SNARE assembly and disassembly are not in equilibrium

As a result of the structural changes occurring during SNARE complex formation, assembly can be described as a folding reaction. Using reversible denaturation protocols, we found that unfolding (disassembly) and refolding (assembly) are not in equilibrium (Fasshauer, 2002). This hysteresis makes the determination of the thermodynamic parameters involved in SNARE assembly challenging. Hence, the energetic contribution of SNARE assembly towards membrane fusion remains to be clarified. In addition, the hysteresis probably indicates that SNARE assembly occurs via a transient intermediate.

Dissociation (disassembly) is not in equilibrium with refolding (assembly) of the neuronal SNARE-complex.
A) Without heating the neuronal SNARE-complex runs as an SDS-resistant band upon SDS-PAGE. The SDS-resistant band disappears only at temperatures above 85 °C (right panel). After unfolding at 95 °C the SDS-resistant band reappears only at temperatures below about 60 °C even after 11 h incubation (left panel). For these experiments the complex were exposed to elevated temperatures in a buffer without SDS. Only for electrophoresis SDS-sample buffer was added.
B) Upon CD-spectroscopy a similar hysteresis in the folding and unfolding transitions was observed. The a-helical complex unfolds at 82 °C independent of the concentration, whereas reassembly is concentration dependent and occurs only at temperatures below about 60 °C.