Biophysical Society Thematic Meeting

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.