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  Binding of His-tagged fluorophores to lipid bilayers and giant vesicles

Pramanik, S., Steinkühler, J., Dimova, R., Spatz, J., & Lipowsky, R. (2022). Binding of His-tagged fluorophores to lipid bilayers and giant vesicles. bioRxiv, 2022.02.01.478643. doi:10.1101/2022.02.01.478643.

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Pramanik, Shreya1, Author              
Steinkühler, Jan2, Author              
Dimova, Rumiana2, Author              
Spatz, Joachim, Author
Lipowsky, Reinhard1, Author              
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1Reinhard Lipowsky, Theorie & Bio-Systeme, Max Planck Institute of Colloids and Interfaces, Max Planck Society, ou_1863327              
2Rumiana Dimova, Theorie & Bio-Systeme, Max Planck Institute of Colloids and Interfaces, Max Planck Society, ou_1863328              

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 Abstract: His-tagged molecules can be attached to lipid bilayers via certain anchor lipids, a method that has been widely used for the biofunctionalization of membranes and vesicles. To measure the coverage by the membrane-bound molecules, it is useful to study molecules that are fluorescent as well. Here, we use two such molecules, green fluorescence protein (GFP) and green-fluorescent fluorescin isothiocyanate (FITC), both of which are tagged with a chain of six histidines that bind to achor lipids within the bilayers. This His-tag is much smaller in size than the GFP molecule but somewhat larger than the FITC dye. The lipid bilayers form giant unilamellar vesicles (GUVs), the behavior of which can be directly observed in the optical microscope. Several protocols for the preparation of GUVs have been developed. We apply and compare three well-established protocols based on polyvinyl alcohol (PVA) hydrogel swelling, electroformation on platinum wires, and electroformation on indium tin oxide (ITO) glass. For the same nanomolar concentration in the exterior solution, the coverage by His-tagged FITC is much lower than the one by His-tagged GFP. However, for both GFP and FITC, we find that the binding of the His-tagged molecules to the anchor lipids depends strongly on the preparation method. The highest binding affinitiy is obtained for electroformation on platinum wires. PVA gel swelling gives rise to a somewhat smaller binding affinity whereas electroformation on ITO glass leads to essentially no binding. Furthermore, the binding affinitiy is also observed to depend on the pH of the aqueous solution, with a relatively weak and strong pH-dependence for His-tagged GFP and His-tagged FITC, respectively.

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Language(s): eng - English
 Dates: 2022-02-032022
 Publication Status: Published in print
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 Identifiers: DOI: 10.1101/2022.02.01.478643
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Title: bioRxiv
Source Genre: Journal
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Publ. Info: Cold Spring Harbor : Cold Spring Harbor Laboratory
Pages: - Volume / Issue: - Sequence Number: 2022.02.01.478643 Start / End Page: - Identifier: ZDB: 2766415-6