) in their full-length and native form has therefore been for long time hampered. HCV is a positive-strand RNA virus building its replication complex on a specific membrane rearrangement (membranous web), which serves as a scaffold for the HCV replicase, and is induced by the concerted action of several HCV non-structural proteins including NS2, NS4B and NSSA. The knowledge of the three- dimensional structure of these proteins and their role in virus replication is still limited. To overcome the limitations that prevent the structural and functional studies of these proteins, a wheat germ cell-free protein expression system has been developed. A production protocol was designed which allows us to directly obtain membrane proteins in a soluble form by adding detergent during the in vitro protein synthesis. A large number of mainly viral proteins were successfully expressed, and full protocols were developed for the full-length NS2, NS4B and NSSA proteins. These membrane proteins were produced and purified by affinity chromatography using a Strep-tag II in the milligram range. These protein samples are homogenous, as shown by gel filtration analysis. Moreover, structural analyses by circular dichroism showed that the proteins produced in the wheat germ cell-free system are well folded. Reconstitution of these proteins in lipids is currently under optimization. The ultimate goal is to determine their structure by solid-state NMR in a native-like membrane lipids environment
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Lv, Guohua [Verfasser], Lange [Akademischer Betreuer], Tittmann [Akademischer Betreuer] et Bert L. de [Akademischer Betreuer] Groot. « Protein Structure Characterization by Solid-State NMR : Structural Comparison of Mouse and Human alpha-Synuclein Fibrils, Sparse 13C Labeling Schemes, and Stereospecific Assignment of Val and Leu Prochiral Methyl Groups / Guohua Lv. Gutachter : Tittmann ; Bert L. de Groot. Betreuer : Lange ». Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2013. http://d-nb.info/1044736720/34.
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MALANHO, DA SILVA JOSE PEDRO. « Ultra-high resolution structure determination of transition metal substituted human carbonic anhydrase 2 – inhibitor complexes ». Doctoral thesis, 2022. https://hdl.handle.net/2158/1291044.
Texte intégralRésumé :
Paramagnetic Nuclear Magnetic Resonance (NMR) is developing to aid the characterization of paramagnetic molecules, whose paramagnetic centers changes the spectroscopic proprieties of said molecules. These paramagnetic centers can be exploited to overcome some troublesome aspects of NMR, such as sensitivity, by increasing the number of experiments. To aid this development, we used human Carbonic Anhydrase II (hCAII), which is a model protein that contains zinc(II) in its active center, which is diamagnetic. hCAII is an enzyme capable of interconverting carbon dioxide to bicarbonate, making it one of the most important proteins in life. Several comprehensive studies have structurally and functionally characterized hCAII making it an excellent model protein. Furthermore, the metal ion in the active center can be substituted by other transition metals ions (see chapter 1), such as cobalt(II) (see chapter 3), nickel(II) (see chapter 4) and copper(II) (see chapter 5), which are paramagnetic that will help answering different problems described in this thesis.
The ion cobalt(II), explored in chapter 3, can induce considerable changes on the NMR observables and is useful to understand the interactions of ligands with proteins. For this we used cobalt(II)-hCAII and used NMR, Electron Paramagnetic Resonance (EPR) and X-ray crystallography to characterize the interaction of thiocyanate under high concentrations with the hCAII. The addition of 500 mM of sodium thiocyanate changes the dynamics of the protein without changing the protein structure.
Solid-state NMR (SSNMR) is another field of NMR where the methodological and practical aspects are currently under development to reach better sensitivity and resolution. We proposed the usage of nickel(II)-hCAII as a paramagnetic molecule to increase the amount of tools in SSNMR, which is explored in chapter 4. The nickel(II) ion is capable of breaking the dipolar bath by changing the frequency of the nuclei close to the paramagnetic center, thus increasing the resolution and sensitivity of the SSNMR experiments. Furthermore, in parallel we discovered that hCAII is capable of binding two nickel(II) ions, one in the active center, as expected and described in literature, and one in the N-terminal site of the protein, a novel discovery.
The description of the paramagnetic effects, such as the Pseudocontact Shifts (PCS), in the NMR observables have been subjected to debate, where different treatments of theoretical equations were clashing. The experimental proof to determine which equation holds true is fully described in chapter 5. For this, we developed copper(II)-hCAII to acquire NMR and EPR data under the same conditions, and determine which equations describe better the PCS. The data interpretation from different techniques (both NMR and EPR) led us to conclude that the original treatment from Kurland and McGarvey equation is the correct one.
