Advanced EPR spectroscopy
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Date
2025
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unraveling the binding behavior and modes of metals and biomolecules
Abstract
Moderne Elektronenparamagnetspektroskopie hat ihren Weg in fast jedes wissenschaftliche Feld gefunden. Wohingegen jedoch die meisten Methoden, die so fachübergreifend Verwendung finden, durch breite Anwendbarkeit und einfache Nutzung charakterisiert sind, ist es bei EPR eher umgekehrt. Unzählige Experimente mit unzähligen Variationen je nach Probe. Und alle zeigen einen etwas anderen Ausschnitt des Gesamtbildes. Diese Unterschiede können genutzt werden, um Eigenschaften komplizierter Systeme mithilfe verschiedener Experimente und Parameter auseinander zu ziehen. Diese Arbeit demonstriert, wie ein kleines Set von EPR-Methoden genutzt werden kann, um in verschiedenen Systemen sonst nur schwierig zugängliche Informationen zu extrahieren.
Obwohl sie wohl unterschiedlicher nicht sein könnten, sind sowohl Ribonukleotidreduktase (RNR) als auch Guanin-Quadruplexe (GQs) instrumental für Leben, wie wir es kennen. Wo die Rolle von RNR darin besteht, als einziges Enzym neue Desoxyribonukleotide, die Bausteine für DNS, herzustellen, ist die Rolle von GQs durch ihre Struktur in DNS und Interaktion mit der Umgebung und anderen GQs gekennzeichnet. Oft spielen diese Interaktionen wichtige Rollen in der Kontrolle und Regulation des Genoms.
Diese Arbeit charakterisiert die β₂-Untereinheit von Klasse Ia RNR, welche ein Tyrosilradikal generiert, was für die Aktivität von RNR essentiell ist. Im Genaueren beschäftigt sich die Arbeit damit, wie sich der zur Radikalgenerierung benötigte Dieisen-Kofaktor bildet, beziehungsweise was passiert, wenn β₂ fälschlicherweise Mangan bindet. Hierfür wurden sowohl mehrere dipolare Spektroskopie-Methoden als auch feldabhängige Messungen kombiniert, um die globalen Bindungsverhältnisse von Mangan in β₂ zu charakterisieren. In diesem Zusammenhang wird eine Fehlinterpretation in der Literatur aufgedeckt, die nur durch Kombination aller dieser Methoden ersichtlich wird. Die zentrale Rolle von RNR in der Zelle macht das Verständnis des Mechanismus von RNR zu einem sehr wichtigen Thema.
Des Weiteren wird in dieser Arbeit das Bindeverhalten von GQs mit sich selbst und mit Interkalatoren untersucht. Diese Arbeit erweitert die vorherige Arbeit mit Kupferlabeln für GQs, welche höchst präzise Distanzmessungen ermöglichen. Dazu wurde ein neues Messschema entwickelt, welches die starke Orientierungsselektivität von dipolaren Distanzmessungen in mit Kupfer gelabelten GQs ausnutzt, um über quantitative Messungen die Dynamik der Systeme zu charakterisieren. Hierbei werden die Einflüsse von Orientierungselektivität auf Modulationsamplitude und quantitative Messungen ausführlich diskutiert. Dies erlaubte, einen unerwarteten Effekt, den das Binden des Interkalators PIPER auf die Stabilität von GQ-dimeren hat, zu ermitteln. Des Weiteren wurde eine schalterartige Änderung im Bindungsmodus von PIPER entdeckt. Diese Forschung legt die Grundlage für die Nutzung von kupfergelabelten GQs jenseits ihrer Anwendung als Modellsystem für EPR-Distanzmessungen, als aktiver Reporter in der Entwicklung von neun Medikamenten.
