Abstract
Proteomic identification of protein interactions with membrane associated molecules in their native membrane environment pose a challenge because of technical problems of membrane handling. We investigate the possibility of employing membrane nanodiscs for harboring the membrane associated molecule to tackle the challenges. Nanodiscs are stable, homogenous pieces of membrane with a discoidal shape. They are stabilized by an encircling amphipatic protein with an engineered epitope tag. In the present study we employ the epitope tag of the nanodiscs for detection and co-immunoprecipitation of interaction partners of the glycolipid ganglioside GM1 harbored by nanodiscs. Highly specific binding activity for nanodisc-GM1 immobilized on sensorchips was observed by surface plasmon resonance in culture media from enterotoxigenic Escherischia coli. To isolate the interaction partner(s) from enterotoxigenic Escherischia coli, GM1-nanodiscs were employed for co-immunoprecipitation. The B subunit of heat labile enterotoxin was identified as a specific interaction partner by mass spectrometry, thus demonstrating that nanodisc technology is useful for highly specific detection and identification of interaction partners to specific lipids embedded in a membrane bilayer.
Highlights
The ability of biomolecules to interact with surrounding substances is essential for their function
In the Biacore system that we used for surface plasmon resonance (SPR) measurements, a continuous flow of running buffer or analyte is directed over a sensor chip to which a ligand is attached
The refractive index change accompanied with injection of a binding analyte is composed of the increment in refractive index caused by the up-concentration of analyte in the vicinity of the sensor chip caused by binding to the immobilized ligand plus the change in refractive index caused by mismatch between the refractive index of the analyte solution and running buffer
Summary
The ability of biomolecules to interact with surrounding substances is essential for their function. Our aim is to develop methods to isolate and identify proteins that associate with membrane embedded molecules. Even nonionic detergents that are often considered nondenaturing can change the properties of membrane embedded biomolecules by stripping off the membrane exposing hydrophobic areas of the molecule. Membrane associated molecules can be reconstituted in phospholipid membranes as part of synthetic vesicles This approach has disadvantages because vesicles are prone to aggregation because of curvature of the membrane, which exposes hydrophobic sites because of cracks in the hydrophilic surface. This may be detrimental to reconstituted integral membrane proteins [1,2,3].
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