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Kinetics of Complexin Binding to the SNARE Complex: Correcting Single Molecule FRET Measurements for Hidden Events

Yulong Li ^*, George J. Augustine ^*  and Keith Weninger 

^* Department of Neurobiology, Duke University Medical Center, Durham, North Carolina; Marine Biological Laboratory, Woods Hole, Massachusetts; and Physics Department, North Carolina State University, Raleigh, North Carolina

Correspondence: Address reprint requests to Keith Weninger, E-mail:keith_weninger{at}ncsu.edu.

Virtually all measurements of biochemical kinetics have been derived from macroscopic measurements. Single-molecule methods can reveal the kinetic behavior of individual molecular complexes and thus have the potential to determine heterogeneous behaviors. Here we have used single-molecule fluorescence resonance energy transfer to determine the kinetics of binding of SNARE (soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptor) complexes to complexin and to a peptide derived from the central SNARE binding region of complexin. A Markov model was developed to account for the presence of unlabeled competitor in such measurements. We find that complexin associates rapidly with SNARE complexes anchored in lipid bilayers with a rate constant of 7.0 x 10⁶ M^–1 s^–1 and dissociates slowly with a rate constant of 0.3 s^–1. The complexin peptide associates with SNARE complexes at a rate slower than that of full-length complexin (1.2 x 10⁶ M^–1 s^–1), and dissociates much more rapidly (rate constant >67 s^–1). Comparison of single-molecule fluorescence resonance energy transfer measurements made using several dye attachment sites illustrates that dye labeling of complexin can modify its rate of unbinding from SNAREs. These rate constants provide a quantitative framework for modeling of the cascade of reactions underlying exocytosis. In addition, our theoretical correction establishes a general approach for improving single-molecule measurements of intermolecular binding kinetics.