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

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-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 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-His₆ 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)-His₆ 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.