Protein-Glycolipid Interactions Studied by Immobilized Lipid Bilayer Nanodiscs.

Description

Nanodiscs are self-assembled soluble discoidal phospholipid bilayers encirculated by an amphipatic protein that together provide a functional stabilized membrane disc for the incorporation of membrane-bound and membrane associated molecules. Integral membrane proteins in nanodiscs adopt a uniform environment that resembles biological membranes and where the lipid composition in the immediate surroundings of the protein can be controlled. Additionally, its oligomerization state can be varied by careful control of protein:MPS stoichometry and by selection of MSP with a suitable length that leads to a disc area that can accommodate the desired oligomerization state. The scope of the present work is to investigate how Nanodiscs and their incorporated membrane receptors can be attached to surface plasmon resonance sensorchips and used to measure the kinetics of the interaction between soluble molecules and membrane receptors inserted in the bilayer of nanodiscs. Cholera Toxin and its glycolipid receptor GM1 constitute a system that can be considered a paradigm for interactions of soluble proteins with membrane receptors. In this work, we have investigated different technologies for capturing nanodisc containing the glycolipid receptor GM1 in lipid bilayers, enabling measurements of binding of its soluble interaction partner Cholera Toxin B subunit to the receptor with the sensorchip based Surface Plasmon Resonance (SPR) technology. The measured stoichiometric and kinetic values of the interaction are in agreement with those reported by previous studies providing evidence that nanodiscs can be employed for kinetic SPR studies.

In addition to immobilization on sensorchips we have used FLAG-tagged GM1 nanodiscs for co-immunoprecipitation with FLAG antibody-coated agarose beads from enterotoxigenic E. coli lysates. The co-immunoprecipitated protein was identified by mass spectrometry as heat-labile toxin, a homolog of cholera toxin.