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RINALDELLI, MAURO. « Development of software tools for protein structural and dynamic characterization ». Doctoral thesis, 2014. http://hdl.handle.net/2158/835698.
Texte intégralRésumé :
In this thesis I focused on the development of novel bioinformatic tools for the structural characterization of proteins. The protein structure of MMP-12 in solid state has been calculated and its crystal packing has been reconstructed using distance restraints and pseudocontact shifts (pcs), by implementing in Cyana a protocol for the simultaneous analysis of the intermolecular and intramolecular contributions to the pcs.
The use of pcs and residual dipolar couplings (rdc) has been implemented in the program REFMAC5, for a joint refinement against X-ray and NMR data.
A web portal for the analysis of paramagnetic data against the molecular structures has been developed.
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PAVELKOVA', ANNA. « NMR Characterization of Protein-Protein Interactions ». Doctoral thesis, 2010. http://hdl.handle.net/2158/484862.
Texte intégral
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Denis, Christopher. « Structural and functional characterization of E2A:KIX interactions in leukemia ». Thesis, 2012. http://hdl.handle.net/1974/7469.
Texte intégralRésumé :
The E2A proteins are transcription factors critical for B-lymphopoiesis. A chromosomal translocation involving the E2A gene promotes acute lymphoblastic leukemia (ALL) through expression of the oncoprotein E2A-PBX1. Two activation domains of E2A-PBX1, AD1 and AD2, have been implicated in transcription mediated by recruitment of the transcriptional co-activator CBP/p300. A motif has been identified within AD1 that is important for recruiting CBP/p300, known as PCET. This recruitment requires an interaction between the activation domains of E2A-PBX1 and the KIX domain of CBP/p300. The KIX domain recognizes a generic ΦXXΦΦ sequence (Φ corresponds to a hydrophobic residue) found in the activation domains of numerous transcription factors. Mutation of leucine 20 in PCET has been shown to abrogate ex vivo immortalization of murine bone marrow and oncogenesis in a murine bone marrow transplantation model. A similar sequence is also found in AD2 and is implicated in E2A transcriptional activity and recruitment of CBP/p300. The structural details of these interactions remain largely unknown.
NMR spectroscopy was used to determine the solution structure of the PCET:KIX complex, and the functional consequences of the Leu20Ala mutation were structurally rationalized. Other PCET mutations informed by this structure were tested and correlations were found between in vitro binding affinities and both transcriptional activation and immortalization. The binding site of the ΦXXΦΦ-containing E2A AD2 peptide was mapped to the same site on the KIX domain used by the PCET motif. A model of this complex was generated and mutations were tested using a similar approach as was used for PCET. E2A AD2 binds the KIX domain with lower affinity than the PCET motif and is not required for immortalizing bone marrow. A mutation that increases the affinity of E2A AD2 for the KIX domain to levels approaching that seen for the PCET:KIX interaction restores transcriptional activation and immortalization, demonstrating that immortalization by E2A-PBX1 is an affinity dependent process involving the KIX domain of CBP/p300. These studies indicate that the activation domains of E2A-PBX1 serve to support the in vivo function of the oncoprotein and that the PCET:KIX complex is a potential target for novel therapeutics in E2A-PBX1+ leukemia.Thesis (Ph.D, Biochemistry) -- Queen's University, 2012-09-13 13:30:48.848
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Seidel, Karsten. « Structural characterization of membrane proteins by solid-state NMR spectroscopy ». Doctoral thesis, 2008. http://hdl.handle.net/11858/00-1735-0000-0006-B46E-1.
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CEROFOLINI, LINDA. « Mechanistic, structural and dynamic characterization of biomolecules : from DNA to membrane proteins ». Doctoral thesis, 2013. http://hdl.handle.net/2158/823331.
Texte intégralRésumé :
In the present research project, an extensive NMR analysis has been carried out on four different biomolecules to clarify the structure/dynamics-function paradigm. NMR data have been integrated with structural and dynamical information provided by other biophysical and computational techniques such as SAXS and metadynamics to better characterize the
structural and dynamical features of the investigated biomolecules in solution. NMR has thus been used i) for the identification of a new DNA structural folding, ii) for the speculation of the mechanism of catalysis of an enzyme, highlighting the most probable conformations in
solution, iii) to underline the functional states of proteins in solution, and iv) to shed light on the mechanism of signal transduction of the Receptor for Advanced Glycation Endproducts.
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Chun-ChunOu et 歐純純. « Characterization and NMR Structure of SPy0985, a Phage-associated Hypothetical Protein from Group A Streptococcus ». Thesis, 2010. http://ndltd.ncl.edu.tw/handle/31008515846420756378.
