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Untersuchung Der Putativen Substratbindungsstelle Von Untersuchung der putativen Substratbindungsstelle von Escherichia coli Parvulin 10 Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Naturwissenschaftlichen Fakultät I – Biowissenschaften – der Martin-Luther-Universität Halle-Wittenberg, vorgelegt von André Diessner geb. am 01.06.1982 in Halle (Saale) Promotion eingereicht am: 07.12.2016 Tag der öffentlichen Verteidigung: 29.05.2017 Gutachter: Prof. Dr. Frank Bordusa Prof. Dr. Mike Schutkowski Prof. Dr. Peter Bayer Meiner Familie. Wenn wir wüssten, was wir tun, würden wir das nicht Forschung nennen. Albert Einstein (1879-1955) Abstract Keywords: Peptidyl-Prolyl-cis/trans-Isomerase (PPIase), Parvulin 10, Alkyl hydroperoxide reductase subunit C (AhpC), pKa, semisynthesis, peptide segment condensation, native chemical synthesis, enzymatic ligation, fluorescent labeling This thesis has a focus on Escherichia coli Parvulin 10 (Par10), the founding member of the parvulin enzyme family, a class of peptidyl-prolyl-cis/trans-isomerases (PPIases). As the prototypical member of the parvulin family, Par10 is particularly suitable to answer basic questions pertaining to substrate specificity and catalytic mechanism of these enzymes. Par10 catalyzes the interconversion of cis and trans conformers of prolyl bonds (Xaa-Pro bonds) in peptides and proteins. Furthermore, some PPIases exhibit chaperone activity and cell regulation functions, which are often mediated by additional protein domains besides the PPIase domain. Members of human Parvulins, e. g. human Pin1 (hPin), are associated with a variety of diseases, including breast cancer, PARKINSON's and ALZHEIMER's disease. This work was thought to contribute to a better understanding of the mode of operation of parvulins by using structural studies and by providing novel, chemically modified parvulin species. The interaction between parvulin 10 and its, so far, only known natural interaction partner, the alkyl hydroperoxide reductase subunit C (AhpC), has been further investigated in this study and has been examined at the atomic level for the first time by means of nuclear magnetic resonance spectroscopy. The direct binding of a partial amino acid sequence of AhpC to the catalytic site of Par10 could be shown as well as the enzymatic conversion of an AhpC segment by Par10. For a long time a highly conserved cysteine residue in parvulins has been a subject of intense studies. Results of the here presented study show significantly lowered pKa values for both cysteine side chains in Par10 compared to cysteines in short, unstructured peptides and a great influence of Cys40 on Par10's overall protein structure. Additionally, more in vitro and in vivo studies are required to understand how Par10 recognizes prolyl bonds in proteins and catalyzes its cis/trans conversion. Beside the interaction and structural studies a vast number of methods were used to provide catalytically active and site-specific fluorescence-labeled Par10 molecules, thus laying the foundation for further inhibition and protein-ligand studies. Kurzzusammenfassung Schlüsselwörter: Peptidyl-Prolyl-cis/trans-Isomerase (PPIase), Parvulin 10, Alkylhydroperoxid- Reduktase Untereinheit C (AhpC) pKS, Semisynthese, Pepetidsegmentkondensation, Natürlich Chemische Ligation, Enzymatische Ligation, Fluoreszenzmarkierung Diese Arbeit befasst sich mit Parvulin 10 (Par10) aus Eschericia coli, dem Namensgeber der Peptidyl- Prolyl-cis/trans-Isomerase-Familie der Parvuline. Als prototypischer Vertreter dieser Enzymfamilie ist Par10 besonders geeignet, um grundlegende Fragen bezüglich der Substratspezifität und des Katalysemechanismus dieser Enzyme zu beantworten. Par10 katalysiert die Umwandlung der cis- und trans-Konformere von Prolylbindungen (Xaa-Pro-Bindungen) in Peptiden und Proteinen. Darüber hinaus besitzen einige Parvuline Chaperonaktivität und Zellzykluskontrollfunktionen, welche oft durch zusätzliche Proteindomänen neben der PPIase-Domäne vermittelt werden. Vertreter humaner Parvuline, wie zum Beispiel humanes Pin1 (hPin), werden mit einer Vielzahl von Erkrankungen, wie dem Brustkrebs, der PARKINSON- und der ALZHEIMER-Krankheit, in Verbindung gebracht. Diese Arbeit sollte durch Strukturuntersuchungen und durch die Bereitstellung neuartiger, chemisch modifizierter Parvulinspezies zum Verständnis der Funktionsweise der Parvuline beitragen. Die Interaktion zwischen Parvulin 10 und dem bislang einzig bekannten natürlichen Inter- aktionspartner, der Alkylhydroperoxid Reduktase Untereinheit C (AhpC), wurde in dieser Arbeit ein- gehender und erstmalig auf atomarer Ebene mittels der Kernspinresonanzspektroskopie untersucht. Es konnte eine direkte Bindung eines AhpC-Segments an das katalytische Zentrum von Par10 und die enzymatische