Biophysikalische und thermodynamische Charakterisierung der neuronalen SNARE-Komplexbildung

Abstract

Membrane fusion events are mediated by a set of conserved protein machinery, the SNARE proteins. SNARE proteins assemble into a very stable α-helical complex between two membranes. The plasma membrane residing SNARE proteins, syntaxin and SNAP-25, form an acceptor complex for the vesicular SNARE protein, synaptobrevin. The formation of the SNARE complex brings the two membranes closer and thus, leads to membrane fusion. The exact process of this reaction and the possible intermediates of complex formation have yet to be described. In order to gain deeper insights into the process of SNARE mediated membrane fusion, I used biophysical methods such as circular dichroism and fluorescence spectroscopy to characterize distinct steps of complex assembly in vitro. Also, I investigated the thermodynamics of the SNARE complex assembly using isothermal titration calorimetry. In the first section, I investigated the interaction of syntaxin and SNAP-25 and their assembly into an acceptor complex for synaptobrevin binding. Previous work has shown that the formation of this Q-SNARE acceptor complex is the rate-limiting step for subsequent steps in SNARE complex formation. Site-directed mutations in SNAP-25 revealed that the N-terminal regions of the Q-SNAREs are critical for the acceptor complex assembly. Further thermodynamic analysis showed that this slow interaction is probably catalyzed in vivo by a priming factor. Next, I investigated the binding of synaptobrevin to the Q-SNARE acceptor complex. In the in vitro setting, a second syntaxin molecule occupies the synaptobrevin bindung site. This technical problem was solved by using a stabilized acceptor complex which consists of SNAP-25, syntaxin and a C-terminal synaptobrevin peptide. This peptide-stabilized acceptor complex provides a free N-terminal binding site for synaptobrevin. Upon synaptobrevin binding, the peptide is displaced and the ternary SNARE complex forms. Using this stabilized complex, I studied the synaptobrevin binding with mutations in the different hydrophobic layers as well as by truncating the single SNARE proteins. The N-terminal region of synaptobrevin proved to be essential in the formation of the ternary SNARE complex. Additionally, contrary to other R-SNARE homologs, the regulatory R-SNARE tomosyn is unable to bind to the synaptobrevin binding site in the stabilized acceptor complex. In the third part, I attempted to study the C-terminal folding of the SNARE complex after binding of synaptobrevin. Thermal melts using circular dichroism revealed that C-terminally mutated SNARE complexes unfold in two phases. Furthermore, based on thermodynamic experiments, it seems that the C-terminal folding step is able to alternate between a folded and unfolded state. This results might indicate the existence of a partially destabilized SNARE complex. Thus, the final assembly step might not provide the driving force for membrane fusion and other regulatory proteins are necessary to fulfill this task.