Dokument: Solid-State NMR Spectroscopy of HIV-1 Vpu and Human CD4 in Lipid Bilayers
| Titel: | Solid-State NMR Spectroscopy of HIV-1 Vpu and Human CD4 in Lipid Bilayers | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=23688 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20131017-130058-8 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Do, Hoa Quynh [Autor] | |||||||
| Dateien: |
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| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibung: | In HIV-infected human cells, the viral protein Vpu of HIV-1 directly interacts with the human T-cell surface glycoprotein CD4 and subsequently induces the degradation of this protein in the endoplasmic reticulum. However, the exact mechanism of the interaction is still under debate, and the binding interface between CD4 and Vpu has not yet been determined precisely. In this work, solid-state NMR spectroscopy with magic angle spinning (MAS) was employed to study the behavior of HIV-1 Vpu and human CD4(372-433) in POPC lipid bilayers.
The uniformly labeled 13C, 15N integral membrane proteins, full-length Vpu and CD4(372-433) were reconstituted in POPC bilayers. The resulting proteoliposomes were then lyophilized, packed into sample containers (the rotors), and rehydrated for MAS solid-state NMR measurements. The resonance assignment was performed on data which were acquired using 2D solid-state NMR spectroscopy. Site specific resonance assignments were obtained for 13 residues in CD4, while for Vpu only type-specific assignments could be obtained. The secondary chemical shifts of the identified amino acids report that the transmembrane part of CD4(372-433) in POPC bilayers has an alpha helical structure. The shifts of the amino acids belonging to transmembrane part of full-length Vpu in the POPC bilayers are also indicative of an alpha helical secondary structure, whereas the cytoplasmic part of this protein indicates a conformational disorder. The mobility of CD4(372-433) and Vpu in POPC bilayers was investigated with the aid of homonuclear 2D solid state NMR techniques at above and below the freezing temperature. Magnetization transfer from mobile protons to the protein utilizing a 3D experiment which is a combination of T2 filter, 1H spin diffusion, cross polarization, and proton driven spin diffusion was employed to probe the topology of the membrane proteins in POPC bilayers. Our results are in agreement with a pore-like structure with water accessibility for Vpu. For both proteins Vpu and CD4(372-433), the transmembrane parts were observed in the vicinity of the lipid side-chains. This indicates that the proteins were reconstituted correctly. Isotope-labeled Vpu was also reconstituted into POPC bilayers in presence of unlabeled CD4(372-433) and investigated using 2D homonuclear C-C correlation spectroscopy. While different mobilities for certain residues of the cytoplasmic domain of Vpu could be observed, it is not clear whether these effects arise from the interaction or are due to the different hydration levels of these samples. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Biologie | |||||||
| Dokument erstellt am: | 17.10.2013 | |||||||
| Dateien geändert am: | 17.10.2013 | |||||||
| Promotionsantrag am: | 10.01.2012 | |||||||
| Datum der Promotion: | 23.02.2012 |
