<p>Multidrug tolerance (MDT) is the ability of pathogenic bacteria to survive killing from exposure to multiple antibiotics, and is a major obstacle in the treatment of infectious disease. A small population of bacteria (0.0001%) termed persisters is the culprit that causes MDT and allows these cells to persist. In <italic>Escherichia coli</italic>, the HipBA toxin&ndash;antitoxin pair plays a role in multidrug tolerance. HipA, a 50 kDa serine protein kinase, is the more stable toxin and abrogates cell growth in the absence of the more labile antitoxin HipB. HipB is a transcription repressor that binds to the four conserved (TATCCN₈GGATA) operator sites of the <italic>hipBA</italic> promoter to autoregulate expression of the <italic>hipBA</italic> operon. Delineation of the molecular mechanism of HipB&ndash;<italic>hipBA</italic>operator binding is critical to understand fully the regulation of persistence by HipB. Thus, we determined the equilibrium dissociation constants (K_d) of HipB for each of the four <italic>hipBA</italic> operators and the paired operator sites <italic>O₁O₂</italic> and <italic>O₃O₄</italic>. We found that the affinity of HipB for binding the <italic>O₁</italic> and <italic>O₃</italic> operators is seven to eight times higher than for the <italic>O₂</italic> and <italic>O₄</italic> operators. In addition, the affinity of HipB for the <italic>O₁O₂</italic> and <italic>O₃O₄</italic> operators is at least four times higher than the <italic>O₁</italic> and <italic>O₃</italic> operators. The HipB&ndash;operator complex structures reveal that HipB makes the same key contacts to the conserved TATCC motifs and bends each operator DNA by the same extent between 50&deg; to 70&deg; implying thus the affinity differences are attributed to indirect readout of the 8 base pair spacer (N₈). Mutational studies on residues involved in HipB&ndash;DNA interaction revealed the contribution of a series of selected residues to binding affinity with residues K38 and Q39 contributing greatly to affinity whereas other base contacting residues S29 and A40 contribute less to affinity. Surprisingly residue S43, which is involved in a hydrogen bond to the DNA phosphate backbone contributes more than expected because S43 forms a hydrogen bond network with nearby water molecules. </p><p>HipA was the first described <italic>bona fide</italic> persistence factor. The <italic>hip</italic> locus was discovered through a mutagenesis screen whereby <italic>hipA7</italic> was isolated. Described herein, biochemical and structure&ndash;function studies on HipA7, the gene product of the high persistent mutant allele having two point mutations G22S and D291A, revealed that the D291A mutation weakens the binding affinity for HipB by 3 to 4 fold. The HipA7 structure revealed the conformational heterogeneity of the P&ndash;loop motif (the ATP binding motif), which suggests a dynamic role of the loop in regulation of the kinase activity of HipA. To identify <italic>in vivo</italic> HipA substrates, we developed a mass spectrometry (MS)&ndash;based kinase assay, which led to identification of a novel phosphorylation site (residue S348) on HipA and a proposed consensus phosphorylation motif +&#981;S, where +, &#966; and S designate a positive, hydrophobic and serine amino acid residue, respectively. Phosphorylation of peptides with this consensus motif, including the S150 (EENDF<bold><underline>RIS</underline></bold>VAGAQEK), S348 (TGI<bold><underline>HIS</underline></bold>DLK) and GltX (GK<bold><underline>KLS</underline></bold>KRH), was confirmed subsequently by the MS&ndash;based kinase assay. Further analysis of the HipA7 structure suggested that HipA might undergo pyrophosphorylation on residue S150, and the MS&ndash;based kinase assay confirmed pyrophosphorylation of HipA. </p><p>Thus, our data support that HipA is a persistence factor via its kinase activity and precise <italic>hipBA</italic> gene regulation through HipB binding tightly to <italic>O₁</italic> and <italic>O₃</italic> is critical for the survival of bacteria in the presence of antibiotics. In addition, we propose a consensus motif for HipA substrates.</p> / Dissertation

Post-translationale Modifikationen (PTMs) sind wesentlich für die Regulierung von zellulären Mechanismen. Um diese Prozesse besser zu verstehen, ist es essentiell Methoden für deren Erforschung zu entwickeln. In dieser Arbeit wurden zwei chemoproteomische Ansätze entwickelt, um die PTMs, Proteinphosphorylierung und Proteinpyrophosphorylierung zu untersuchen. Die Proteom-weite Erforschung von Proteinphosphorylierungen beruht gewöhnlich auf der LC-MS/MS-Analyse von enzymatisch verdauten Proteomen und da die Phosphorylierung von niedriger Abundanz ist, wird ein Phosphopeptid-Anreicherungsschritt benötigt. Die Identifizierung von bestimmten Phosphopeptiden ist allerdings abhängig von der gewählten Anreicherungsmethode. Die Entwicklung von neuen Prozeduren ist daher bedeutsam, um neue Phosphorylierungsstellen zu identifizieren. Im ersten Projekt wurde eine milde und selektive Phosphopeptid-Anreicherungsmethode entwickelt und optimiert. Die Methode zeigte die Fähigkeit Phosphopeptide anzureichern und somit das Potential, das Repertoire der vorherigen Methoden zu erweitern, um neue Phosphorylierungsstellen zu identifizieren. Proteinpyrophosphorylierung ist eine unlängst identifizierte PTM, die nicht-enzymatisch an Proteine angefügt wird und es ist nur wenig ist über ihre Funktion bekannt. Vorherige Studien wiesen darauf hin, dass diese Modifikation enzymatisch entfernt wird, allerdings sind die verantwortlichen Enzyme („Proteinpyrophosphatasen“) nicht bekannt. Hier wurde eine Peptidaffinitätsmethode entwickelt, um potentielle Pyrophosphatasen und weitere interagierende Proteine aus humanen Zellen zu identifizieren. Damit