Two-Dimensional Trap for Ultrasensitive Quantification of Transient Protein Interactions

被引:7
作者
Beutel, Oliver [1 ]
Roder, Friedrich [1 ]
Birkholz, Oliver [1 ]
Rickert, Christian [2 ]
Steinhoff, Heinz-Juergen [2 ]
Grzybek, Michal [3 ,4 ]
Coskun, Uenal [3 ,4 ]
Piehler, Jacob [1 ]
机构
[1] Univ Osnabruck, Dept Biol, D-49074 Osnabruck, Germany
[2] Univ Osnabruck, Dept Phys, D-49076 Osnabruck, Germany
[3] Tech Univ Dresden, Univ Clin Carl Gustav Carus, Helmholtz Ctr Munich, Paul Langerhans Inst Dresden, D-01307 Dresden, Germany
[4] German Ctr Diabet Res DZD, D-85764 Neuherberg, Germany
关键词
protein-protein interaction; polymer-supported membrane; lipid phase separation; fluorescence microscopy; signaling complexes; protein-lipid interaction; BIMOLECULAR FLUORESCENCE COMPLEMENTATION; POLYMER-SUPPORTED MEMBRANES; GROWTH-FACTOR RECEPTOR; LIVING CELLS; EGF RECEPTOR; PLASMA-MEMBRANE; LIPID-BILAYERS; BINDING-SITE; DOMAIN; ACTIVATION;
D O I
10.1021/acsnano.5b02696
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
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.
引用
收藏
页码:9783 / 9791
页数:9
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