Texte intégralRésumé :
碩士國立成功大學
生物化學研究所
98
Streptococcus pyogenes (group A Streptococcus [GAS]) is one of the most common human bacterial pathogens. GAS causes a variety of human diseases, including pharyngitis, impetigo, scarlet fever, cellulitis, toxic shock syndrome, necrotizing fasciitis. The genomes of many GAS strains have been sequenced, and they contained ~2000 genes. However, the functions of ~33% GAS genes are still unknown. In particular, most of these unknown genes are bacteriophage and transposon genes. It has been known that phage infection can enhance bacterial virulence, including bacterial adhesion, colonization, invasion; resistance to immune defenses; exotoxin production; sensitivity to antibiotics; and transmissibility among humans. In this study we identified a phage-associated hypothetical protein, SPy0985, which contains 88 amino acids residues with a superantigen signature sequence at the position of 49-75. SPy0985 was successfully expressed in E. coli and purified to homogeneity with a yield of 30 mg/L. We also found that of SPy0985 in the presence of 50 mM of arginine and glutamate (R+E) was properly folded with less aggregation. Molecular weight determination by size-exclusion chromatography analysis showed that SPy0985 existed as a shape of dimeric size under R+E and physiological conditions. The result of peripheral blood mononuclear cell assay showed that SPy0985 was a superantigen. NMR analysis showed that SPy0985 exhibited stable structures at N- and C-terminal regions with positive NOE and consisted of two α-helices packed against each other at C-terminus. It also formed a hydrophobic core packed by F8, Y17, Y23, L31 and Y36 at N terminal region. NMR relaxation studied showed that residues G39-D51 exhibited low NOE values, indicating the formation of a flexible loop in this region. Interestingly, the N-terminus and the loop ranging from K3 to T46 exhibited two resonances in 2D 1H-15N HSQC spectrum, indicating a chemical exchange process found from this region. The average NOE value of Spy0985 was 0.58, showing that SPy0985 was highly flexible. The R2/R1 ratio indicated that SPy0985 existed as a monomer. The underlying functional and structural details of SPy0985 remain to be elucidated.
30
Shojania, Shaheen. « Nuclear magnetic resonance and dynamic characterization of the intrinsically disordered HIV-1 Tat protein ». 2007. http://hdl.handle.net/1993/2814.
Texte intégralRésumé :
The HIV-1 transactivator of transcription (Tat) is a protein essential for both viral gene expression and virus replication. Tat is an RNA-binding protein that, in cooperation with host cell factors cyclin T1 and cyclin-dependent kinase 9, regulates transcription at the level of elongation. Tat also interacts with numerous other intracellular and extracellular proteins, and is implicated in a number of pathogenic processes. The Tat protein is encoded by two exons and is 101 residues in length. The first exon encodes a 72-residue molecule that activates transcription with the same proficiency as the full-length protein. The physico-chemical properties of Tat make it a particularly challenging target for structural studies: Tat contains seven cysteine residues, six of which are essential for transactivation, and is highly susceptible to oxidative cross-linking and aggregation. In addition, a basic segment (residues 48-57) gives the protein a high net positive charge of +12 at pH 7, endowing it with a high affinity for anionic polymers and surfaces. In order to study the structure of Tat, both alone and in complex with partner molecules, we have developed a system for the bacterial expression and purification of polyhistidine-tagged and isotopically enriched (in 15N and 15N /13C) recombinant HIV-1 Tat1-72 (BH10 isolate) that yields large amounts of protein. These preparations have facilitated the assignment of 95% of the non-proline backbone resonances using heteronuclear 3-dimensional nuclear magnetic resonance (NMR) spectroscopy. Analysis by mass spectrometry and NMR demonstrate that the cysteine-rich Tat protein is unambiguously reduced and monomeric in aqueous solution at pH 4. NMR chemical shifts and coupling constants suggest that it exists in a disordered conformation. Line broadening and multiple peaks in the cysteine-rich and core regions suggest that transient folding occurs in two of the five sequence domains. NMR relaxation parameters were measured and analysed by spectral density and model-free approaches both confirming the lack of structure throughout the length of the molecule. The absence of a fixed conformation and the observation of fast dynamics are consistent with the ability of the Tat protein to interact with a wide variety of proteins and nucleic acid lending further support to the concept that Tat exists as an intrinsically disordered protein.October 2007
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Wu, Kuen-Phon. « NMR characterization of intrinsically disordered alpha-synuclein implication for aggregation in Parkinson's disease / ». 2010. http://hdl.rutgers.edu/1782.2/rucore10001600001.ETD.000052165.
