| dc.contributor.advisor | Steinem, Claudia Prof. Dr. | |
| dc.contributor.author | Kramer, Kristina | |
| dc.date.accessioned | 2022-11-22T16:02:06Z | |
| dc.date.available | 2022-11-29T00:50:11Z | |
| dc.date.issued | 2022-11-22 | |
| dc.identifier.uri | http://resolver.sub.uni-goettingen.de/purl?ediss-11858/14354 | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-9566 | |
| dc.format.extent | 999 | de |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by/4.0/ | |
| dc.subject.ddc | 571.4 | de |
| dc.title | Droplet-based giant unilamellar vesicles as a tool for pore-spanning membrane preparation and transmembrane protein reconstitution | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Steinem, Claudia Prof. Dr. | |
| dc.date.examination | 2022-04-22 | de |
| dc.description.abstracteng | Model membranes represent a central tool to decipher complex processes of living cells. Giant unilamellar vesicles (GUVs) are of particular interest as they are cell-like in size, observable with optical microscopy and starting point for the formation of pore-spanning membranes (PSMs). The latter combines merits of solid-supported and free-standing membranes as they provide high stability, separate two aqueous compartments and offer optimal conditions for studying e.g. transmembrane proteins. Thus, developing strategies for GUV production and simultaneous protein reconstitution under physiological conditions is of great interest. In this work, droplet-based microfluidics were employed as a tool for producing droplet-stabilized GUVs (dsGUVs) by encapsulation of large unilamellar vesicles (LUVs) in water-in-oil droplets. Subsequent de-emulsification allowed to release GUVs in an aqueous phase. Basically, a defined interplay of surfactants, ions and lipids was found to be essential for dsGUV assembly. It was shown that the method ensures flexible choice of the constituents allowing to adjust physiological conditions. Moreover, microfluidic-GUVs served for the formation of PSMs. Investigating the biophysical properties like lipid mobility and mechanics of microfluidic-derived membranes revealed that the lipid diffusion coefficient and bending modulus were unaltered compared to membranes derived from electroformation. In contrast, the lateral membrane tension of microfluidic-PSMs was found to be 1.5-fold decreased suggesting an impact of residual surfactants in the membranes. Moreover, reconstitution of two model transmembrane proteins (TFOF1 ATP synthase, synaptobrevin 2) in (ds)GUVs was probed. Initial detergent-mediated reconstitution in LUVs revealed that the choice of detergent influences the success of the following dsGUV assembly from the proteo-LUVs. Analysing the protein activity and reconstitution efficiency revealed loss of the reconstituted proteins during the microfluidic preparation and de-emulsification. Thus, the microfluidic approach appears to be promising for GUV production under biomimetic conditions, yet the reconstitution of proteins requires further adaptions. Besides, activity of TFOF1 was characterized in depth after functional reconstitution in LUVs by luminescence-based ATP synthesis assays as a function of the applied ΔpH across the lipid bilayer and thus of the proton motive force (pmf). Resulting turnover rates ranged from 0.042 s-1 to 13.0 s-1. Quantification of the acidification step of the ATP synthesis assay (required to generate the pmf) was performed by a pyranine-based proton influx assay and revealed incomplete acidification of the intravesicular volume. Hereby, it was shown that misinterpretation of the pmf and thus the protein activity may occur by applying common acid-base transition. | de |
| dc.contributor.coReferee | Grubmüller, Helmut Prof. Dr. | |
| dc.contributor.thirdReferee | Brose, Nils Prof. Dr. | |
| dc.contributor.thirdReferee | Walla, Peter Jomo Prof. Dr. | |
| dc.contributor.thirdReferee | Geil, Burkhard Prof. Dr. | |
| dc.contributor.thirdReferee | Simeth, Nadja Jun.-Prof. Dr. | |
| dc.subject.eng | TFOF1 ATP synthase | de |
| dc.subject.eng | droplet-based microfluidics | de |
| dc.subject.eng | pore-spanning membranes | de |
| dc.subject.eng | giant unilamellar vesicles | de |
| dc.subject.eng | protein reconstitution | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-ediss-14354-3 | |
| dc.affiliation.institute | Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB) | de |
| dc.subject.gokfull | Biologie (PPN619462639) | de |
| dc.description.embargoed | 2022-11-29 | de |
| dc.identifier.ppn | 1823235255 | |
| dc.notes.confirmationsent | Confirmation sent 2022-11-23T06:15:01 | de |