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Strukturelle Analyse Der Dynamischen Konformationsänderung Des Prion-Proteins in Proteinmissfaltungskrankheiten

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Strukturelle Analyse Der Dynamischen Konformationsänderung Des Prion-Proteins in Proteinmissfaltungskrankheiten Strukturelle Analyse der dynamischen Konformationsänderung des Prion-Proteins in Proteinmissfaltungskrankheiten DISSERTATION zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.) Eingereicht im Fachbereich Chemie der Fakultät für Mathematik, Informatik und Naturwissenschaften an der Universität Hamburg vorgelegt von Anne Sommer Hamburg 2015 Die praktischen Arbeiten wurden im Zeitraum von Februar 2010 bis September 2014 in der Nachwuchsgruppe von Dr. Lars Redecke im Laboratorium für Strukturbiologie von Infektion und Entzündung der Universitäten Hamburg und Lübeck sowie am Institut für Biochemie und Molekularbiologie des Fachbereichs Chemie der Universität Hamburg in den Laboratorien der Arbeitsgruppe von Prof. Dr. Christian Betzel durchgeführt. 1. Gutachter: Prof. Dr. Ch. Betzel 2. Gutachter: Prof. Dr. B. Meyer Tag der Disputation: 07.08.2015 I. Inhaltsverzeichnis I I. Inhaltsverzeichnis II. Abkürzungsverzeichnis ...................................................................................................... V III. Zusammenfassung ........................................................................................................... IX IV. Summary ........................................................................................................................... XI 1. Einleitung .............................................................................................................................. 1 1.1. Prionen-Erkrankungen .................................................................................................... 2 1.2. Der Erreger und die „Protein-only“-Theorie ................................................................... 4 1.3. Struktur und Funktion des Prion-Proteins ....................................................................... 5 1.4. Aggregation des Prion-Proteins ...................................................................................... 8 1.5. Therapie von Prionen-Erkrankungen ............................................................................ 11 1.6. Dynamische Lichtstreuung (DLS) ................................................................................ 13 2. Zielsetzung der Doktorarbeit ............................................................................................ 15 3. Ergebnisse ........................................................................................................................... 17 3.1. Klonierung der Prion-Protein-Konstrukte ..................................................................... 18 3.2. Optimierung der Prion-Protein-Herstellung .................................................................. 19 3.2.1. Rekombinante Genexpression in E. coli ................................................................ 19 3.2.2. Rückfaltung, Reinigung und Entfernung der Hexahistidinsequenz ....................... 20 3.3. Charakterisierung der Prion-Proteine ............................................................................ 23 3.3.1. CD-spektroskopische Analyse ............................................................................... 24 3.3.2. Hydrodynamische Radien und Proteinase K Sensitivität ....................................... 24 3.3.3. Bestimmung der Schmelzpunkte ............................................................................ 28 3.3.4. Strukturelle Charakterisierung von hPrP90-230+EFEA mit Hexahistidinsequenz in Lösung .............................................................................................................................. 31 3.4. Oxidativ-induzierte Umwandlung ................................................................................. 33 3.4.1. Validierung des bestehenden UV-Versuchsaufbaus .............................................. 33 3.4.1.1. Validierung des integrierten Online-DLS-Systems ......................................... 34 3.4.1.2. Anpassung der UV-Bestrahlungsparameter .................................................... 39 3.4.1.3. Charakterisierung der entstandenen oligomeren Spezies ................................ 42 3.4.1.4. Einfluss der Laserleistung auf die UV-induzierte Prion-Protein-Umwandlung ...................................................................................................................................... 48 3.4.2. Herstellung der intermediären Zwischenzustände ................................................. 55 3.4.2.1. Produktionsläufe im Kreislaufsystem ............................................................. 58 3.4.2.2. Produktionsläufe im Durchflusssystem ........................................................... 59 3.4.2.3. Isolierung des „dimeren“ Zwischenzustandes ................................................. 61 I. Inhaltsverzeichnis II 3.4.2.4. Charakterisierung des Prion-Spezies-Gemisches ............................................ 63 3.4.3. Metallkatalysierte oxidative Umwandlung ............................................................ 69 3.4.3.1. Analyse der Kupferbindung nach metallkatalysierter Umwandlung .............. 70 3.5. Inhibition der oxidativen Umwandlung von Prion-Proteinen ....................................... 73 3.5.1. Inhibition der metallkatalysierten oxidativen Umwandlung .................................. 73 3.5.1.1. Bestimmung der Peptid-Bindungsaffinitäten mittels Oberflächenplasmonen- resonanz (SPR)-Spektroskopie ..................................................................................... 79 3.5.1.2. Untersuchung des Faltungszustandes der PrP-Peptid-Komplexe nach metall- katalysierter Oxidation ................................................................................................. 81 3.5.2. Untersuchungen zur Inhibition der UV-induzierten Umwandlung ........................ 82 3.6. Kristallisation ................................................................................................................ 83 3.6.1. In-vivo-Kristallisation ............................................................................................. 87 4. Diskussion ........................................................................................................................... 89 4.1. UV-Bestrahlungssystem mit integriertem Online-DLS-System ................................... 90 4.2. Anwendung des UV-Bestrahlungssystems zur Induktion der Umwandlung und Aggregation des Prion-Proteins ........................................................................................... 91 4.3. Das Prion-Protein als Übergangsmetall-bindendes Protein .......................................... 96 4.4. Einfluss des GPI-Ankers auf die Umwandlung des Prion-Proteins .............................. 97 4.5. Inhibition der Prion-Protein-Umwandlung und Aggregation ....................................... 98 4.6. Kristallisationsversuche .............................................................................................. 102 4.7. Ausblick ...................................................................................................................... 104 5. Material und Methoden ................................................................................................... 107 5.1. Material ....................................................................................................................... 107 5.1.1. Geräte ................................................................................................................... 107 5.1.2. Verbrauchsmaterialien ......................................................................................... 109 5.1.3. Chemikalien ......................................................................................................... 110 5.1.4. Kommerzielle Kristallisationslösungen, Kits und Enzyme .................................. 111 5.1.5. DNA und Proteingrößenmarker ........................................................................... 111 5.1.6. Plasmide und Oligonukleotide ............................................................................. 111 5.1.7. Bakterienstämme .................................................................................................. 114 5.1.8. Insektenzellen ....................................................................................................... 115 5.1.9. Medien, Puffer und Lösungen .............................................................................. 115 5.2. Methoden ..................................................................................................................... 119 5.2.1. Molekularbiologische Methoden .......................................................................... 119 I. Inhaltsverzeichnis III 5.2.1.1. Polymerase-Kettenreaktion (PCR) ................................................................ 119 5.2.1.2. Agarose-Gelelektrophorese ........................................................................... 120 5.2.1.3. Reinigung von PCR-Fragmenten .................................................................. 120 5.2.1.4. Restriktionsspaltung ...................................................................................... 120 5.2.1.5. Bestimmung von DNA-Konzentrationen ...................................................... 121 5.2.1.6. Ligation ......................................................................................................... 121 5.2.1.7. Herstellung chemisch kompetenter Zellen .................................................... 121 5.2.1.8. Transformation von E. coli-Zellen ...............................................................
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