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

Biochimica Et Biophysica Acta
Franziska A ThomasPetra Schwille

Abstract

The affinity of peripheral membrane proteins for a lipid bilayer can be described using the partition coefficient (KP). Although several methods to determine KP 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 KP, and a simple measurement standard employing hexahistidine-tagged enhanced green fluorescent protein (eGFP-His6) 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 KP. 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 KP. Both modes were compared by determining the affinity of our standard, resulting in equivalent KP values. As observed in other systems, eGFP-His6 affinity for membranes containing increasing amounts of NTA(Ni) lipids rises in a stronger-than-line...Continue Reading

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Citations

Nov 4, 2016·Journal of Molecular Biology·Ilaria ViscoPetra Schwille
Feb 25, 2018·Nature Communications·Henri G FranquelimPetra Schwille
Mar 23, 2021·Nature Communications·Konstantin GavriljukPhilippe I H Bastiaens
Sep 27, 2018·Langmuir : the ACS Journal of Surfaces and Colloids·Alena KhmelinskaiaPetra Schwille
Nov 23, 2019·Analytical Chemistry·Alison SuAmy E Herr

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