Incorporation of membrane proteins into giant unilamellar vesicles: Towards complex cell mimetic systems

Schick, Thomas (2019). Incorporation of membrane proteins into giant unilamellar vesicles: Towards complex cell mimetic systems (Unpublished). (Dissertation, University of Bern, Faculty of Science of the University of Bern)

Text PhD Thesis Thomas Schick _ Incorporation of soluble and membrane proteins into giant unilamellar vesicles.pdf - Submitted Version Restricted to registered users only Available under License BORIS Standard License. Download (4MB)

All cells share certain structural features. These are a nucleus (eukaryotes) or nucleoid (prokaryotes) and a plasma membrane. The plasma membrane is composed of lipids and proteins and defines the periphery of the cell. It enables the accumulation of molecules in a defined area, a crucial feature for all domains of life. Being capable to create artificial membranes enables to study the properties of membrane proteins and membrane properties.
Artificial membranes can vary in size from a few nanometers up to several micrometers. Vesicles with diameters of at least 1 micron can easily be observed using light microscopy. This opens the possibility of doing single vesicle measurements and gives several advantages such as requiring reduced amounts of protein to investigate them and enabling kinetic measurements in vesicles. It further enables encapsulation of smaller compartments, a crucial feature of all eukaryotic cells. One of the aims of this PhD thesis is to establish the formation of giant unilamellar vesicles (GUVs) as a model system in our laboratory to study membrane proteins. We decided to focus on the electroformation technique which is based on swelling thin, dried lipid films in presence of an electric
field. We established a new reconstitution method for membrane proteins into GUVs using oppositely charged lipids to induce membrane fusion. This fusion strategy should allow coreconstitution of a large number of different proteins by inserting each protein first into small liposomes, followed by fusion of different liposome populations to GUVs. We observed enzymatic activity of the ATP synthase from E. coli and the sodium/proton transporter NapA from Thermus thermophilus in real time using light microscopy.
In the second part of this thesis we aimed to characterise the human antiporter NHA2. Based on genetics and sequence similarity, it is suggested that NHA2 is an endosomal sodium/proton antiporter.
We overexpressed and purified different isoforms of NHA2 including a functionally inactive mutant with the aim to characterise them using a fluorescence-based assays. Therefore, NHA2 was incorporated into liposomes and proton or sodium gradients were applied to induce cation exchange. Additionally, we wanted to test specific inhibitors of NHA2, synthesized by the collaborating group of Prof. Jean-Louis Reymond. However, we were not able to detect significant cation exchange of NHA2 compared to the inactive mutant.

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