Probing 2-dimensional protein-protein interactions on model membranes.
This protocol describes an in vitro approach for measuring the kinetics and affinities of interactions between membrane-anchored proteins. This method is particularly established for dissecting the interaction dynamics of cytokines with their receptor subunits. For this purpose, the receptor subunits are tethered in an orientated manner onto solid-supported lipid bilayers by using multivalent chelator lipids. Interaction between the ligand with the receptor subunits was probed by a combination of surface-sensitive spectroscopic detection techniques. Label-free detection by reflectance interferometry is used for following assembly of the membrane and tethering of the receptor subunits in quantitative terms. Total internal reflection spectroscopy is used for monitoring ligand binding to the membrane-anchored receptor, for monitoring ligand-receptor interactions by FRET and for monitoring ligand-exchange kinetics. These assays can be used for determining the affinities and stabilities of ligand-receptor complexes in plane of the membrane. The techniques described in this protocol can be established in 2-3 months.
Gavutis
Martynas
M
Institute of Biochemistry, Biocenter N210, Johann Wolfgang Goethe-University, Max-von-Laue Strasse 9, 60438 Frankfurt am Main, Germany.
Lata
Suman
S
Piehler
Jacob
J
D007368:Interferometry / Q000379:methods; D007700:Kinetics; D008024:Ligands; D008027:Light; D008567:Membranes, Artificial; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D008024:Ligands; D008567:Membranes, Artificial; D011956:Receptors, Cell Surface