Receptor-Ligand Interactions: Binding Affinities Studied by Single-Molecule and Super-Resolution Microscopy on Intact Cells

• Published in: Chemphyschem Vol. 15; no. 4; pp. 671 - 676
• Main Authors: Dietz, Marina S., Fricke, Franziska, Krüger, Carmen L., Niemann, Hartmut H., Heilemann, Mike
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 17.03.2014; WILEY‐VCH Verlag; Wiley; Wiley Subscription Services, Inc
• Subjects: Binding Sites; Chemistry; Colloidal state and disperse state; Exact sciences and technology; fluorescence microscopy; General and physical chemistry; HeLa Cells; Humans; Ligands; Membranes; MET receptor; Microscopy - methods; protein-protein interactions; Receptors, Cytoplasmic and Nuclear - chemistry; Signal transduction; single-molecule studies; TNF receptor 1; Tumor Cells, Cultured; Binding; MET receptor; Dissociation constant; Protein; TNF receptor 1; protein-protein interactions; single-molecule studies; Fluorophore; Imaging; Affinity; Membrane; Chemical properties; Fluorescence microscopy
• ISSN: 1439-4235; 1439-7641
• DOI: 10.1002/cphc.201300755

Protein–ligand interactions play an important role in many biological processes. Notably, membrane receptors are the starting point for a huge variety of cellular signal transduction pathways. Quantifying the binding affinity of a ligand for its transmembrane receptor is of great importance as it provides information on the potency of the ligand. We developed a new experimental procedure to determine binding affinities of ligands for their membrane receptors directly on intact single cells using super‐resolution imaging. Dissociation constants were determined by titrating fluorophore‐labelled ligand against cells expressing the target protein and applying single‐molecule imaging. Single‐molecule and super‐resolution fluorescence microscopy are applied to determine receptor copy numbers and binding affinities on intact cells. The authors demonstrate the validity of the approach, by addressing MET receptor and TNF receptor 1, and determine dissociation constants in the nM range.