Accumulation and Separation of Membrane-Bound Proteins Using Hydrodynamic Forces

Abstract

The separation of molecules residing in the cell membrane remains a largely unsolved problem in the fields of bioscience and biotechnology. We demonstrate how hydrodynamic forces can be used to both accumulate and separate membrane-bound proteins in their native state. A supported lipid bilayer (SLB) was formed inside a microfluidic channel with the two proteins streptavidin (SA) and cholera toxin (CT) coupled to receptors in the lipid bilayer. The anchored proteins were first driven toward the edge of the lipid bilayer by hydrodynamic forces from a flowing liquid above the SLB, resulting in the accumulation of protein molecules at the edge of the bilayer. After the concentration process, the bulk flow of liquid in the channel was reversed and the accumulated proteins were driven away from the edge of the bilayer. Each type of protein was found to move at a characteristic drift velocity, determined by the frictional coupling between the protein and the lipid bilayer, as well as the size and shape of the protein molecule. Despite having a similar molecular weight, SA and CT could be separated into monomolecular populations using this approach. The method also revealed heterogeneity among the CT molecules, resulting in three subpopulations with different drift velocities. This was tentatively attributed to multivalent interactions between the protein and the monosialoganglioside G(M1) receptors in the lipid bilayer.

Hydrodynamic trapping of molecules in lipid bilayers

“…The diffusivity of the protein molecules was determined to D ¼ 1 μm 2 ∕s, obtained by fitting the relative intensity as a function of time after the hydrodynamic trap was turned off to simulated data with different values of D. No immobile fraction of SA molecules or formation of SA aggregates could be observed in the experiments. The obtained diffusivity is of the same magnitude as the diffusivity of SA in SLBs previously measured by fluorescence recovery after photobleaching (FRAP) in a lipid bilayer with a homogeneous surface coverage of SA molecules (17). However, compared to FRAP, we are measuring on accumulated and not on bleached molecules.…”

“…1B. The liquid flow will exert a hydrodynamic force on the molecules in the SLB causing them to move in the direction of the liquid flow (16,17). Molecules that protrude from the SLB will typically experience a higher hydrodynamic force than that felt by a lipid molecule in the SLB, and will therefore accumulate in the area beneath the pipette (see Fig.…”

Nanofabrication for the Analysis and Manipulation of Membranes

“…44 In particular, the recently developed methodology of incorporating native cell membranes into continuous SLBs will allow for analysis and sorting of components within complex membranes with these nanofabricated structures. 82 These techniques have great potential to advance membrane biology through the on-chip manipulation of membrane components for diverse scientific and medical applications.…”

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

“…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.…”