Biophysical Society Thematic Meeting | Ascona, Switzerland

Liposomes, Exosomes, and Virosomes: From Modeling Complex Membrane Processes to Medical Diagnostics and Drug Delivery

Poster Abstracts

9-POS Board 5 Fusion of Oppositely Charged Proteoliposomes as a Method for Membrane Protein Co-Reconstitution Olivier Biner , Thomas Schick, Christoph Von Ballmooos. University of Bern, Bern, Switzerland. In order to investigate the functional interplay of several membrane proteins (MP) on a molecular basis, they have to be extracted from their complex native environment and reconstituted into a well-defined membrane mimicking system such as liposomes. A good example for a functional interplay is oxidative phosphorylation in bacteria and mitochondria by the members of the respiratory chain (complex I-IV). Since every MP requires its own reconstitution protocol, we split the procedure in two steps. First, we reconstitute each purified MP into liposomes and in a second step, fuse the proteoliposomes. Different strategies to achieve liposome fusion have been described such as the use of SNARE proteins, fusogenic peptides, DNA oligomers, or oppositely charged lipids. We recently successfully applied SNARE-mediated fusion of proteoliposomes, but this method is limited to one round of fusion and therefore only interactions of two MPs can be studied. To investigate more complex systems, more than one round of liposome fusion might be necessary. We are therefore currently testing liposome fusion by oppositely charged lipids (DOPG/ DOTAP) as an alternative to SNARE-dependent fusion. Using fluorescent lipid mixing assays and liposome size determination, we established protocols for charge mediated liposome fusion in our hands and applied the optimised conditions to fuse liposomes containing respiratory chain enzymes. Using this strategy, it was possible to co-reconstitute different terminal oxidases and the E. coli ATP synthase, imitating the last step of oxidative phosphorylation. The oxidase thereby creates an electrochemical proton gradient that energizes the ATP synthase, requiring an intact (proton tight) lipid bilayer after the fusion process. We applied the same technology to incorporate purified ATP synthase into inverted membrane vesicles from an ATP synthase deficient E. coli strain, successfully restoring respiratory driven ATP synthesis in these vesicles.

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