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Saudino, Giovanni. « Protein expression and characterization for systems involved in the biogenesis of iron sulfur proteins ». Doctoral thesis, 2021. http://hdl.handle.net/2158/1251249.
Texte intégralRésumé :
Iron-sulfur clusters are essential cofactors found in all kingdoms of life; they had unique functional roles throughout evolution. These clusters, which are the second major form of complex iron cofactors in biology, are ubiquitous in all organisms, playing a key role in several biological pathways. Mutations on the protein involved in the iron-sulfur clusters biosynthesis pathway are associated with a group of multiple mitochondrial dysfunction syndromes (MMDS). These severe diseases could give infantile encephalopathy, lactic acidosis, leukodystrophy and death in early childhood. In human cells, cluster biosynthesis involves three different types of machinery: ISC (iron-sulfur cluster assembly machinery) located in the mitochondria, CIA (cytosolic iron-sulfur cluster assembly machinery) located in the cytosol and ISC export machinery located in the mitochondria inner membrane. The aim of the thesis was the deep investigation of the third step of the ISC assembly machinery. Indeed, using NMR, UV-vis and CD-vis spectroscopies in combination with size exclusion chromatography and multi-angle light scattering we assessed the key role of NFU1, ISCA1, ISCA2, FDX2 and LIAS in the above-mentioned machinery. Moreover, clinical BOLA3 Cys59Tyr mutation involved in the MMDS diseases has investigated at the atomistic and molecular levels. The gained data elucidated fundamental molecular details in the [4Fe-4S] cluster maturation and transfer to apo recipient proteins along the third step of ISC assembly machinery.
33
Hu, Tony Ken. « Biophysical analysis and NMR structural characterization of the binding between peptidomimetic drug CN2097 and scaffolding protein PSD-95 ». Thesis, 2019. https://hdl.handle.net/2144/36537.
Texte intégralRésumé :
BACKGROUND: At the postsynaptic membrane of neurons there is a dense network of
proteins called the postsynaptic density (PSD). One such protein is the postsynaptic
density protein 95 (PSD-95), which functions as a molecular scaffold for forming protein
complexes at the PSD. PSD-95 is composed of three PDZ domains, which studies have
shown to be sequentially and structurally similar. Studies have shown that PSD-95 plays
a role in regulating signaling of glutamatergic neurons, as well as the induction of longterm
potentiation through an association with TrkB receptors. PSD-95 may be a
promising target for treatment of a number of neurological disorders such as depression,
epilepsy, and cognitive dysfunction. The cyclic peptidomimetic drug CN2097 was
designed based on the PDZ-binding motif of the CRIPT protein that binds to PDZ3.
While CN2097 has been shown to affect the binding of PSD-95 to different synaptic
proteins, no NMR studies have been performed to characterize the binding of CN2097 to
PDZ3. Furthermore, few studies have characterized the inter-domain interactions
between PDZ domains or whether the binding of calmodulin (CaM) to the N-terminal
region of PSD-95 has any effect on the binding between the PDZ domains and CN2097.
OBJECTIVE: To use isothermal titration calorimetry and nuclear magnetic resonance
spectroscopy to analyze and characterize how CN2097 binds to PDZ domains and
whether inter-domain interactions exist between PDZ domains.
METHODS: The gene sequences for the PDZ domains were inserted into the pET28a(+)
vector by subcloning. E. coli bacteria were then transformed with the different PDZ
plasmids. The bacterial cells were grown and induced to express the proteins of interest,
followed by lysis and purification using affinity chromatography and fast protein liquid
chromatography (FPLC). Isothermal titration calorimetry (ITC) was used to measure the
dissociation constant and thermodynamic binding parameters between the
peptidomimetic drug CN2097 and each isolated PDZ domain. Nuclear magnetic
resonance (NMR) spectroscopy was used to study how CN2097 binds to PDZ1 and
PDZ3, and how the PDZ domains interact with each other.
RESULTS: The ITC data showed the dissociation constant between CN2097 and PDZ3 to
be 5.12 ± (1.65) μM, and that of PDZ2+3 to be 42.63 ± (6.11)μM. ITC data on other
domains was inconclusive. The NMR data showed no interaction between N-terminal
region and PDZ1, and between PDZ2 and PDZ3 but significant interaction was seen
between PDZ1 and PDZ2, as well as between PDZ3 and the inter-domain linker
connecting it to PDZ2. NMR data showed that CN2097 binding perturbs PDZ3 more
strongly than PDZ1 and that CN2097 does not bind to PDZ1 in the presence of CaM.
