title: Determination of binding curves via protein micropatterning in vitro and in living cells.
abstract: Quantification of protein interactions in living cells is of key relevance for understanding cellular signaling. With current techniques, however, it is difficult to determine binding affinities and stoichiometries of protein complexes in the plasma membrane. We introduce here protein micropatterning as a convenient and versatile method for such investigations. Cells are grown on surfaces containing micropatterns of capture antibody to a bait protein, so that the bait gets rearranged in the live cell plasma membrane. Upon interaction with the bait, the fluorescent prey follows the micropatterns, which can be readout with fluorescence microscopy. In this study, we addressed the interaction between Lck and CD4, two central proteins in early T-cell signaling. Binding curves were recorded using the natural fluctuations in the Lck expression levels. Surprisingly, the binding was not saturable up to the highest Lck expression levels: on average, a single CD4 molecule recruited more than nine Lck molecules. We discuss the data in view of protein- and lipid-mediated interactions.
authors.affiliation: Biophysics Institute, Johannes Kepler University Linz, A-4040 Linz, Austria.
authors.forename: Stefan
authors.initials: S
authors.lastname: Sunzenauer
authors.forename: Verena
authors.initials: V
authors.lastname: Zojer
authors.forename: Mario
authors.initials: M
authors.lastname: Brameshuber
authors.forename: Andreas
authors.initials: A
authors.lastname: Tröls
authors.forename: Julian
authors.initials: J
authors.lastname: Weghuber
authors.forename: Hannes
authors.initials: H
authors.lastname: Stockinger
authors.forename: Gerhard J
authors.initials: GJ
authors.lastname: Schütz
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D015704:CD4 Antigens / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D025941:Protein Interaction Mapping
chemical_list: D001426:Bacterial Proteins; D015704:CD4 Antigens; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)
keywords: CD4; Lck; Micropatterning; equilibrium binding constant; plasma membrane; single molecule microscopy