Single molecule diffusion of membrane-bound proteins: window into lipid contacts and bilayer dynamics

Biophys J. 2010 Nov 3;99(9):2879-87. doi: 10.1016/j.bpj.2010.08.046.


Membrane targeting proteins are recruited to specific membranes during cell signaling events, including signals at the leading edge of chemotaxing cells. Recognition and binding to specific lipids play a central role in targeting reactions, but it remains difficult to analyze the molecular features of such protein-lipid interactions. We propose that the surface diffusion constant of peripheral membrane-bound proteins contains useful information about protein-lipid contacts and membrane dynamics. To test this hypothesis, we use single-molecule fluorescence microscopy to probe the effects of lipid binding stoichiometry on the diffusion constants of engineered proteins containing one to three pleckstrin homology domains coupled by flexible linkers. Within error, the lateral diffusion constants of these engineered constructs are inversely proportional to the number of tightly bound phosphatidylinositol-(3,4,5)-trisphosphate lipids. The same trend is observed in coarse-grained molecular dynamics simulations and hydrodynamic bead calculations of lipid multimers connected by model tethers. Overall, single molecule diffusion measurements are found to provide molecular information about protein-lipid interactions. Moreover, the experimental and computational results independently indicate that the frictional contributions of multiple, coupled but well-separated lipids are additive, analogous to the free-draining limit for isotropic fluids--an insight with significant implications for theoretical description of bilayer lipid dynamics.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Biophysical Phenomena
  • Facilitated Diffusion
  • Hydrodynamics
  • Lipid Bilayers / chemistry*
  • Lipid Bilayers / metabolism*
  • Membrane Proteins / chemistry*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Microscopy, Fluorescence
  • Molecular Dynamics Simulation
  • Protein Binding
  • Protein Engineering
  • Receptors, Cytoplasmic and Nuclear / chemistry
  • Receptors, Cytoplasmic and Nuclear / genetics
  • Receptors, Cytoplasmic and Nuclear / metabolism
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism


  • Lipid Bilayers
  • Membrane Proteins
  • Receptors, Cytoplasmic and Nuclear
  • Recombinant Fusion Proteins
  • phosphatidylinositol receptors