Significant NMR chemical shift perturbations are seen on the second α-helix, second β-
sheet, and β2-β3 loop.
CONCLUSIONS: There are no significant contacts between the N-terminal α-helix and
PDZ1. There is inter-domain conformational exchange and interaction between PDZ1
and PDZ2. PDZ3 interacts with the second inter-domain linker. CN2097 binds tighter to
PDZ3 than to PDZ1, and does not bind to PDZ1 in the presence of CaM. The β2-β3 loop
is a prime target for future development of CN2097.
34
Bajaj, Rakhi. « Residue level characterization of molecular interactions of intermembrane space domains governing the preprotein import into the mitochondrial matrix ». Doctoral thesis, 2013. http://hdl.handle.net/11858/00-1735-0000-0023-9958-7.
Texte intégral
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Fallas, Valverde Jorge. « Design and Structural Characterization of Self-Assembling Triple Helical Heterotrimers ». Thesis, 2012. http://hdl.handle.net/1911/71303.
Texte intégralRésumé :
Design of self-assembling ABC-type collagen triple helical heterotrimers is challenging due to the number of competing species that can be formed in ternary mixture of peptides with a high propensity to fold into triple helices and the fact that well understood rules for pair-wise amino acid stabilization of the canonical collagen triple helix have remained elusive. Given the required one amino acid stagger between adjacent peptide strands in this fold, a ternary mixture of peptides can form as many as 27 triple helices with unique composition or register. Previously we have demonstrated that electrostatic interactions can be used to bias the helix population towards a desired target but the presence of competing states in mixtures has remained an outstanding problem. In this work we use high-resolution structural biology techniques to do a detailed study of stabilizing pair-wise interactions between positively and negatively charged amino acids in triple helices. Two types of contacts with distinct sequence requirements depending on the relative stagger of the interacting chains are observed: axial and lateral. Such register-specific interactions are crucial for the understanding of the registration process of collagens and the overall stability of proteins in this family. Using this knowledge we developed distinct design strategies to improve the specificity of our designed systems towards the desired ABC heterotrimeric target state. We validate our strategies through the synthesis and characterization of the designed sequences and show that they self-assemble into a highly stable ABC triple helices with control over composition in the case of the rational approach and with control over both composition and register in the case of the computational approach.
36
Basehore, Heather Kenney. « Characterization of the early interactions on the folding pathway of the ileal lipid-binding protein by ¹⁹F-NMR ». 2007. http://etda.libraries.psu.edu/theses/approved/WorldWideIndex/ETD-2147/index.html.
Texte intégral
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Yao, Xuejun. « Solution NMR-based characterization of the structure of the outer mitochondrial membrane protein Tom40 and a novel method for NMR resonance assignment of large intrinsically disordered proteins ». Doctoral thesis, 2013. http://hdl.handle.net/11858/00-1735-0000-0022-5EA9-4.
Texte intégral
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Lukhele, Sabelo. « Characterization of Structural and Binding Properties of 4E-BP2 ». Thesis, 2012. http://hdl.handle.net/1807/35545.
Texte intégralRésumé :
Eukaryotic initiation factor-4E (eIF4E) controls the rate of cap-dependent translation initiation and is in turn exquisitely regulated by 4E-BPs. 4E-BP2 binds eIF4E with the highest affinity and is implicated in cancer, and metabolic and neurological disorders. Herein we use NMR, ITC and fluorescence to characterize 4E-BP2 structural and binding properties. Isolated 4E-BP2 is intrinsically disordered, but possesses some transient secondary structural propensities. eIF4E, however, is folded but has a disordered N-terminus. The eIF4E:4E-BP2 interaction is tight (Kd = 10-9 nM) and involves 4E-BP2 C-terminal and canonical binding regions, and the disordered eIF4E N-terminus. 4E-BP2 remains largely disordered upon binding to eIF4E. Noteworthy, high affinity interactions are not necessarily mediated by static structures, and 4E-BP2 binding is not the simple “disorder-to-order” transition observed in many interactions involving disordered proteins. This study offers molecular insights into 4E-BP2 functionality, and lays a foundation for development of novel therapies for cancer and neurological disorders.
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Sharifahmadian, Mahzad. « Structural and Biochemical Characterization of VirB8 Protein in Type IV Secretion Systems ». Thèse, 2017. http://hdl.handle.net/1866/19323.
