Interactions between Synaptic Vesicle Fusion Proteins Explored by Atomic Force Microscopy

• Published in: Proceedings of the National Academy of Sciences - PNAS 2003-07, Vol.100 (15), p.8736-8741
• Main Authors: Yersin, A., Hirling, H., Steiner, P., Magnin, S., Regazzi, R., Hüni, B., Huguenot, P., De Los Rios, P., Dietler, G., Catsicas, S., Kasas, S.
• Format: Article
• Language: English
• Subjects: Antigens, Surface - chemistry; Antigens, Surface - physiology; Atomic interactions; Atoms & subatomic particles; Biological Sciences; Biophysical Phenomena; Biophysics; Cytoplasm; Histograms; In Vitro Techniques; Kinetics; Loading rate; Macromolecular Substances; Membrane Fusion - physiology; Membrane Proteins - chemistry; Membrane Proteins - physiology; Mica; Microscopy, Atomic Force; Munc18 Proteins; Nerve Tissue Proteins - chemistry; Nerve Tissue Proteins - pharmacology; Nerve Tissue Proteins - physiology; P branes; Protein Binding; Proteins; R-SNARE Proteins; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; SNARE Proteins; Synaptic Vesicles - physiology; Synaptosomal-Associated Protein 25; Syntaxin 1; Tetanus; Tetanus Toxin - pharmacology; Vesicular Transport Proteins - pharmacology; Vesicular Transport Proteins - physiology
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1533137100

Measuring the biophysical properties of macromolecular complexes at work is a major challenge of modern biology. The protein complex composed of vesicle-associated membrane protein 2, synaptosomal-associated protein of 25 kDa, and syntaxin 1 [soluble N-ethyl-maleimide-sensitive factor attachment protein receptor (SNARE) complex] is essential for docking and fusion of neurotransmitter-filled synaptic vesicles with the presynaptic membrane. To better understand the fusion mechanisms, we reconstituted the synaptic SNARE complex in the imaging chamber of an atomic force microscope and measured the interaction forces between its components. Each protein was tested against the two others, taken either individually or as binary complexes. This approach allowed us to determine specific interaction forces and dissociation kinetics of the SNAREs and led us to propose a sequence of interactions. A theoretical model based on our measurements suggests that a minimum of four complexes is probably necessary for fusion to occur. We also showed that the regulatory protein neuronal Sec1 injected into the atomic force microscope chamber prevented the complex formation. Finally, we measured the effect of tetanus toxin protease on the SNARE complex and its activity by on-line registration during tetanus toxin injection. These experiments provide a basis for the functional study of protein microdomains and also suggest opportunities for sensitive screening of drugs that can modulate protein-protein interactions.