Synthesis and Analysis of Modified SNARE Proteins with Respect to Assembly and Disassembly of the SNARE Complex
von Meike Pauline Wilhelmine Junius
Datum der mündl. Prüfung:2016-08-26
Betreuer:Prof. Dr. Ulf Diederichsen
Gutachter:Prof. Dr. Ulf Diederichsen
Gutachter:Prof. Dr. Reinhard Jahn
EnglischIn this thesis, the first aim was to synthesise an inhibitor for the alpha-SNAP/NSF-mediated disassembly reaction of the SNARE complex. Based on identified interaction sites between alpha-SNAP and Synaptobrevin-2, suitable sequence positions (D51, E55, Q58, E62, D65) of Synaptobrevin-2 were chosen for modification. One or more sterically demanding side chains were introduced on the surface of the SNARE complex to prevent or at least weaken the alpha-SNAP binding which is essential for the disassembly reaction. For the site-specific modification of the Synaptobrevin-2 SNARE motif by SPPS, the adamantyl-modified amino acid building block Fmoc-Asn(Ad)-OH was synthesised. SNARE assembly and disassembly properties were investigated by time-dependent fluorescence anisotropy. The second aim was to study the influence of the adamantyl-modified Syb-2 analogues on the Complexin binding to the SNARE complex, due to the suggested competitive binding of alpha-SNAP and Complexin to the SNARE complex surface. The binding reaction was investigated by time-dependent fluorescence anisotropy and monitored by an increase of anisotropy upon addition of the SNARE complex to OregonGreen-labelled Complexin. In a third part of this study, the SNARE complex formation itself, the SNARE zippering process was investigated. By introducing photocleavable protecting groups, a soluble caged Synaptobrevin-2 derivative was designed and synthesised which was inactive towards SNARE complex zippering, and could be reactivated upon irradiation. In the last part of this study, Syntaxin transmembrane domains (TMDs) were easily accessible by SPPS and attachment of fluorophores to the N-terminal end of the sequence on the solid-support were successfully performed.
Keywords: Synaptobrevin; SNARE Complex; SNARE Disassembly; caged Synaptobrevin; Fluorescence Anisotropy; Complexin; alpha-SNAP; NSF; SPPS