Method development and state of the art EPR techniques for new insights into apoptosis

Diese Arbeit strebt danach eine Brücke zwischen Elektronenspinresonanz (EPR) -Methodenentwicklung und der Anwendung modernster EPR-Techniken auf ein komplexes Proteinsystem zu bauen. Die Sensitivität und Signaltreue des Doppel-Elektron-Elektron-Resonanz (DEER) Experiments kann durch die Verwendung von gaußförmigen Pulsen erheblich verbessert werden. Außerdem wird experimentell gezeigt wie Signalübersprechen zwischen DEER Kanälen in Proben entsteht, die orthogonal mit Nitroxiden und Gadolinium spinmarkiert sind. Dazu werden Identifikations- und Unterdrückungsstrategien eingeführt und diskutiert. Die Anwendung von orthogonalen Spinmarkierungsstrategien zur Erforschung der Bcl-2-Familie, die der primäre Regulator des mitochondrialen Pfades der Apoptose ist, bietet neue Einblicke in das Bcl-2-Interaktom. Dem Bestreben folgend Proteine in physiologischeren Umgebungen zu messen, wird untersucht, wie geeignete Protein/Spinmarkierungs-Kombinationen für In-Zell-Studien gefunden werden können.This thesis strives to span a bridge between electron paramagnetic resonance (EPR) method development and the application of state of the art EPR techniques to a complex protein system. Double electron-electron resonance (DEER) sensitivity and signal fidelity can be considerably improved by the utilization of Gaussian pulses that allow to remove the "2+1" pulse train artifact. Moreover, it is shown experimentally how DEER channel cross-talk signals appear in samples that are orthogonally spin-labeled with nitroxide and gadolinium labels. Both identification and suppression strategies are introduced and discussed. The introduction of orthogonal spin labeling strategies to the Bcl-2 family, which is the primary regulator of the mitochondrial pathway of apoptosis, offers new insights into the Bcl-2 interactome. Following the aspiration to measure proteins in more physiological environments, it is addressed how suitable protein/spin label combinations can be found for in-cell studies

Strategies to identify and suppress crosstalk signals in double electron–electron resonance (DEER) experiments with gadoliniumIII^{III} and nitroxide spin-labeled compounds

Double electron-electron resonance (DEER) spectroscopy applied to orthogonally spin-labeled biomolecular complexes simplifies the assignment of intra- and intermolecular distances, thereby increasing the information content per sample. In fact, various spin labels can be addressed independently in DEER experiments due to spectroscopically nonoverlapping central transitions, distinct relaxation times, and/or transition moments; hence, they are referred to as spectroscopically orthogonal. Molecular complexes which are, for example, orthogonally spin-labeled with nitroxide (NO) and gadolinium (Gd) labels give access to three distinct DEER channels that are optimized to selectively probe NO–NO, NO–Gd, and Gd–Gd distances. Nevertheless, it has been previously recognized that crosstalk signals between individual DEER channels can occur, for example, when a Gd–Gd distance appears in a DEER channel optimized to detect NO–Gd distances. This is caused by residual spectral overlap between NO and Gd spins which, therefore, cannot be considered as perfectly orthogonal. Here, we present a systematic study on how to identify and suppress crosstalk signals that can appear in DEER experiments using mixtures of NO–NO, NO–Gd, and Gd–Gd molecular rulers characterized by distinct, nonoverlapping distance distributions. This study will help to correctly assign the distance peaks in homo- and heterocomplexes of biomolecules carrying not perfectly orthogonal spin labels