Texte intégralRésumé :
Secretion is the passage of macromolecules across cellular membranes. In bacteria, secretion is essential for virulence and survival. Gram-negative bacteria use specialized envelope-spanning multiprotein complexes to secrete macromolecules called type IV secretion system (T4SS). T4SSs mediate the secretion of monomeric proteins, multisubunit protein toxins and nucleoprotein complexes. Also, they contribute to the horizontal spread of plasmid-encoded antibiotic resistance genes. Consequently, they are potential targets for antivirulence drugs. Gram- negative bacteria have two membranes that the secretion complex spans. As a result, the T4SS consists of proteins inserted in the membranes and of soluble proteins that face into or out of the bacterial cell. The details of channel assembly and structure are not known, although recent advances have revealed the structure of the core secretion channel. VirB8 is an inner membrane protein of the complex that interacts with many other T4SS subunits and works as nucleation factor for T4SS channel assembly. Biophysical studies and NMR experiments in particular were conducted to characterize the structural aspects of VirB8 interactions. Dynamic regions of VirB8 during monomer-to-dimer transition were identified by NMR spectroscopy. X-ray crystal and NMR analyses revealed structural differences at the helical regions (α-1 and α-4) of wild-type VirB8 and its monomeric variant VirB8M102R. Fragment screening identified small molecules binding to the wild-type and monomeric variant. In silico docking analyses suggested that the surface groove in the VirB8 structure is important for effective binding of the small molecules. NMR experiments and biochemical assays demonstrated that the β-sheet domain (β1 in particular) is the binding interface of VirB8 for the interaction with VirB10. The identified interface has functional importance for T4SS-mediated conjugation. In addition, I used NMR spectroscopy to identify changes in the structure of VirB8 upon interaction with VirB5. Altogether, structural and biochemical studies on periplasmic and full length VirB8 enabled us to characterize the sequence of interactions between VirB8 and other VirB proteins during T4SS complex assembly and function. The results of this research may lead to an innovative strategy for the development of novel antimicrobial drugs.La sécrétion est le passage de macromolécules à travers les membranes cellulaires. Chez les bactéries, la sécrétion est essentielle pour la virulence et la survie. Les bactéries à Gramnégatif utilisent le système de sécrétion de type IV (SST4) pour la sécrétion de toxines et de nucléoprotéines. Les SST4 contribuent notamment à la propagation des gènes de résistance aux antibiotiques. Pour cette raison, les composants du SST4 sont des cibles potentielles pour le développement de médicaments antivirulence. Le SST4 est un complexe protéique qui s’étend entre la double membrane de la bactérie à Gram-négatif. Les protéines qui le composent sont insérées dans les membranes cellulaires ou solubles. Bien que la structure du pore central du SST4 ait été résolue récemment, les détails de l'assemblage et la structure de ce complexe ne sont pas connus. VirB8 est une protéine de la membrane interne qui interagit avec de nombreuses autres sous-unités du SST4. Il s’agit d’un acteur central de l'assemblage du SST4. Des études biophysiques, et notamment des expériences de RMN ont ainsi été réalisées pour caractériser les aspects structuraux des interactions avec VirB8. Des regions dynamiques dans la structure de VirB8 ont été identifiées par spectroscopie RMN lors de la transition entre la forme monomérique et dimérique. Les analyses de cristallographie et de RMN ont révélé des différences structurales dans les régions hélicoïdales (α1 et α4) de VirB8 wild-type et du variant monomérique VirB8M102R. Le criblage de fragments a permis d’identifier de petites molécules capables de se lier à VirB8 ainsi qu’au variant monomérique. Les analyses d’arrimage moléculaire in silico suggèrent que la rainure de surface dans la structure VirB8 est importante pour laliaison de ces petites molécules. Les expériences de RMN et les essais biochimiques révèlent que le feuillet β (β1 en particulier) constitue l'interface d’interaction entre VirB8 et VirB10. Cette interface d’interaction est d’ailleurs importante pour la conjugaison du SST4. De plus, j'ai identifié des changements dans la structure de VirB8 lors de l'interaction avec VirB5. Les études sur la protéine VirB8 nous ont permis de caractériser la séquence d'événements entre VirB8 et d'autres protéines VirB, régulant l'assemblage et la fonction du SST4.
40
Silva, Marta Alexandra Fernandes. « Characterization of novel heme-containing sensor proteins from Geobacter sulfurreducens ». Doctoral thesis, 2014. http://hdl.handle.net/10362/14390.
