Molecular handles for the mechanical manipulation of single-membrane proteins in living cells.
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.
Gorostiza
Pau
P
Departments of Physics and Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720, USA. pau@berkeley.edu
Tombola
Francesco
F
Verdaguer
Albert
A
Smith
Steven B
SB
Bustamante
Carlos
C
Isacoff
Ehud Y
EY
D000818:Animals; D001696:Biomechanical Phenomena / Q000379:methods; D019921:Biotinylation; D002478:Cells, Cultured; D003432:Cross-Linking Reagents; D008565:Membrane Proteins / Q000502:physiology*; D008846:Micromanipulation / Q000379:methods*; D018625:Microscopy, Atomic Force / Q000379:methods*; D015336:Molecular Probe Techniques; D036103:Nanotechnology / Q000379:methods*; D009865:Oocytes / Q000502:physiology*; D011485:Protein Binding; D051637:Shaker Superfamily of Potassium Channels / Q000502:physiology*; D014982:Xenopus laevis
D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D051637:Shaker Superfamily of Potassium Channels