Manipulation and charge determination of proteins in photopatterned solid supported bilayers.
This work demonstrates the use of deep UV micropatterned chlorotrimethylsilane (TMS) monolayers to support lipid membranes on SiO(2) surfaces. After immersing such a patterned surface into a solution containing small unilamellar vesicles of egg PC, supported bilayer lipid membranes were formed on the hydrophilic, photolyzed regions and lipid monolayer over the hydrophobic, non-photolyzed regions. A barrier between the lipid monolayer and bilayer regions served to stop charged lipids migrating between the two. This allows the system to be used to separate charged lipids or proteins by electrophoresis. Either oppositely charged fluorescence labeled lipids [Texas Red DHPE (negative charge) and D291 (positive charge)] or lipids with different charge numbers [Texas Red DHPE (one negative charge) and NBD PS (two negative charges)] can be separated. We have also studied the migration of streptavidin attached to a biotinylated lipid. Negatively charged streptavidin responds to the applied electric field by moving in the direction of electroosmotic flow, i.e. towards the negative electrode. However the direction of streptavidin movement can be controlled by altering the difference in zeta potential between that of the streptavidin (zeta(1)) and the lipid membrane (zeta(2)). If zeta(1) > zeta(2), streptavidin moves to the negative electrode, while if zeta(1) < zeta(2), streptavidin moves to the positive electrode. This balance was manipulated by adding positively charged lipid DOTAP to the membrane. After measuring the average drift velocity of streptavidin as a function of DOTAP concentration, the point where zeta(1) approximately zeta(2) was found. At this point zeta(1) was calculated to be -9.8 mV which is in good agreement with the value of -13 mV from force measurements and corresponds to a charge of -2e per streptavidin, thus demonstrating the applicability of this method for determining protein charge.
Han
Xiaojun
X
School of Physics and Astronomy, University of Leeds, Leeds, UK.
Cheetham
Matthew R
MR
Sheikh
Khizar
K
Olmsted
Peter D
PD
Bushby
Richard J
RJ
Evans
Stephen D
SD
D036681:Fluorescence Recovery After Photobleaching; D008051:Lipid Bilayers / Q000138:chemical synthesis / Q000737:chemistry*; D008560:Membrane Fluidity / Q000502:physiology*; D008565:Membrane Proteins / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D008856:Microscopy, Fluorescence; D019809:Streptavidin / Q000737:chemistry*
D008051:Lipid Bilayers; D008565:Membrane Proteins; D019809:Streptavidin