Texte intégralRésumé :
The focus of this Thesis was the study of the sensor domains of two heme-containing methyl-accepting chemotaxis proteins (MCP) from Geobacter sulfurreducens: GSU0582 and GSU0935. These domains contain one c-type heme, form swapped dimers with a PAS-like fold and are the first examples of a new class of heme sensors.
NMR spectroscopy was used to assign the heme and polypeptide signals in both sensors, as a first step to probe conformational changes in the vicinity of the hemes. However, the presence of two conformations in solution impaired the confident assignment of the polypeptide signals.
To understand how conformational changes and swapped dimerization mechanism can effectively modulate the function of the two sensor domains and their signal transduction process, the sensor domains folding and stability were studied by circular dichroism and UV-visible spectroscopy. The results showed differences in the thermodynamic stability of the sensors, with GSU0582 displaying higher structural stability. These studies also demonstrated that the heme moiety undergoes conformational changes matching those occurring at the global protein structure and that the content of intrinsically disordered segments within these proteins (25% for GSU0935; 13% for GSU0582) correlates with the stability differences observed.
The thermodynamic and kinetic properties of the sensor domains were determined at different pH and ionic strength by visible spectroscopy and stopped-flow techniques. Despite the remarkably similar spectroscopic and structural features of the two sensor domains, the results showed that their properties are quite distinct. Sensor domain GSU0935 displayed more negative reduction potentials and smaller reduction rate constants, which were more affected by pH and ionic strength. The available structures were used to rationalize these differences.
Overall, the results described in this Thesis indicate that the two G. sulfurreducens MCP sensor domains are designed to function in different working potential ranges, allowing this bacterium to trigger an adequate cellular response in distinct anoxic subsurface environments.Fundação para a Ciência e a Tecnologia (FCT)- Bolsa de Doutoramento SFRH/BD/61952/2009, do Projecto PTDC/BBB-BEP/0753/2012 e ao Projecto Estratégico PEst-C/EQB/LA0006/2013 concedido ao REQUIMTE Laboratório
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Bologna, Sara. « Expression and characterization of human proteins involved in neurological disorders ». Doctoral thesis, 2019. http://hdl.handle.net/2158/1179619.
Texte intégralRésumé :
My Ph.D. research project, entitled “Expression and characterization of human proteins involved in neurodegenerative diseases”, was focused on the application of molecular biology and proteomics methodologies to prepare samples of proteins involved in neurodegenerative diseases. The target proteins were α-synuclein (α-syn) and the amyloid-beta peptides (Aβ), involved in the pathogenesis of Parkinson’s disease (PD) and Alzheimer’s disease (AD), respectively. The aim of my research activity was the optimization of the expression and purification of the neurodegeneration-associated proteins to carry out the following projects: “NMR analysis of the aggregation kinetics of Aβ1-40”, to reveal aggregation mechanisms of Aβ1-40 and to develop a kinetic model describing the formation of oligomeric and fibrillary species. “NMR analysis of the assembly of Aβ42: Aβ40 mixed fibrils”. This project is in the frame of an integrative study aimed to characterize the structure of the mixed fibrils (containing Aβ1-42 and Aβ1-40 peptides) at atomic detail. “Development of a protein aggregation assays for the diagnosis of synucleinopathies” This project was focused on the development of a protocol tool for the diagnosis PD, dementia with Lewy bodies (DLB) and multiple system atrophy (MSA) based on α-synuclein aggregation assays (SAA-seeding aggregation assays) starting from aliquots of Cerebrospinal fluid (CSF). Study of the interaction between alpha-synuclein and human biofluids components” This project concerned the study of the interaction of α-synuclein with lipoproteins, proteins and other constituents of CSF and plasma.
42
Bramkamp, Marc. « Characterization of the KdpFABC complex from Escherichia coli, of soluble subdomains from KdpB, and of a homologous protein of Methanococcus jannaschii ». Doctoral thesis, 2003. https://repositorium.ub.uni-osnabrueck.de/handle/urn:nbn:de:gbv:700-2003071014.