Modern Electron Paramagnetic Resonance (EPR) has found applications in nearly every field of science. However, while many methods spread into other fields through their high applicability and ease of use, EPR tends to be the opposite. Countless experiments with countless variations for different samples, all requiring different considerations and giving slightly different parts of the full picture. These differences between the experiments can be leveraged to untangle complex systems by combining different EPR techniques. This thesis shows how a small group of EPR experiments can be used in entirely different paradigms to extract otherwise difficult-to-collect data. Although they are as different as can be, both ribonucleotide reductase (RNR) and guanine quadruplexes (GQs) form cornerstones of life as we know it. Where RNR’s role is catalytic, being the only enzyme capable of creating new deoxynucleotides, the building blocks of DNA, the function of guanine quadruplexes is primarily in their structure, and the interaction of that structure with its surroundings, often to create regulatory effects in the genome. This thesis examines the β₂-subunit of class Ia RNR, which generates a tyrosyl radical necessary for subsequent RNR activity. Specifically, the generation of the necessary di-iron cofactors, or more precisely, what happens when manganese is incorporated in place of iron, is studied. It combines multiple dipolar spectroscopy methods as well as different types of field-sweeps to gain information on global binding modes of manganese, uncovering a previous misinterpretation of data by combining the results of all these methods. Given the central importance RNR has in cells, deepening our understanding of how RNR functions is a top priority. Furthermore, this thesis examines the interaction of GQ-dimers and the binding of intercalators within GQs. This work builds on previous work with highly rigid copper labels for GQs, which can be used for high-precision dipolar distance measurements. To study the dynamics of GQ systems, a new measurement scheme for quantitative dipolar distance measurements is created, which exploits the large orientation selection of copper rather than recording complete orientation-averaged spectra. To this end, the implications and treatment of orientation selection as it influences quantitative dipolar spectroscopy measurements are discussed for different types of measurements in different systems. The application of the new measurement scheme was able to uncover an unexpected effect of the intercalator PIPER onto GQ-dimers, as well as a switch-like behavior between different binding modes not seen before. It furthermore lays the foundation for the usage of copper-labeled GQs not only as an EPR ruler but also as playing an active role in future drug development.
Modern Electron Paramagnetic Resonance (EPR) has found applications in nearly every field of science. However, while many methods spread into other fields through their high applicability and ease of use, EPR tends to be the opposite. Countless experiments with countless variations for different samples, all requiring different considerations and giving slightly different parts of the full picture. These differences between the experiments can be leveraged to untangle complex systems by combining different EPR techniques. This thesis shows how a small group of EPR experiments can be used in entirely different paradigms to extract otherwise difficult-to-collect data. Although they are as different as can be, both ribonucleotide reductase (RNR) and guanine quadruplexes (GQs) form cornerstones of life as we know it. Where RNR’s role is catalytic, being the only enzyme capable of creating new deoxynucleotides, the building blocks of DNA, the function of guanine quadruplexes is primarily in their structure, and the interaction of that structure with its surroundings, often to create regulatory effects in the genome. This thesis examines the β₂-subunit of class Ia RNR, which generates a tyrosyl radical necessary for subsequent RNR activity. Specifically, the generation of the necessary di-iron cofactors, or more precisely, what happens when manganese is incorporated in place of iron, is studied. It combines multiple dipolar spectroscopy methods as well as different types of field-sweeps to gain information on global binding modes of manganese, uncovering a previous misinterpretation of data by combining the results of all these methods. Given the central importance RNR has in cells, deepening our understanding of how RNR functions is a top priority. Furthermore, this thesis examines the interaction of GQ-dimers and the binding of intercalators within GQs. This work builds on previous work with highly rigid copper labels for GQs, which can be used for high-precision dipolar distance measurements. To study the dynamics of GQ systems, a new measurement scheme for quantitative dipolar distance measurements is created, which exploits the large orientation selection of copper rather than recording complete orientation-averaged spectra. To this end, the implications and treatment of orientation selection as it influences quantitative dipolar spectroscopy measurements are discussed for different types of measurements in different systems. The application of the new measurement scheme was able to uncover an unexpected effect of the intercalator PIPER onto GQ-dimers, as well as a switch-like behavior between different binding modes not seen before. It furthermore lays the foundation for the usage of copper-labeled GQs not only as an EPR ruler but also as playing an active role in future drug development.
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Keywords
EPR Spektroskopie, G-Quadruplex, Ribonukleotidreduktase, Physikalische Chemie
Subjects based on RSWK
EPR-Spektroskopie, G-Quadruplex