Characterisation of synaptic proteins and their complexes using structural mass spectrometry

Abstract

In this thesis, individual SNARE proteins, binary SNARE sub-complexes and intermediates as well as the fully assembled SNARE complex were structurally characterised. In addition, interactions of the SNAREs with Complexin-1 at all stages of the SNARE complex assembly were investigated and lipid binding preferences determined. Using native mass spectrometry (MS), formation, stoichiometry and stability of oligomers and complexes were analysed. Specific interaction sites between the interacting proteins were further identified by chemical cross-linking. To address protein-lipid interactions, binding of the proteins to immobilised lipids as well as membranes in form of liposomes was analysed. Binding affinities of the proteins to solubilised lipids were determined by native MS. In summary, the stoichiometry of intermediates and off-pathway complexes was unravelled and a road map of SNARE complex assembly including regulation by Complexin-1 was provided.