Texte intégralRésumé :
The KdpFABC complex is a P-type ATPase. Several features make the inducible Kplus-transporting ATPase a unique member of this enzyme family. Aspects of structure and function of KdpB, the catalytic subunit of the complex, were examined here. Site-directed mutagenesis of the charged residues aspartate 583 and lysine 586 in the putative transmembrane helix 5 of KdpB revealed that these charges are involved in the coupling of ATP hydrolysis to ion translocation. The binding of FITC was shown to be specific and the binding site is within the nucleotide-binding domain of KdpB, most probably at lysine 395. Modification of KdpB with FITC was affected by adenosine nucleotides. A Mg2plus-dependent hydrolysis of p-nitrophenolphosphate was observed, which was inhibited by micromolar concentrations of ortho-vanadate and FITC. Low concentrations of ATP stimulated pNPP hydrolysis, while higher concentrations of ATP were inhibitory. ADP, AMP and Pi inhibited the pNPP hydrolysis. The catalytic modules of KdpB were separately synthesized, purified and biochemically characterized. It was found that KdpBN was highly soluble and could be concentrated up to 1 mM and higher. Therefore, the KdpBN domain was used for further structural analysis using nuclear magnetic resonance spectroscopy. The KdpBN domain could be purified from cells grown in minimal medium with 15N-ammonium sulfate and 13C1-6 glucose as sole nitrogen and carbon sources, respectively. The purified, labeled KdpBN protein was applied to NMR analysis. High quality multidimensional NMR spectra were obtained (M. Haupt and H. Kessler, TU Munich, personal communication) and structure calculations leading to backbone assignments were carried out. An ortholog of the H4H5 domain of KdpB from the thermopilic archaeon Methanococcus jannaschii, Mj0968, was cloned, expressed, purified, and characterized.
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Dantas, Joana Margarida Franco. « Characterization of extracellular electron transfer networks in Geobacter sulfurreducens, a key bacterium for bioremediation and bioenergy applications ». Doctoral thesis, 2017. http://hdl.handle.net/10362/27867.
Texte intégralRésumé :
Geobacter bacteria have awakened significantly attention because of their impact on natural environments and biotechnological applications that include the bioremediation of organic and inorganic contaminants, bioenergy production and bioelectronics. In addition to electron transfer towards extracellular terminal acceptors, Geobacter cells can also accept electrons from electrodes, in currentconsuming biofilms, a process that is currently explored in microbial electrosynthesis. These practical applications rely on an efficient transfer of electrons between the cell and its exterior, a process
designated extracellular electron transfer (EET). However, the precise mechanisms underlying EET processes are still under debate. Genetic and proteomics studies have identified several c-type cytochromes as key components for EET in G. sulfurreducens. These proteins are located at the innermembrane (IM), periplasm and outer-membrane (OM). Examples of such cytochromes include the IMassociated cytochrome MacA, periplasmic cytochromes PpcA-E and PccH, as well as, the OM cytochrome OmcF, which were studied in this Thesis.
Molecular interactions between PpcA-E and their putative redox partners, including a humic substance analogue molecule, MacA or PccH, were probed by NMR spectroscopy, stopped-flow kinetics and molecular docking. For the interacting pairs, their binding affinity was also determined by NMR chemical shift perturbation experiments. The results obtained showed that the interacting molecules establish reversible low-binding affinity complexes in specific regions of the proteins to warrant a rapid and selective electron transfer, a typical feature observed for electron transfer reactions between redox partners.
In addition, NMR spectroscopy was also used to determine the solution structure of OmcF in the reduced state, its pH-dependent conformational changes and backbone dynamics.
A biochemical and structural characterization of the cytochrome PccH was also carried out using circular dichroism, UV-visible and NMR spectroscopic techniques. The structure of PccH determined by X-ray crystallography showed that it is unique among the monoheme c-type cytochromes. The reduction potentials determined for PccH at different pH values by visible redox titrations are unusually low compared to those reported for other monoheme c-type cytochromes. Considering the structural and
functional features of PccH it was proposed that this protein represents a first characterized example of a new subclass of monoheme c-type cytochromes.
Overall, the results obtained constitute an important contribute to the current understanding of the G. sulfurreducens extracellular electron transfer mechanisms.
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Fernandes, Tomás Monteiro. « Characterization of extracellular electron transfer components of Geobacter bacteria ». Master's thesis, 2018. http://hdl.handle.net/10362/52580.
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D'Silva, Loyola [Verfasser]. « Monitorng the effects of antagonists on protein-protein interactions with NMR spectroscopy and structural characterization of the major intermediate in the oxidative folding of the leech carboxypeptidase inhibitor / Loyola D'Silva ». 2006. http://d-nb.info/978966600/34.
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Lv, Guohua. « Protein Structure Characterization by Solid-State NMR : Structural Comparison of Mouse and Human alpha-Synuclein Fibrils, Sparse 13C Labeling Schemes, and Stereospecific Assignment of Val and Leu Prochiral Methyl Groups ». Doctoral thesis, 2013. http://hdl.handle.net/11858/00-1735-0000-0015-983C-F.
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