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Analysis of Interactions between the Epidermal Growth Factor Receptor and Soluble Ligands on the Basis of Single‐Molecule Diffusivity in the Membrane of Living Cells
Author(s) -
Kim DoHyeon,
Zhou Kai,
Kim DongKyun,
Park Soyeon,
Noh Jungeun,
Kwon Yonghoon,
Kim Dayea,
Song Nam Woong,
Lee JongBong,
Suh PannGhill,
Lee Nam Ki,
Ryu Sung Ho
Publication year - 2015
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201500871
Subject(s) - biophysics , membrane , chemistry , receptor , cooperativity , thermal diffusivity , ligand (biochemistry) , cell surface receptor , cooperative binding , molecule , membrane protein , diffusion , epidermal growth factor , biochemistry , binding site , biology , physics , organic chemistry , quantum mechanics , thermodynamics
We present a single‐molecule diffusional‐mobility‐shift assay (smDIMSA) for analyzing the interactions between membrane and water‐soluble proteins in the crowded membrane of living cells. We found that ligand–receptor interactions decreased the diffusional mobility of ErbB receptors and β‐adrenergic receptors, as determined by single‐particle tracking with super‐resolution microscopy. The shift in diffusional mobility was sensitive to the size of the water‐soluble binders that ranged from a few tens of kilodaltons to several hundred kilodaltons. This technique was used to quantitatively analyze the dissociation constant and the cooperativity of antibody interactions with the epidermal growth factor receptor and its mutants. smDIMSA enables the quantitative investigation of previously undetected ligand–receptor interactions in the intact membrane of living cells on the basis of the diffusivity of single‐molecule membrane proteins without ligand labeling.