Article abstract
Nature Methods 5, 1053 - 1060 (2008)
Published online: 9 November 2008 | doi:10.1038/nmeth.1268
Micropatterning for quantitative analysis of protein-protein interactions in living cells
Michaela Schwarzenbacher1, Martin Kaltenbrunner1, Mario Brameshuber1, Clemens Hesch1, Wolfgang Paster2, Julian Weghuber1, Bettina Heise3, Alois Sonnleitner4, Hannes Stockinger2 & Gerhard J Schütz1
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.
- Biophysics Institute, Johannes Kepler University Linz, Altenbergerstr. 69, A-4040 Linz, Austria.
- Department of Molecular Immunology, Center for Physiology, Pathophysiology and Immunology, Medical University of Vienna, Lazarettgasse 19, A-1090 Vienna, Austria.
- Department of Knowledge-based Mathematical Systems, Johannes Kepler University Linz, Altenbergerstr. 69, A-4040 Linz, Austria.
- Center for Biomedical Nanotechnology, Upper Austrian Research GmbH, Scharitzerstr. 6-8, A-4020 Linz, Austria.
Correspondence to: Gerhard J Schütz1 e-mail: gerhard.schuetz@jku.at
Correspondence to: Hannes Stockinger2 e-mail: hannes.stockinger@meduniwien.ac.at
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