Manipulation and charge determination of proteins in photopatterned solid supported bilayers

Abstract

This work demonstrates the use of deep UV micropatterned chlorotrimethylsilane (TMS) monolayers to support lipid membranes on SiO₂ surfaces. After immersing such a patterned surface into a solution containing small unilamellar vesicles of egg PC, supported bilayerlipid membranes were formed on the hydrophilic, photolyzed regions and lipidmonolayer over the hydrophobic, non-photolyzed regions. A barrier between the lipidmonolayer 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 (ζ₁) and the lipid membrane (ζ₂). If ζ₁ > ζ₂, streptavidin moves to the negative electrode, while if ζ₁ < ζ₂, streptavidin moves to the positive electrode. This balance was manipulated by adding positively charged lipidDOTAP to the membrane. After measuring the average drift velocity of streptavidin as a function of DOTAP concentration, the point where ζ₁ ≈ ζ₂ was found. At this point ζ₁ 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.