Single-molecule fluorescence imaging of peptide binding to supported lipid bilayers

Anal Chem. 2009 Jul 1;81(13):5130-8. doi: 10.1021/ac9007682.


Single-molecule fluorescence imaging techniques have been adapted to the quantitative characterization of peptide-binding to lipid bilayers. Peptide-membrane interactions are important in therapeutics, diagnostics, and membrane permeation and for understanding of the structure and function of membrane-bound proteins. Total-internal reflection fluorescence (TIRF) imaging is capable of determining membrane-binding equilibrium constants through the reliable counting of individual peptide molecules in order to report their surface density in the membrane. The residence times of the individual molecules in the membrane can also be determined and the rates of unbinding determined from a histogram of residence times. A combination of the unbinding kinetics and the equilibrium constant allows the binding rate of a peptide to the membrane also to be reported. We apply this method to characterize the lipid membrane affinity of glucagon-like peptide-1 (GLP-1), a 30-residue membrane-active peptide that is involved in glycemic control. Using single-molecule TIRF imaging, we have measured the affiliation of GLP-1 with a supported, phospholipid bilayer and determined its binding equilibrium constant. Two rates of dissociation were observed, suggesting strongly and weakly bound states of the peptide. The rate of membrane association was much slower than diffusion-controlled, indicating a significant kinetic barrier to membrane binding. The data were interpreted using a heterogeneous, surface-reaction model analogous to electron-transfer kinetics at an electrode. To our knowledge, these results are the first example of using single-molecule counting to quantify peptide-lipid bilayer binding equilibria and kinetics.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Fluorescent Dyes / chemistry*
  • Glucagon-Like Peptide 1 / analysis
  • Glucagon-Like Peptide 1 / chemistry*
  • Kinetics
  • Lipid Bilayers / chemistry*
  • Microscopy, Fluorescence / methods*
  • Molecular Sequence Data
  • Protein Binding
  • Staining and Labeling


  • Fluorescent Dyes
  • Lipid Bilayers
  • Glucagon-Like Peptide 1