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Strukturaufklärung gehört zu den wichtigsten Gebieten der Grundlagenforschung, da sie direkte Einblicke in biologische Systeme und ihre Mechanismen liefert. Der NMR Spektroskopie kommt dabei eine besondere Bedeutung zu, denn sie ermöglicht Forschung unter physiologischen Bedingungen. Dementsprechend ist die Entwicklung neuer Techniken zur Verbesserung dieser Methode weiterhin ein zentrales Forschungsgebiet. Paramagnetische Markierung von Biomolekülen ermöglicht die Bestimmung von NMR Parametern, wie z.B. residuale dipolare Restkopplungen (RDCs) oder Pseudokontaktverschiebungen (PCSs), die für die Strukturaufklärung wertvolle Winkel- und Abstandsinformationen über das Zielmolekül beinhalten. In diesem Zusammenhang wurden Lanthanoidionen-koordinierende Tags entwickelt und erfolgreich an Proteinen angebracht. Durch die paramagnetischen Eigenschaften der Lanthanoidionen wird eine partielle Ausrichtung des Zielmoleküls im Magnetfeld des NMR Spektrometers induziert und somit das Messen residualer dipolarer Kopplungen ermöglicht. Zusätzlich werden die NMR Signale durch eine Dipol-Dipol-Wechselwirkung zwischen dem Lanthanoidion und den Kernen verschoben (PCS). In der konventionellen NMR Spektroskopie werden diese Effekte, aufgrund der Brownschen Molekularbewegung und dem Fehlen eines Metallions, nicht beobachtet. In der Fachliteratur ist ein Transfer dieser Methode auf Oligonukleotide nicht bekannt, obwohl DNA und RNA zu den wichtigsten Biomolekülen überhaupt zählen. In dieser Arbeit wurde mit Hilfe des kürzlich entwickelten Cys-Ph-TAHA Tags ein Protokoll zur Bestimmung von paramagnetischen Effekten in der DNA entwickelt. Dafür wurde eine modifizierte Nukleobase synthetisiert, die eine passende Bindungsstelle für den Tag aufweist. Mit der neu entwickelten Methode wurden zwei paramagnetische und eine diamagnetische Referenzprobe hergestellt. Mittels hochauflösender NMR Spektroskopie konnten paramagnetisch-induzierte PCSs und RDCs gemessen werden. Die Auswertung zeigte eine hohe Qualität der gemessenen PCSs in beiden paramagnetischen Proben. Die RDCs wiesen einen signifikanten Fehler auf. Die in der NMR Spektroskopie übliche Isotopenmarkierung (13C/15N) ist bei im DNA-Synthesizer hergestellten Oligonukleotiden auf Grund der teuren Ausgangsmaterialien nicht möglich, sodass die hergestellten NMR Proben eine natürliche Isotopenhäufigkeit aufwiesen. In den NMR Spektren zur Bestimmung der RDCs ist damit das Verhältnis von Signal-zu-Rausch relativ niedrig, was zusammen mit der paramagnetischen Relaxationsverstärkung zu einem größeren Messfehler führt. Dennoch konnten die erhaltenen paramagnetischen Daten mit einem Ensemblemodell beschrieben werden. In der vorliegenden Arbeit wurde die Methode der paramagnetischen Markierung erfolgreich auf die Stoffklasse der Oligonukleotide übertragen. Dabei wurde ein reproduzierbares Protokoll entwickelt, mit dem eine Bindungsstelle in einen DNA Strang eingebaut und das Zielmolekül anschließend mit dem Cys-Ph-TAHA Tag markiert wurde. Die erfolgreiche Anwendung der Methode konnte durch die erhaltenen paramagnetischen Messwerte von hoher Qualität verifiziert werden.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第23460号 / 理博第4754号 / 新制||理||1681(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 杤尾 豪人, 教授 森 和俊, 教授 望月 敦史 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

<p>Nuclear magnetic resonance (NMR) spectroscopy is an established technique for macromolecular structure determination at atomic resolution. However, the majority of the current structure determination approaches require a large set of experiments and use large amount of data to elucidate the three dimensional protein structure. While current structure determination protocols may perform well in data-rich settings, protein structure determination still remains to be a difficult task in a sparse-data setting. Sparse data arises in high-throughput settings, for larger proteins, membrane proteins, and symmetric protein complexes; thereby requiring novel algorithms that can compute structures with provable guarantees on solution quality and running time.</p><p>In this dissertation project we made an effort to address the key computational bottlenecks in NMR structural biology. Specifically, we improved and extended the recently-developed techniques by our laboratory, and developed novel algorithms and computational tools that will enable protein structure determination from sparse NMR data. An underlying goal of our project was to minimize the number of NMR experiments, hence the amount of time and cost to perform them, and still be able to determine protein structures accurately from a limited set of experimental data. The algorithms developed in this dissertation use the global orientational restraints from residual dipolar coupling (RDC) and residual chemical shift anisotropy (RCSA) data from solution NMR, in addition to a sparse set of distance restraints from nuclear Overhauser effect (NOE) and paramagnetic relaxation enhancement (PRE) measurements. We have used tools from algebraic geometry to derive analytic expressions for the bond vector and peptide plane orientations, by exploiting the mathematical interplay between RDC- or RCSA-derived sphero-conics and protein kinematics, which in addition to improving our understanding of the geometry of the restraints from these experimental data, have been used by our algorithms to compute the protein structures provably accurately. Our algorithms, which determine protein backbone global fold from sparse NMR data, were used in the high-resolution structure determination protocol developed in our laboratory to solve the solution NMR structures of the FF Domain 2 of human transcription elongation factor CA150 (RNA polymerase II C-terminal domain interacting protein), which have been deposited into the Protein Data Bank. We have developed a novel, sparse data, RDC-based algorithm to compute ensembles of protein loop conformations in the presence of a moderate level of dynamics in the loop regions. All the algorithms developed in this dissertation have been tested on experimental NMR data. The promising results obtained by our algorithms suggest that our algorithms can be successfully applied to determine high-quality protein backbone structures from a limited amount of experimental NMR data, and hence will be useful in automated NOE assignments and high-resolution protein backbone structure determination from sparse NMR data. The algorithms and the software tools developed during this project are made available as free open-source to the scientific community.</p> / Dissertation

In der Kernspinresonanzspektroskopie (NMR) treten drei Effekte auf, die paramagnetische und diamagnetische Moleküle in isotroper Lösung unterscheiden: residuale dipolare Kopplung (RDC), Pseudokontaktverschiebung (PCS) und paramagnetische Relaxationsverstärkung (PRE). Alle drei Effekte sind abhängig von intermolekularen Winkeln und Abständen und können daher Informationen über die Struktur und Dynamik des Moleküls liefern. Um diese Informationen zu erhalten, muss das Molekül paramagnetische Eigenschaften aufweisen. Eine der heutzutage gebräuchlichen Methoden verwendet kleine molekulare Tags, die paramagnetische Metallionen koordinieren. Die meisten dieser Tags binden über eine Disulfidbrücke an Cysteine an der Proteinoberfläche. Um diese Methode für DNA anzuwenden werden daher neue Taggingstrategien benötigt. Im Rahmen dieser Arbeit wurde eine modifizierte Nukleobase synthetisiert, mit der ein Schwefelatom in die DNA eingebracht werden kann. Diese Methode erlaubt es, jeden Tag an die DNA zu binden, der als Verbindungsmethode eine Disulfidbrücke nutzt. Mit der Nukleobase wird eine Kohlenstoff-Dreifachbindung in die DNA eingefügt und mit Hilfe einer dipolaren Cycloaddition wird die freie Thiolgruppe eingebracht. Die modifizierte Nukleobase wurde erfolgreich an einem selbstkomplementären DNA-Strang (24 Nukleobasen) getestet. Die Nukleobase wurde während der Synthese der DNA eingefügt und der mit Lutetium, Terbium oder Thulium vorbeladene Cys-Ph-TAHA Tag wurde über eine Disulfidbrücke an die DNA gebunden. Die Beladung des Tags und die Taggingreaktion verliefen hierbei quantitativ. Nach diesem Erfolg war es ein Hauptaspekt dieser Arbeit, eine verlässliche und reproduzierbare Aufreinigungs- und Probenvorbereitungsmethode zu entwickeln. Diesem Punkt kommt besondere Bedeutung zu, da das Phosphatrückgrat der DNA, im Gegensatz zu Proteinen, Metallionen koordinieren kann. Im Theorieteil dieser Arbeit ist eine komplette Herleitung der drei Hauptmerkmale paramagnetischer NMR gegeben. Diese Herleitung beginnt bei Grundbegriffen des Magnetismus und neben den Gleichungen für RDCs, PCSs und PREs werden Ausdrücke für den dipolaren Hamiltonoperator, Kreuzrelaxationsraten, kreuzkorrelierte Relaxationsraten, durch Alignment induzierte RDCs, Korrelationsfunktionen und spektrale Dichten gegeben. Das zweite Thema dieser Arbeit basiert auf einem weiteren paramagnetischen Effekt. Um der reduzierten Empfindlichkeit der Kernspinresonanzspektroskopie verglichen mit anderen Spektroskopiemethoden entgegenzuwirken, wurden viele Methoden entwickelt, die auf eine Erhöhung der Polarisierung der Atomkerne zielen, d.h. um sogenannte hyperpolarisierte Kerne zu erzeugen. Eine dieser Methoden, die photochemisch erzeugte dynamische Kernpolarisierung (photo CIDNP), basiert auf kurzlebigen Radikalen, die durch direkte Laserbestrahlung der Probe im Magneten erzeugt werden. Im Rahmen dieser Arbeit wurde ein photo CIDNP Aufbau entworfen, gebaut und getestet. Die ersten Experimente und Resultate mit Triethylendiamin, L-Tyrosin und 3-Fluor-L-tyrosin zeigen die Vorteile und Grenzen dieser Methode auf. Für 3-Fluor-L-tyrosin wurde eine komplette Analyse des Relaxationsverhaltens, einschließlich der Kreuzrelaxation und der kreuzkorrelierten Relaxation, durchgeführt.

