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Liposomes, Exosomes, and Virosomes: From Modeling Complex
Membrane Processes to Medical Diagnostics and Drug Delivery
Poster Abstracts
102
24-POS
Board 12
From Model to Product: Using Proteorhodopsin to Drive a Molecular Hoover
Roland Goers
1,2
, Johannes Thoma
2
, Alfredo Di Silvestro
1
, Noah Ritzmann
2
, Claudio Alter
1
,
Dimitrios Fotiadis
3
, Daniel Müller
2
, Wolfgang Meier
1
.
1
University of Basel, Basel, Switzerland,
2
ETH Zürich, Basel, Switzerland,
3
University of Bern,
Bern, Switzerland.
The creation of biomimetic reaction compartments is part of the bottom-up approach in synthetic
biology. In order to fulfil a desired task, these systems often require transport of substrates and
products to/from their interior. Passive diffusion can be enabled by pores, whereas active and
controllable transport requires energy. Light-driven proton pumps such as proteorhodopsin (PR)
generate a fundamental electrochemical gradient (proton motif force) upon illumination. Modern
detergent-mediated membrane protein reconstitution procedures allow the integration of
membrane proteins into synthetic membranes, however, they usually lack control over the final
orientation of the proteins which is especially crucial for directional transporters like PR.
Furthermore, this process relies on the self-assembly of the membrane components without
direct control. Predetermined outcomes are only achieved by changing the starting conditions,
which requires detailed knowledge about key parameters.
We bypassed this issue by fusing green fluorescent protein (GFP) to PR, as the hydrophilic
nature of GFP drives its orientation upon reconstitution into preformed lipid and polymer
vesicles. Statistical modelling ‘Design of experiments (DoE)’ was used to identify significant
factors and further allows their optimization towards a desired outcome. We applied this
methodology rationally to find conditions, which lead to proper formation of proteolipo- and
proteopolymersomes.
The fluorescence of GFP allowed us to detect PR-GFP inside the membrane by fluorescence
correlation spectroscopy. It turned out that lipid and polymer membranes require different
treatments for successful reconstitution. Subsequently, the parameter space was narrowed down
by setting boundary conditions for the highest pumping activity. The internal pH of the
proteovesicles was measured via fluorescence spectroscopy and the final model was able to
predict the activity with a high precision. The model allows optimization of the process and
provides the relevant knowledge of key parameters at the same time.