Determination of binding curves via protein micropatterning in vitro and in living cells

• Published in: Cytometry. Part A 2013-09, Vol.83 (9), p.847-854
• Main Authors: Sunzenauer, Stefan, Zojer, Verena, Brameshuber, Mario, Tröls, Andreas, Weghuber, Julian, Stockinger, Hannes, Schütz, Gerhard J.
• Format: Article
• Language: English
• Subjects: Antibodies; Bacterial Proteins - genetics; CD4; CD4 Antigens - metabolism; Cell Line, Tumor; Cell Membrane - metabolism; equilibrium binding constant; HEK293 Cells; Humans; Lck; Luminescent Proteins - genetics; Lymphocyte Specific Protein Tyrosine Kinase p56(lck) - metabolism; Micropatterning; Microscopy, Fluorescence; plasma membrane; Protein Binding; Protein Interaction Mapping; single molecule microscopy
• ISSN: 1552-4922; 1552-4930
• DOI: 10.1002/cyto.a.22225

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. © 2012 International Society for Advancement of Cytometry