A fosfatase Cdc25B atua na progressão do ciclo celular através da ativação de complexos Cdk/Ciclina. Atualmente, nos modelos estruturais propostos do domínio catalítico da Cdc25B não estão incluídos os últimos 16 resíduos da região C-terminal. Este segmento tem importante papel no reconhecimento do substrato proteico e pode estar envolvido na complexação de pequenas moléculas com a Cdc25B. Assim, o principal objetivo desta tese foi avaliar a flexibilidade conformacional do domínio catalítico completo da Cdc25B em solução através de simulações computacionais e por medidas experimentais de ressonância magnética nuclear (RMN). A similaridade entre as estruturas cristalográficas e em solução foi confirmada pela previsão de ângulos diedrais &#966;/&#968; da cadeia principal a partir dos deslocamentos químicos (CS) e pela concordância entre os acoplamentos dipolares residuais (RDC) medidos e calculados a partir da geometria cristalina. Medidas de parâmetros de relaxação de 15N e RDC evidenciaram a presença de desordem conformacional na região C-terminal, em acordo com a ausência de densidade eletrônica desse segmento no experimento de difração de raios-X. Através da comparação entre CS experimentais e previstos de simulações de dinâmica molecular (DM) longas (total de 6&#181;s de duração) foram apontados artefatos de cristalização, possíveis erros nos campos de força usados nas simulações, falhas na composição do sistema simulado e estados conformacionais populados pela Cdc25B em solução distintos da geometria cristalográfica. De maneira geral, os CS previstos a partir das simulações para a flutuação estrutural dos resíduos da região C-terminal desordenada estão em acordo com os valores experimentais, sugerindo que os estados conformacionais deste segmento foram razoavelmente bem amostrados nas simulações. Em particular, verificou-se que o contato tipo cátion-&#960; entre as cadeias laterais dos resíduos 550W do C-terminal desordenado e 482R do núcleo proteico, ausente na estrutura cristalográfica, pode ser importante em solução. A formação desse contato na simulação de DM também está de acordo com medidas experimentais de perturbação de deslocamentos químicos (CSP) entre construções completa e truncada do domínio catalítico da Cdc25B. Assim, através do uso conjunto de simulações computacionais e medidas experimentais foi possível obter uma representação mais completa e realista da flexibilidade conformacional do domínio catalítico da Cdc25B em solução, incluindo a determinação de possíveis contatos intramoleculares entre a região C-terminal desordenada e o núcleo proteico. Essas informações poderão ser usadas na construção de um ensemble conformacional da Cdc25B. / Cdc25B phosphatase acts on the progression of cell cycle through the activation of Cdk/Cyclin complexes. Currently, the proposed structural models of Cdc25B catalytic domain lack the last 16 residues from the C-terminal region. This segment is important for protein substrate recognition and might be involved in small molecule binding to Cdc25B. Thus, the main goal of this thesis was to evaluate the conformational flexibility of the complete catalytic domain from Cdc25B through computer simulations and experimental nuclear magnetic resonance (NMR) measurements. Similarity between crystal and in solution structures was confirmed by the prediction of backbone &#966;/&#968; dihedral angles from chemical shifts (CS) and by the agreement between observed and back-calculated residual dipolar couplings (RDC). 15N relaxation and RDC measurements pointed to the conformational disorder of the C-terminal region, in agreement with the X-ray diffraction experiment where this segment showed no electronic density. Comparison between experimental and predicted CS from long molecular dynamics (MD) simulations (6&#181;s total running time) pointed to the presence of crystallographic artifacts, possible deficiencies in simulation force fields, inaccurate composition of the simulated system and conformational states visited by Cdc25B in solution that were not observed in the crystallographic geometry. Generally, CS predicted from simulations for the structural fluctuation of the disordered C-terminal region were in agreement with experimental values, suggesting that the simulations sampled the conformational states populated by this segment reasonably well. In particular, a cation-&#960; contact not observed in the crystal structure between side chains of residue 550W from the disordered C-terminal tail and residue 482R from the protein core might be important in solution. This contact is also in agreement with experimental chemical shift perturbations (CSP) measured between complete and truncated constructs of Cdc25B catalytic domain. Therefore, the joint use of computer simulations and experimental measurements allowed the achievement of a more complete and realistic representation of the conformational flexibility of the Cdc25B catalytic domain in solution, including intramolecular contacts between the disordered C-terminal region and the protein core. This information might be used to obtain a conformational ensemble of Cdc25B.