wurden 6 Phosphatasen als potentielle Pyrophosphatase-Kandidaten identifiziert und weitere interagierende Proteine gaben Aufschlüsse über die Funktion der Proteinpyrophosphorylierung. Dadurch wurde das Potential der Methode aufgezeigt, interagierende Proteine der Proteinpyrophosphorylierung zu identifizieren, um die zelluläre Rolle zu verstehen. / Post-translational modifications (PTMs) are crucial for the regulation of cellular mechanisms. To better understand these processes, the development of chemical tools to investigate them is of high importance. In this thesis, two chemoproteomics approaches were established to investigate the PTMs protein phosphorylation and protein pyrophosphorylation. The proteome-wide study of protein phosphorylation usually relies on LC-MS/MS analysis of enzymatically digested proteomes, requiring a phosphopeptide enrichment step, due to the low abundance of phosphorylation. However, the identification of certain sets of phosphopeptides is dependend on the choice of enrichment method. Therefore, the development of new workflows is important to identify new phosphorylation sites. In the first project, a mild and selective phosphopeptide enrichment method was developed and optimized. The method was able to enrich phosphopeptides and therefore, showed the potential to complement the repertoire of current methods to identify new phosphorylation sites. Protein pyrophosphorylation is a recently discovered PTM, which is non-enzymatically attached to proteins and there is only sparse knowledge about the function. Previous studies have indicated the enzymatic removal of this modification, but the responsible enzymes (‘protein pyrophosphatases’) are unknown. Here, a peptide affinity capture method was developed to identify potential pyrophosphatases and further interacting proteins from human cells. Therewith, 6 phosphatases were identified as potential pyrophosphatase candidates and further interacting proteins gave insights into the function and mechanisms of protein pyrophosphorylation. Thereby, the potential of this method was demonstrated to identify interacting proteins of protein pyrophosphorylation to understand the cellular role.

Kovalente posttranslationale Modifikationen (PTMs) beeinflussen die Struktur und Funktion von Proteinen. Zu den bedeutendsten PTMs zählt die Proteinphosphorylierung. Labile Phosphorylierungen an Cystein- und Lysinresten, sowie Pyrophosphorylierungen an Serin- und Threoninbausteinen sind vermehrt in den Fokus der Wissenschaft gerückt. Trotz großer Fortschritte auf dem Gebiet der Massenspektrometrie (MS) bleibt die Analyse dieser empfindlichen Modifikationen mittels Tandem-MS eine große Herausforderung. In der vorliegenden Arbeit wird gezeigt, dass Elektronentransferdissoziation (ETD) in Kombination mit zusätzlicher HCD Aktivierung (EThcD) in der Lage ist, Peptide mit labilen Phosphorylierungen in der Seitenkette unter Erhalt der Modifikation zu fragmentieren. In verschiedenen proteomischen Ansätzen wird demonstriert, dass EThcD eine zweifelsfreie Identifizierung natürlich vorkommender Cysteinphosphorylierungen ermöglicht. Darüber hinaus wurde unter dem Gesichtspunkt der Labilität von Lysinphosphorylierungen ein bottom-up-Phosphoproteomikansatz etabliert. Das MS-Verfahren beruht auf der Generierung eines diagnostischen Phospholysinimmoniumions, welches im zweiten Schritt die Erfassung eines zusätzlichen EThcD-Spektrums desselben Precursorions veranlässt (triggert). Darüber hinaus wird im Zuge dieser Arbeit gezeigt, dass sich pyrophosphorylierte Peptide unter CID-Bedingungen in ihrem Neutralverlustmuster von isobaren diphosphorylierten Peptiden unterscheiden. Dieses Verhalten stellt einen Schlüsselschritt in einer neutralverlustgetriggerten EThcD Methode dar, welche die zweifelsfreie Identifizierung von Pyrophosphorylierungen ermöglicht. Darauf basierend konnten in Hefezellen und humanen embryonalen Nierenzellen die ersten Proteinpyrophosphorylierungen, einer neuen endogenen posttranslationalen Modifikation, nachgewiesen werden. / Covalent posttranslational modifications (PTMs) influence the structure and function of proteins. Protein phosphorylations belong to the most important PTMs. Rarely characterized labile phosphorylations, for instance phosphorylations of cysteine and lysine and pyrophosphorylations of serine and threonine residues got into the focus of science. However, the analysis of those delicate modifications via tandem mass spectrometry remains a challenge. In the present work, it is shown that electron-transfer dissociation (ETD) combined with HCD supplemental activation (EThcD) is able to fragment peptides with labile phosphorylations at the side chains without losing the modification. In several bottom-up proteomic approaches, EThcD allowed the reliable identification of a naturally occurring cysteine phosphorylation. Moreover, methods for identification of lysine phosphorylations were developed. For the proteome wide analysis of lysine phosphorylations, considering the lability, a bottom-up phosphoproteomic approach with a highly selective mass spectrometry method was established. The MS-method relies on the generation of diagnostic phospholysine immonium ions during HCD, which trigger in a second step an additional EThcD spectrum of the same precursor ion. This strategy ensures the confident identification of lysine phosphorylated peptides. Furthermore, the present work shows that isobaric pyro- and diphosphorylated peptides differ in their neutral loss pattern during CID. This behavior was a key step in a specific neutral loss triggered EThcD method, which enabled the reliable identification of pyrophosphorylations. This method allowed the identification of the first protein pyrophosphorylations, a new endogenous PTM, in yeast and human embryonic kidney cells.