Two-Dimensional Trap for Ultrasensitive Quantification of Transient Protein Interactions.

Oliver Beutel, Friedrich Roder, Oliver Birkholz, Christian Rickert, Heinz-Jürgen Steinhoff, Michał Grzybek, Ünal Coskun, Jacob Piehler

Index: ACS Nano 9 , 9783-91, (2015)

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Abstract

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.