A fosfatase Cdc25B atua na progressão do ciclo celular através da ativação de complexos Cdk/Ciclina. Atualmente, nos modelos estruturais propostos do domínio catalítico da Cdc25B não estão incluídos os últimos 16 resíduos da região C-terminal. Este segmento tem importante papel no reconhecimento do substrato proteico e pode estar envolvido na complexação de pequenas moléculas com a Cdc25B. Assim, o principal objetivo desta tese foi avaliar a flexibilidade conformacional do domínio catalítico completo da Cdc25B em solução através de simulações computacionais e por medidas experimentais de ressonância magnética nuclear (RMN). A similaridade entre as estruturas cristalográficas e em solução foi confirmada pela previsão de ângulos diedrais &#966;/&#968; da cadeia principal a partir dos deslocamentos químicos (CS) e pela concordância entre os acoplamentos dipolares residuais (RDC) medidos e calculados a partir da geometria cristalina. Medidas de parâmetros de relaxação de 15N e RDC evidenciaram a presença de desordem conformacional na região C-terminal, em acordo com a ausência de densidade eletrônica desse segmento no experimento de difração de raios-X. Através da comparação entre CS experimentais e previstos de simulações de dinâmica molecular (DM) longas (total de 6&#181;s de duração) foram apontados artefatos de cristalização, possíveis erros nos campos de força usados nas simulações, falhas na composição do sistema simulado e estados conformacionais populados pela Cdc25B em solução distintos da geometria cristalográfica. De maneira geral, os CS previstos a partir das simulações para a flutuação estrutural dos resíduos da região C-terminal desordenada estão em acordo com os valores experimentais, sugerindo que os estados conformacionais deste segmento foram razoavelmente bem amostrados nas simulações. Em particular, verificou-se que o contato tipo cátion-&#960; entre as cadeias laterais dos resíduos 550W do C-terminal desordenado e 482R do núcleo proteico, ausente na estrutura cristalográfica, pode ser importante em solução. A formação desse contato na simulação de DM também está de acordo com medidas experimentais de perturbação de deslocamentos químicos (CSP) entre construções completa e truncada do domínio catalítico da Cdc25B. Assim, através do uso conjunto de simulações computacionais e medidas experimentais foi possível obter uma representação mais completa e realista da flexibilidade conformacional do domínio catalítico da Cdc25B em solução, incluindo a determinação de possíveis contatos intramoleculares entre a região C-terminal desordenada e o núcleo proteico. Essas informações poderão ser usadas na construção de um ensemble conformacional da Cdc25B. / Cdc25B phosphatase acts on the progression of cell cycle through the activation of Cdk/Cyclin complexes. Currently, the proposed structural models of Cdc25B catalytic domain lack the last 16 residues from the C-terminal region. This segment is important for protein substrate recognition and might be involved in small molecule binding to Cdc25B. Thus, the main goal of this thesis was to evaluate the conformational flexibility of the complete catalytic domain from Cdc25B through computer simulations and experimental nuclear magnetic resonance (NMR) measurements. Similarity between crystal and in solution structures was confirmed by the prediction of backbone &#966;/&#968; dihedral angles from chemical shifts (CS) and by the agreement between observed and back-calculated residual dipolar couplings (RDC). 15N relaxation and RDC measurements pointed to the conformational disorder of the C-terminal region, in agreement with the X-ray diffraction experiment where this segment showed no electronic density. Comparison between experimental and predicted CS from long molecular dynamics (MD) simulations (6&#181;s total running time) pointed to the presence of crystallographic artifacts, possible deficiencies in simulation force fields, inaccurate composition of the simulated system and conformational states visited by Cdc25B in solution that were not observed in the crystallographic geometry. Generally, CS predicted from simulations for the structural fluctuation of the disordered C-terminal region were in agreement with experimental values, suggesting that the simulations sampled the conformational states populated by this segment reasonably well. In particular, a cation-&#960; contact not observed in the crystal structure between side chains of residue 550W from the disordered C-terminal tail and residue 482R from the protein core might be important in solution. This contact is also in agreement with experimental chemical shift perturbations (CSP) measured between complete and truncated constructs of Cdc25B catalytic domain. Therefore, the joint use of computer simulations and experimental measurements allowed the achievement of a more complete and realistic representation of the conformational flexibility of the Cdc25B catalytic domain in solution, including intramolecular contacts between the disordered C-terminal region and the protein core. This information might be used to obtain a conformational ensemble of Cdc25B.

No description available.