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

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 × 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 × 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.