Micropatterning for quantitative analysis of protein-protein interactions in living cells

Michaela Schwarzenbacher, Martin Kaltenbrunner, Mario Brameshuber, Clemens Hesch, Wolfgang Paster, Julian Weghuber, Bettina Heise, Alois Sonnleitner, Hannes Stockinger, Gerhard J. Schütz

Research output: Contribution to journalArticlepeer-review

87 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)1053-1060
Number of pages8
JournalNature Methods
Volume5
Issue number12
DOIs
Publication statusPublished - 2008

Keywords

  • Biological Assay/methods
  • Cell Membrane/metabolism
  • Membrane Proteins/metabolism
  • Microscopy, Fluorescence/methods
  • Protein Interaction Mapping/methods
  • Surface Properties

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