Dokument: Untersuchung zu Struktur-Funktionsbeziehungen von Membranproteinen mittels Kraftspektroskopie
| Titel: | Untersuchung zu Struktur-Funktionsbeziehungen von Membranproteinen mittels Kraftspektroskopie | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=15849 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20100830-103549-3 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Deutsch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Oberbarnscheidt, Leoni [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Oesterhelt, Filipp [Gutachter] Prof. Dr. Schmitt, Lutz [Gutachter] Prof. Dr. Wachtveitl, Josef [Gutachter] | |||||||
| Stichwörter: | Kraftspektroskopie, Einzelmolekülspektroskopie, Sensorrhodopsin | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 540 Chemie | |||||||
| Beschreibungen: | Membranproteine spielen eine wichtige Rolle zur Aufrechterhaltung vieler essentieller Bedingungen in der Zelle und ihre Struktur-Funktionsbeziehungen sind von großem Interesse um ihre Wirkungsweise zu verstehen. Mittels Rasterkraftspektroskopie lassen sich sowohl die inter- und intramolekularen Kräfte eines Proteins in der Membran, als auch die Bindungsraten von Rezeptor – Ligand Interaktionen bestimmen, so dass sie sich als geeignete Technik erweist, die gängigen strukturbiologischen Untersuchungen zu ergänzen.
Im ersten Teil dieser Arbeit wurde durch Entfaltungsexperimente an Sensorhodopsin II der Einfluss von Lichtaktivierung und Komplexbildung auf seine inter- und intramolekularen Kräfte untersucht. Hierzu wurde die Analysemethode zur Bestimmung der Stabilität einer Bindung oder Proteinregion weiter entwickelt. Ein Ansatz ist es, die Durchschnittskraft der Stabilität einer Proteinregion anzugeben, indem die Daten der Einzelmolekülmessung als Ensemble-Messung betrachtet werden. In einem anderen Ansatz wurde die Stabilität einer Proteinregion über die Abrisshäufigkeit einer Bindung in dieser Region bestimmt und die Auflösung der Histogramme erhöht. Beide Ansätze identifizieren Regionen, in denen sich die Stabilität in Abhängigkeit von Lichtaktivierung und Komplexbildung ändert, was dazu beiträgt, den Reaktionsmechanismus besser zu verstehen. Um den Einfluss der Kraft auf das Energiepotential der Bindung zu berücksichtigen, wurde eine Analysemethode entwickelt, bei der die Dissoziationskonstante aus dem Verhältnis der Anzahl der Datenpunkte bei einer bestimmten Kraft zu der Anzahl der Abrisse bei dieser Kraft bestimmt werden kann. Im zweiten Teil der Arbeit wurden Rezeptor – Ligand Interaktionen gemessen, für die die Interaktionspartner gezielt an die Oberfläche gebunden wurden, um eine kontrollierte Interaktion zu messen. Zum einen wurde der Einfluss der Multivalenz auf den Ni-NTA-Histag Komplex untersucht. Der Vergleich des Tris-NTA/His6 Systems mit dem Mono-NTA/His2 System zeigt, dass die Affinität mit zunehmender Multivalenz deutlich ansteigt, aber die mechanische Stabilität beider Systeme gleich bleibt. Dies lässt sich auf die Geometrie des Komplexes zurückführen und trägt damit zum Verständnis multivalenter Bindungen bei. Des Weiteren wurde die Interaktion des Präpeptids des Lantibiotikums Nisin mit dem ABC-Exporter NisT, der Bestandteil der Modifizierungsmaschinerie zur Produktion von Nisin ist, untersucht. Fluoreszenzmessungen sowie Kraftspektroskopie zeigen spezifisches Binden des Präpeptids und erlauben somit Einblicke in die Interaktion.Inside the cell membrane proteins play a crucial role to keep up many essential conditions and thus, the investigation of their structure-function relationship is of great interest to understand their mechanism. Atomic force spectroscopy proves to be a suitable technique to complement other established methods in structural biology, as it not only determines inter- and intramolecular forces of a protein but also binding kinetics of receptor – ligand interactions. In the first part of this work the influence of light activation and complex formation on the inter- and intramolecular forces of sensory rhodopsin II has been investigated by unfolding experiments and the analysis to determine the stability of a protein region has been improved. In one approach, the stability is given as average fore derived by treating the data form single molecule measurements as if they were derived from ensemble measurements. In another approach the stability of a protein region was determined by the frequency of rupture events in the respective region. Also, the resolution of the histograms was increased. In both approaches regions are identified, in which light activation and complex formation induce changes in stability, which leads to a better understanding of the reaction mechanism. Furthermore, an analysis which considers the influence of the forces was developed. Here, the dissociation constant is derived from the relation of the number of data points at a specific given force and the number of rupture events at this force. In the second part of this work receptor – ligand interactions were investigated, which requires the controlled and specific binding of two interaction partners towards the surface. First, the influence of the multivalency on the Ni-NTA-histag complex was probed. Comparison of the behaviour of the tris-NTA/His6 system and the mono-NTA/His2 system reveals that affinity increases with increasing multivalency, but mechanical stability does not change. This might be explained by the geometry of the complex and thus leads to a deeper understanding of multivalent binding. Another application of force spectroscopic measurements of a receptor – ligand interaction was the investigation of the interaction between the prepeptid of the lantibiotic nisin and the ABC-exporter NisT. NisT is part of the modification machinery in the production of nisin. Fluorescent measurements as well as force spectroscopic data show a specific binding of prenisin, which allows deeper insights in this interaction. | |||||||
| Quelle: | G.I. Bell, 1978
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| Lizenz: |  Urheberrechtsschutz | |||||||
| Bezug: | 10/2006 - 07/2010 | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Chemie » Physikalische Chemie und Elektrochemie | |||||||
| Dokument erstellt am: | 30.08.2010 | |||||||
| Dateien geändert am: | 17.08.2010 | |||||||
| Promotionsantrag am: | 07.05.2010 | |||||||
| Datum der Promotion: | 13.07.2010 |
