Micropatterning for quantitative analysis of protein-protein interactions in living cells.
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.
Schwarzenbacher
Michaela
M
Biophysics Institute, Johannes Kepler University Linz, Altenbergerstr. 69, A-4040 Linz, Austria.
Kaltenbrunner
Martin
M
Brameshuber
Mario
M
Hesch
Clemens
C
Paster
Wolfgang
W
Weghuber
Julian
J
Heise
Bettina
B
Sonnleitner
Alois
A
Stockinger
Hannes
H
Schütz
Gerhard J
GJ
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
D008565:Membrane Proteins