Establishment of a fluorescence assay for characterization of protein-mediated vesicle fusion and acidification
von Miriam Schwamborn
Datum der mündl. Prüfung:2017-05-24
Betreuer:Prof. Dr. Claudia Steinem
Gutachter:Prof. Dr. Claudia Steinem
Gutachter:Prof. Dr. Mikael Simons
EnglischMembrane proteins mediate a manifold of essential transport processes across the lipid membrane of cells and organelles, therewith representing important drug targets. To investigate protein-mediated transport in vitro, model membrane systems separating two aqueous compartments are required. Pore-spanning lipid bilayers consist of an array of defined cavities in a solid substrate, sealed by a solvent-free lipid membrane, thus meeting the condition for observation of membrane protein mediated active transport. In the current preparation of pore-spanning lipid bilayers physiological conditions, which are crucial to retain the functionality of most membrane proteins, are not permanently maintained. SNARE-proteins mediate membrane fusion and may be used for protein reconstitution into pore-spanning lipid bilayers under physiological conditions. Therefore, the feasibility of a physiological reconstitution method based on SNARE-protein mediated fusion was examined in this work. For this approach the robust FoF1-ATPase (ATP synthase) from a thermophilic bacterium was chosen as an exemplary proton pumping protein. After isolation of the recombinant protein from Escherichia coli, its reconstitution efficiency into lipid vesicles was characterized and the ATPase induced vesicle acidification was detected with a membrane-permeable fluorophore. The lipid-coupled fluorophore Oregon Green 488 allowed to quantify the average luminal pH-value of acidified vesicles to ΔpH = −0.45 (pH 7.3 → pH 6.85). After successful detergent-mediated ATPase reconstitution, a combined fusion and acidification assay for small proteoliposomes, based on the lipid-coupled Oregon Green 488, was introduced. First, the fusion of SNARE-ATPase-vesicles with SNARE-Oregon Green 488-vesicles was monitored by a dequenching of the fluorophore. This phenomenon resulted from the fluorophore dilution by fusion and allowed to quantify the lipid mixing efficiency to 59 %. Through successful fusion the FoF1-ATPase should come in contact with the Oregon Green 488, thus allowing to detect acidification in the fusion products. However, acidification of the fusion products was not detectable with Oregon Green 488 in any case, while a general ATPase activity after the fusion process could be proven with the membrane permeable fluorophore. To assess whether a heterogeneous protein distribution in the fusing vesicle populations might explain the undetectable acidification of fusion products, a fluorescence microscopy based single vesicle assay was established. In this assay, FoF1-ATPase was reconstituted into biotinylated, Oregon Green 488-doped vesicles that were immobilized on a NeutrAvidin functionalized glass surface. Only 5 % of all singles vesicles showed a clearly distinguishable ATPase-induced acidification (ΔpH = −0.5 to −2.0, average: ΔpH ≈ −1). Averaging the luminal acidification of all immobilized vesicles resulted in ΔpH = −0.47, which was in good agreement with the average luminal pH decrease (ΔpH = −0.45) obtained from the bulk acidification experiments. In order to use SNARE-protein mediated fusion as a reconstitution tool, the functional reconstitution of ATPase itself has to be improved towards a more homogeneous distribution of active protein. For this purpose, the established single vesicle assay will be very helpful.
Keywords: lipid-coupled pH indicator; FOF1-ATPase induced vesicle acidification; pH-quantification in vesicles; proton pump; single vesicle assay