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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.