title: Micropatterning for quantitative analysis of protein-protein interactions in living cells.
abstract: We present a method to identify and characterize interactions between a fluorophore-labeled protein ('prey') and a membrane protein ('bait') in live mammalian cells. Cells are plated on micropatterned surfaces functionalized with antibodies to the bait extracellular domain. Bait-prey interactions are assayed through the redistribution of the fluorescent prey. We used the method to characterize the interaction between human CD4, the major co-receptor in T-cell activation, and human Lck, the protein tyrosine kinase essential for early T-cell signaling. We measured equilibrium associations by quantifying Lck redistribution to CD4 micropatterns and studied interaction dynamics by photobleaching experiments and single-molecule imaging. In addition to the known zinc clasp structure, the Lck membrane anchor in particular had a major impact on the Lck-CD4 interaction, mediating direct binding and further stabilizing the interaction of other Lck domains. In total, membrane anchorage increased the interaction lifetime by two orders of magnitude.
authors.affiliation: Biophysics Institute, Johannes Kepler University Linz, Altenbergerstr. 69, A-4040 Linz, Austria.
authors.forename: Michaela
authors.initials: M
authors.lastname: Schwarzenbacher
authors.forename: Martin
authors.initials: M
authors.lastname: Kaltenbrunner
authors.forename: Mario
authors.initials: M
authors.lastname: Brameshuber
authors.forename: Clemens
authors.initials: C
authors.lastname: Hesch
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Paster
authors.forename: Julian
authors.initials: J
authors.lastname: Weghuber
authors.forename: Bettina
authors.initials: B
authors.lastname: Heise
authors.forename: Alois
authors.initials: A
authors.lastname: Sonnleitner
authors.forename: Hannes
authors.initials: H
authors.lastname: Stockinger
authors.forename: Gerhard J
authors.initials: GJ
authors.lastname: Schütz
mesh_terms: D001681:Biological Assay / Q000379:methods*; D002462:Cell Membrane / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D025941:Protein Interaction Mapping / Q000379:methods*; D013499:Surface Properties
chemical_list: D008565:Membrane Proteins