Single-molecule study of a protein complex using magnetic tweezers

Abstract

In neural communications, the formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex provides mechanical thrust for the synaptic membrane fusion to release neurotransmitters. Although the SNARE complex acts intrinsically as a molecular ‘transducer’ from the Brownian motion of a vesicle to the directional propulsion, but its practical mechanism in the mechanical perspective is still unclear owing to the experimental limitation. This thesis presents the mechanical responses of a single neuronal SNARE complex1 using magnetic-tweezers technique. The single SNARE complexes are observed as being unzipped with over 30 pN force. When rezipping is induced by lowering the force to ~10 pN, only a partially assembled state results, with the C-terminal half of the SNARE complex remaining disassembled. Reassembly of the C-terminal half occurs only when the force is further lowered below 10 pN. Thus, the mechanical hysteresis, characterized by the unzipping and rezipping cycle of a single SNARE complex, produces the partially assembled state. In this metastable state, unzipping toward the N-terminus is suppressed while zippering toward the C-terminus is initiated as a steep function of force. This ensures the directionality of SNARE complex formation, making the SNARE complex a robust force-generating machine.

Further, known as 20S complex, the interactions of SNARE complex with N-ethylmaleimide-sensitive factor (NSF) and one of soluble NSF attachment proteins (α-SNAP) were examined. In neurons, the SNARE complexes are disassembled by the coordinated action of NSF and α-SNAP and the disassembled SNARE proteins are recycled for saving the chemical energy. Hence, the disassembly stage of the SNARE complex has very high significance but its molecular mechanism is uncertain. From the observation of extension changes in SNARE complexes with sub-nm accuracy, it is supposed that the α-SNAP destabilize the linker domains of SNARE comp...