Concentrating Membrane Proteins Using Asymmetric Traps and AC Electric Fields

Abstract

Membrane proteins are key components of the plasma membrane and are responsible for control of chemical ionic gradients, metabolite and nutrient transfer, and signal transduction between the interior of cells and the external environment. Of the genes in the human genome, 30% code for membrane proteins (Krogh et al. J. Mol. Biol. 2001, 305, 567). Furthermore, many FDA-approved drugs target such proteins (Overington et al. Nat. Rev. Drug Discovery 2006, 5, 993). However, the structureÀfunction relationships of these are notably sparse because of difficulties in their purification and handling outside of their membranous environment. Methods that permit the manipulation of membrane components while they are still in the membrane would find widespread application in separation, purification, and eventual structureÀfunction determination of these species (Poo et al. Nature 1977, 265, 602). Here we show that asymmetrically patterned supported lipid bilayers in combination with AC electric fields can lead to efficient manipulation of charged components. We demonstrate the concentration and trapping of such components through the use of a "nested trap" and show that this method is capable of yielding an approximately 30-fold increase in the average protein concentration. Upon removal of the field, the material remains trapped for several hours as a result of topographically restricted diffusion. Our results indicate that this method can be used for concentrating and trapping charged membrane components while they are still within their membranous environment. We anticipate that our approach could find widespread application in the manipulation and study of membrane proteins.

Optimization of Brownian ratchets for the manipulation of charged components within supported lipid bilayers

“…7 Through the use of asymmetric ratchets, this has been the basis for several different applications, some noteworthy examples including DNA transport and analysis, 8,9 particle separation, 10,11 and sorting or directed motion in lipid bilayers. [12][13][14] Lipid membranes are ubiquitous in eukaryotic cells for compartmentalizing internal organelles and for separating the interior of the cell from the external environment. Perhaps unsurprisingly, lipid membranes and proteins contained within them are the target of numerous drugs and there is therefore significant interest in understanding how the function of such proteins relates to their structure and spatio-temporal behavior.…”

“…Since many membrane components are electrically charged, external electric (and hydrodynamic flow) fields can provide a driving force for the motion of components based on electrophoresis, electroosmosis, and hydrodynamic flow. [14][15][16][17][18] This provides scope for the "in-membrane" transport and separation based on the charge of a component. It is still difficult however to sort based upon other properties such as mobility or diffusion coefficient.…”

Novel biomedical applications of supported lipid bilayers

“…To complete the bilayer formation the sample is pushed through the interface and kept submerged while the supported bilayer sandwich is removed from the trough. 37 Another main technique to form SLBs uses a solution of unilamellar lipid vesicles or proteoliposomes that are fused with solid supports (Figure 1.4b). Such vesicles can be obtained using standard liposome preparation or protein reconstitution methods i.e.…”

“…This effect can be retarded by topographically restricting lateral diffusion by introduction of surface ratchets. 37 Still, the lipids in these systems display lateral mobility that restrict the temporal stability of the surface gradients. This lack of temporal control limits the use of SLB platforms for studying biological processes.…”

“…The use of AC electric fields in combination with surface ratchets proved effective in retarding the lateral mobility of lipids in the SLB as shown for micro-contact printed ratchets and a chip-based device ( Figure 1.11c). 195,197,198 On the other hand, Cremer and co-workers demonstrated lipid fixation by freezing the surface after a complex SLB was used for membrane electrophoresis. Freezedried SLBs can be used in set-ups such as MALDI mass spectroscopy for further chemical analyses.…”