Umsetzung dieses Segments durch Par10 gezeigt werden. Seit langem ist ein in Parvulinen hoch konservierter Cysteinrest immer wieder Ziel intensiver Studien. Ergebnisse der vorliegenden Arbeit zeigen deutlich abgesenkte pKS-Werte für die beiden in Par10 vorkommenden Cysteinseitenketten verglichen mit Cysteinseitenketten in kurzen, unstruk- turierten Peptiden und einen starken Einfluss des Cys40 auf die Raumstruktur von Parvulin 10. Um zu verstehen, wie Par10 Prolylbindungen in Proteinen erkennt und ihre cis/trans-Isomerisierung katalysiert, sind weitere in vitro und in vivo Studien notwendig. Neben Interaktions- und Strukturstudien wurde eine Vielzahl von Methoden für die Bereit- stellung katalytisch aktiver und positionsspezifisch fluoreszenzmarkierter Par10-Moleküle ange- wendet und damit die Grundlage für tiefergehende Inhibierungs- und Protein-Ligand-Studien geschaffen. I INHALTSVERZEICHNIS I Inhaltsverzeichnis I Inhaltsverzeichnis .................................................................................... I II Abkürzungsverzeichnis ........................................................................... IV 1 Einleitung ............................................................................................... 1 1.1 Enzyme – die Biokatalysatoren des Lebens ........................................................................ 1 1.2 Prolylbindungen und Peptidyl-Prolyl-cis/trans-Isomerasen ............................................... 2 1.3 Parvulin 10 und die Familie der Parvuline .......................................................................... 5 1.4 Alkylhydroperoxid-Reduktase Untereinheit C .................................................................. 11 1.5 Gewinnung und Modifizierung von Proteinen ................................................................. 13 2 Problemstellung und Zielsetzung .......................................................... 16 3 Materialien und Methoden ................................................................... 18 3.1 Liste der Hersteller und Zulieferer .................................................................................... 18 3.2 Materialien ........................................................................................................................ 19 3.2.1 Chemikalien....................................................................................................................... 19 3.2.2 Verbrauchsmaterialien ..................................................................................................... 20 3.2.3 PCR-Primer, Plasmide und E. coli-Stämme ....................................................................... 20 3.2.4 Enzyme .............................................................................................................................. 21 3.2.5 Nährmedien, Stammlösungen und Puffer ........................................................................ 22 3.2.6 Standards und Reagenzienzusammenstellungen ............................................................. 22 3.2.7 Säulenmaterialien ............................................................................................................. 23 3.2.8 Allgemeine Geräte ............................................................................................................ 23 3.2.9 Computerprogramme und Datenbanken ......................................................................... 24 3.3 Molekularbiologische Methoden ...................................................................................... 25 3.3.1 Polymerasekettenreaktion ............................................................................................... 25 3.3.2 Enzymatische Modifizierung von DNA .............................................................................. 25 3.3.3 Auftrennung, Isolierung und Reinigung von DNA ............................................................. 25 3.3.4 Transformation von E. coli-Zellen mit Plasmid-DNA ......................................................... 26 3.3.5 DNA-Sequenzierung .......................................................................................................... 26 3.3.6 Anlegen von Glycerinkryokulturen ................................................................................... 26 3.3.7 Kultivierung von E. coli-Stämmen und Genexpression ..................................................... 26 3.4 Präparative Methoden ...................................................................................................... 27 3.4.1 Zellaufschluss, Proteinreinigung und Proteinkonzentrierung .......................................... 27 3.4.2 Intein-vermittelte Peptidthioestersynthese ..................................................................... 29 3.4.3
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