Biophysical Society Thematic Meeting | Ascona, Switzerland

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

Poster Abstracts

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.

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