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 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 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.
Li
Yulong
Y
Department of Neurobiology, Duke University Medical Center, Durham, NC, USA.
Augustine
George J
GJ
Weninger
Keith
K
D000818:Animals; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D031541:Fluorescence Resonance Energy Transfer; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D008081:Liposomes; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D046912:Multiprotein Complexes; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050600:SNARE Proteins / Q000737:chemistry* / Q000378:metabolism*
D008024:Ligands; D008081:Liposomes; D046912:Multiprotein Complexes; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins