Direct Measurement of Protein Translocation across Droplet Interface Bilayers

Abstract

We introduce a new method for monitoring and quantitating the transport of materials across a model cell membrane. As a proof of concept, we investigate how a small cell-penetrating peptide called Pep-1 is able to carry a large object, such as a protein, across a lipid bilayer. To create a membrane, two sub-microliter, lipid-encased aqueous droplets are contacted - termed a droplet interface bilayer (DIB). The peptides adsorb to the protein cargo non-covalently and somehow “carry” the protein from one droplet to the other through the membrane. We then assay the translocated cargo through a fluorogenic assay. The DIB method recapitulates the findings of earlier studies involving Pep-1, including the dependence of protein transport on voltage and membrane charge, while also contributing new insights. Specifically, we found that the symmetry of the bilayer membrane may play a role in Pep-1-mediated protein translocation. In addition, we used a newly developed peptide transduction domain mimic (PTDM) as a protein carrier, which exhibited distinct differences compared to Pep-1's mechanism. We've also used the DIB system to monitor the translocation of proteins through pores, such as the anthrax toxin. We anticipate that the DIB method may be useful for a variety of transport-based studies; in particular those which must make use of tiny quantities of purified species.