Two-Dimensional Trap for Ultrasensitive Quantification of Transient Protein Interactions.
We present an ultrasensitive technique for quantitative protein-protein interaction analysis in a two-dimensional format based on phase-separated, micropatterned membranes. Interactions between proteins captured to lipid probes via an affinity tag trigger partitioning into the liquid-ordered phase, which is readily quantified by fluorescence imaging. Based on a calibration with well-defined low-affinity protein-protein interactions, equilibrium dissociation constants >1 mM were quantified. Direct capturing of proteins from mammalian cell lysates enabled us to detect homo- and heterodimerization of signal transducer and activator of transcription proteins. Using the epidermal growth factor receptor (EGFR) as a model system, quantification of low-affinity interactions between different receptor domains contributing to EGFR dimerization was achieved. By exploitation of specific features of the membrane-based assay, the regulation of EGFR dimerization by lipids was demonstrated.
Department of Biology, University of Osnabrück , 49074 Osnabrück, Germany.
Oliver
O
Beutel
Department of Biology, University of Osnabrück , 49074 Osnabrück, Germany.
Friedrich
F
Roder
Department of Biology, University of Osnabrück , 49074 Osnabrück, Germany.
Oliver
O
Birkholz
Department of Physics, University of Osnabrück , 49076 Osnabrück, Germany.
Christian
C
Rickert
Department of Physics, University of Osnabrück , 49076 Osnabrück, Germany.
Heinz-Jürgen
HJ
Steinhoff
Paul Langerhans Institute Dresden of the Helmholtz Centre Munich at the University Clinic Carl Gustav Carus TU Dresden , 01307 Dresden, Germany.|German Center for Diabetes Research (DZD) , 85764 Neuherberg, Germany.
Michał
M
Grzybek
Paul Langerhans Institute Dresden of the Helmholtz Centre Munich at the University Clinic Carl Gustav Carus TU Dresden , 01307 Dresden, Germany.|German Center for Diabetes Research (DZD) , 85764 Neuherberg, Germany.
Ünal
Ü
Coskun
Department of Biology, University of Osnabrück , 49074 Osnabrück, Germany.
Jacob
J
Piehler
D000818:Animals; D004867:Equipment Design; D066246:ErbB Receptors / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D008567:Membranes, Artificial; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D008958:Models, Molecular; D061848:Optical Imaging / Q000295:instrumentation* / Q000379:methods; D044367:Phase Transition; D025941:Protein Interaction Mapping / Q000295:instrumentation* / Q000379:methods; D060066:Protein Interaction Maps; D055503:Protein Multimerization; D015398:Signal Transduction
D008055:Lipids; D008567:Membranes, Artificial; D066246:ErbB Receptors
fluorescence microscopy; lipid phase separation; polymer-supported membrane; protein-lipid interaction; protein−protein interaction; signaling complexes