Rasterkraftmikroskopische Untersuchungen von Membraneigenschaften und Membran-Protein Wechselwirkungen
| Autor: | Roes, Stefanie |
|---|---|
| Přispěvatelé: | Seydel, U., Hansen, U. P. |
| Jazyk: | němčina |
| Rok vydání: | 2004 |
| Předmět: |
LPS-binding Protein
Polarisationsmessungen LPS Abschlussarbeit CD spectroscopy Lipopolysaccharide Faculty of Mathematics and Natural Sciences doctoral thesis Rasterkraftmikroskopie Lipid A LBP AFM Rasterkraftmikroskopie LPS-binding Protein LBP Lipid A Lipopolysaccharide LPS CD spectroscopy Polarisationsmessungen lipids (amino acids peptides and proteins) ddc:610 AFM Mathematisch-Naturwissenschaftliche Fakultät ddc:6XX |
| Popis: | The cell wall of Gram-negative bacteria consists of the cytoplasmic and an additional outer membrane. This outer membrane is an extremely asymmetric bilayer with respect to the lipid composition, the outer leaflet is composed of glycolipids, mainly lipopolysaccharides (LPSs), and the inner leaflet of a phospholipid mixture. When released from bacterial surface into the blood circulation of the host, LPS plays an important role in the pathogenesis and manifestation of gram-negative inflammation, in general, and of septic shock, in particular. In this dissertation, the AFM-technique was used to investigate the properties of membranes, their cooperative processes and lipid-protein interactions with high molecular lateral resolution. Two complementing techniques were used: fluorescence spectroscopy with polarized light to determine phase transition temperatures of lipids and circular dichroism (CD) - spectroscopy to investigate physicochemical properties of the LPS-binding protein (LBP). Experiments were done on reconstituted model membranes - lipid monolayers and -bilayers - which were solid-supported for the AFM-measurements. Two biological questions were important in this dissertation: (i) function of the LPS-layer in Gram-negative bacteria as a permeability barrier and its interaction with antibacterial peptides and (ii) activation of immune cells (mononuclear cells, MNC) of the host immune system by released LPS. To answer the first question, LPS monolayers were prepared. Properties such as topography and phase behaviour and their interaction with antibacterial peptides were investigated. With regard to the second question, the lateral organization of LBP in reconstituted phospholipid membranes mimicking the membrane of MNC was characterized. LBP is involved in the signalling cascade in the activation of MNC and has been described in the literature as a shuttle protein. Here it is shown that LBP has a function as a fusion protein for phospholipid membranes and LPS. Furthermore, force spectroscopy was successfully used for measuring binding forces between LBP and its antibody. This is the first time that a protein was detected in a lipid membrane utilizing antibodies attached to the tip of an AFM-cantilever. |
| Databáze: | OpenAIRE |
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