Investigation of the Linker Region of Coiled Coil SNARE-Analoga and Membrane Composition on Vesicle Fusion

by Mike Christopher Groth
Doctoral thesis
Date of Examination:2021-01-11
Date of issue:2021-03-09
Advisor:Prof. Dr. Ulf Diederichsen
Referee:Prof. Dr. Ulf Diederichsen
Referee:Prof. Dr. Claudia Steinem
Persistent Address: http://dx.doi.org/10.53846/goediss-8480

 

 

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Abstract

English

SNARE (soluble N-ethylmaleimide-sensitive-factor attachment receptor) proteins play a major role in membrane fusion processes in many organisms. As these proteins form a sophisticated tetrameric coiled coil complex, many artificial models have been developed to mimic the natural system and reduce its complexity. The model system used in this work uses a combination of a coiled-coil (E3/K3) pair as the recognition unit and the native sequence of syntaxin 1A and synaptobrevin 2 for the linker and transmembrane region. These two peptides form a stable dimer upon interaction with each other and are capable of performing membrane fusion, as it has been shown previously. In this thesis, improvements of the peptide synthesis, purification and employed fusion assays have been established. DLS measurements have been introduced as a reliable technique for observing vesicle quality. Additionally, this work focusses on the effects on the fusion efficiency of specific amino acids of the linker domains. Strategic substitution of charged and aromatic amino acids showed a decrease in the fusion activity of the system. Furthermore, mechanistical studies were performed targeting the role of the linker domain by the introduction of a sterically demanding photo cage. Fusion activity was reduced by the photo cage, but could be successfully restored after the photo cleaving process. In addition, introduction of charged lipids (PIP2, DOPS) into the vesicle membranes lead to a complete stop of fusion events.
Keywords: SNARE; coiled coil; Fluorescence Spectroscopy; Vesicle fusion assays; Dynamic light scattering (DLS); lipid membrane; photo cage; fluorescence resonance energy transfer (FRET)
 
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