Abstract
The three key players in the exocytotic release of neurotransmitters from synaptic vesicles are the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins synaptobrevin 2, syntaxin 1a, and SNAP-25. Their assembly into a tight four-helix bundle complex is thought to pull the two membranes into close proximity. It is debated, however, whether the energy generated suffices for membrane fusion. Here, we have determined the thermodynamic properties of the individual SNARE assembly steps by isothermal titration calorimetry. We found extremely large favorable enthalpy changes counterbalanced by positive entropy changes, reflecting the major conformational changes upon assembly. To circumvent the fact that ternary complex formation is essentially irreversible, we used a stabilized syntaxin-SNAP-25 heterodimer to study synaptobrevin binding. This strategy revealed that the N-terminal synaptobrevin coil binds reversibly with nanomolar affinity. This suggests that individual, membrane-bridging SNARE complexes can provide much less pulling force than previously claimed.
Highlights
Plasma membrane proteins syntaxin and SNAP-25 provide the binding site for the vesicular synaptobrevin
To find out whether the SNARE complex assembly is enough for membrane fusion, only the amount of energy released during complex formation and the amount of energy needed for membrane fusion need to be compared
Because neuronal SNARE complex formation occurs by interaction of the three molecules, it was necessary to investigate each individual binary binding step first
Summary
Plasma membrane proteins syntaxin and SNAP-25 provide the binding site for the vesicular synaptobrevin. The role of fusion proteins is thought to lower the energy barrier for membrane fusion, but understanding how they modulate the lipid membrane and how their conformational changes are translated into a mechanical force is still in its infancy It is not clear, for instance, whether SNARE-catalyzed fusion proceeds through a stalk-like structure or just locally alters the membranes, a mechanism that might need much less activation energy. To avoid the quasi-irreversibility of the process, the problem has been elegantly tackled by atomic force microscopy by two different research groups (26 – 28) In these experiments individual complexes affixed to solid supports were ruptured, yielding energy values of 43 and 33 kBT. Our results suggest that individual SNARE complexes might provide much less pulling energy than previously claimed
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