Kinetic analysis of neuronal SNARE protein interactions
by Sonja Pribićević
Date of Examination:2021-11-03
Date of issue:2022-08-08
Advisor:Prof. Dr. Reinhard Jahn
Referee:Prof. Dr. Reinhard Jahn
Referee:Prof. Dr. Tobias Moser
Referee:Prof. Dr. Marina Rodnina
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EnglishWhen an action potential arrives at the axon terminal, it causes a local and transient increase in the Ca2+ concentration that starts a cascade of molecular interactions eventually leading to the fusion of synaptic vesicles with the plasma membrane. The key proteins in this process – syntaxin-1A, SNAP25 and synaptobrevin 2 – assemble into a SNARE complex that directly drives the membrane fusion. The interaction of the SNARE proteins and mechanism by which this interaction mediates membrane fusion is intensively investigated. Despite numerous studies, the answers to the core questions regarding the molecular mechanism, the guidance and the speed of SNARE complex formation remain unclear. In this work, I address one of these core questions, namely, the molecular mechanism of SNARE assembly as it represents a fundamental problem that underlies the understanding of any other process regarding SNARE-mediated fusion. Using purified, soluble SNARE proteins and kinetic measurements under different conditions, I identified some of the most likely intermediates of the SNARE assembly reaction and estimated the rate constants of their formation. Accordingly, I confirmed that the syntaxin-1A-SNAP25 interaction represents the first of the SNARE complex assembly reaction. Size exclusion chromatography experiments provided further details on the possible intermediates by showing that stable complexes can form between syntaxin-1A, synaptobrevin 2 and the individual SNARE domains of SNAP25. I conclude that SNARE complex formation occurs in at least two steps, and is initiated by the interaction between syntaxin-1A and SNAP25. The results and the experimental approach developed in this work represent a backbone for elucidating the complicated interactions that control synaptic vesicle fusion.
Keywords: SNARE; kinetics; syntaxin-1A; SNAP25; synaptobrevin 2; FRET; stopped-flow