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. uorescence 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 10(6) 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 10 (6) M-1 s(-1)), and dissociates much more rapidly ( rate constant > 67 s(-1)). Comparison of single-molecule. uorescence 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.