Molecular handles for the mechanical manipulation of single-membrane proteins in living cells

IEEE Trans Nanobioscience. 2005 Dec;4(4):269-76. doi: 10.1109/tnb.2005.859552.

Abstract

We have developed a procedure to selectively biotinylate a specific membrane protein, enabling its attachment to external force probes and thus allowing its mechanical manipulation within its native environment. Using potassium channels as model membrane proteins in oocytes, we have found that Maleimide-PEG3400-biotin is the crosslinker with highest conjugation selectivity and accessibility to external probes. Neutravidin-coated beads provide for directed attachment while avoiding nonspecific interactions with the cell. The technology was successfully tested by mechanical manipulation of biotinylated extracellular residues of channels in oocytes using an atomic force microscope under conditions which preserve function of the channels. Binding forces of approximately 80 pN at 100 nN/s were measured.

Publication types

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

MeSH terms

  • Animals
  • Biomechanical Phenomena / methods
  • Biotinylation
  • Cells, Cultured
  • Cross-Linking Reagents
  • Membrane Proteins / physiology*
  • Micromanipulation / methods*
  • Microscopy, Atomic Force / methods*
  • Molecular Probe Techniques*
  • Nanotechnology / methods*
  • Oocytes / physiology*
  • Protein Binding
  • Shaker Superfamily of Potassium Channels / physiology*
  • Xenopus laevis

Substances

  • Cross-Linking Reagents
  • Membrane Proteins
  • Shaker Superfamily of Potassium Channels