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  Introducing a fluorescence-based standard to quantify protein partitioning into membranes

Thomas, F. A., Visco, I., Petrasek, Z., Heinemann, F., & Schwille, P. (2015). Introducing a fluorescence-based standard to quantify protein partitioning into membranes. Biochimica et Biophysica Acta-Biomembranes, 1848(11), 2932-2941. doi:10.1016/j.bbamem.2015.09.001.

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 Creators:
Thomas, Franziska A.1, Author           
Visco, Ilaria1, Author           
Petrasek, Zdenek1, Author           
Heinemann, Fabian1, Author           
Schwille, Petra1, Author           
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1Schwille, Petra / Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, Max Planck Society, ou_1565169              

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Free keywords: SURFACE-GENERATED FLUORESCENCE; ALPHA-SYNUCLEIN BINDING; CORRELATION SPECTROSCOPY; UNILAMELLAR VESICLES; BILAYER INTERACTIONS; PERIPHERAL PROTEINS; LIPID INTERACTIONS; HEXAHISTIDINE-TAG; DIFFUSION; DYNAMICSPartition coefficient; Giant unilamellar vesicles; Fluorescence correlation spectroscopy; Confocal imaging;
 Abstract: The affinity of peripheral membrane proteins for a lipid bilayer can be described using the partition coefficient (K-P). Although several methods to determine K-P are known, all possess limitations. To address some of these issues, we developed both: a versatile method based on single molecule detection and fluorescence imaging for determining K-P, and a simple measurement standard employing hexahistidine-tagged enhanced green fluorescent protein (eGFP-His(6)) and free standing membranes of giant unilamellar vesicles (GUVs) functionalized with NTA(Ni) lipids as binding sites. To ensure intrinsic control, our method features two measurement modes. In the single molecule mode, fluorescence correlation spectroscopy (FCS) is applied to quantify free and membrane associated protein concentrations at equilibrium and calculate K-P. In the imaging mode, confocal fluorescence images of GUVs are recorded and analyzed with semi-automated software to extract protein mean concentrations used to derive K-P. Both modes were compared by determining the affinity of our standard, resulting in equivalent K-P values. As observed in other systems, eGFP-His(6) affinity for membranes containing increasing amounts of NTA(Ni) lipids rises in a stronger-than-linear fashion. We compared our dual approach with a FCS-based assay that uses large unilamellar vesicles (LUVs), which however fails to capture the stronger-than-linear trend for our NTA(Ni)-His(6) standard. Hence, we determined the K-P of the MARCKS effector domain with our FCS approach on GUVs, whose results are consistent with previously published data using LUVs. We finally provide a practical manual on how to measure K-P and understand it in terms of molecules per lipid surface. (C) 2015 Elsevier B.V. All rights reserved.

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Language(s): eng - English
 Dates: 2015
 Publication Status: Published in print
 Pages: 10
 Publishing info: -
 Table of Contents: Referred to by
Franziska A. Thomas, Ilaria Visco, Zdeněk Petrášek, Fabian Heinemann, Petra Schwille
Diffusion coefficients and dissociation constants of enhanced green fluorescent protein binding to free standing membranes
Data in Brief, Volume 5, December 2015, Pages 537-541
 Rev. Type: Peer
 Degree: -

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Title: Biochimica et Biophysica Acta-Biomembranes
Source Genre: Journal
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Publ. Info: Amsterdam : Elsevier
Pages: - Volume / Issue: 1848 (11) Sequence Number: - Start / End Page: 2932 - 2941 Identifier: ISSN: 0005-2736
CoNE: https://pure.mpg.de/cone/journals/resource/954926938702