Single Molecule Studies of Synaptotagmin and Complexin Binding to the SNARE Complex

¹ Stanford University/HHMI
² University of North Carolina
³ Genentech, Inc.
⁴ Lawrence Berkeley National Lab
⁵ Stanford Univ

^* To whom correspondence should be addressed. E-mail: brunger{at}stanford.edu.

Submitted on October 5, 2004
Revised on November 15, 2004
Accepted on 4 April 2005

	Abstract

The assembly of multi-protein complexes at the membrane interface governs many signaling processes in cells. The lack of suitable methods for obtaining biophysical information about protein complex formation at the membrane has limited study to isolated cytoplasmic fragments. We used single molecule fluorescence resonance energy transfer to study complexin and synaptotagmin interactions with the SNARE complex in deposited lipid bilayers. Using total internal reflectance (TIR) microscopy, individual binding events at the membrane could be resolved despite an excess of unbound protein in solution. Fluorescence resonance energy transfer (FRET)-efficiency derived distances for the complexin-SNARE interaction were consistent with the crystal structure of the complexin-SNARE complex. The unstructured N-terminal region of complexin showed broad distributions of FRET efficiencies to the SNARE complex suggesting that information on conformational variability can be obtained from FRET efficiency distributions. The low-affinity interaction of synaptotagmin with the SNARE complex changed dramatically upon addition of Ca2+ with high FRET efficiency interactions appearing between the C2B domain and linker domains of synaptotagmin and the membrane proximal portion of the SNARE complex. These results demonstrate that single molecule FRET can be used as a "spectroscopic ruler" to simultaneously gain structural and kinetic information about transient multi-protein complexes at the membrane interface.

Key Words: membrane fusion, single molecule fluorescence spectroscopy, synaptic neurotransmission, vesicle trafficking

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