Coiled-Coil Tag−Probe System for Quick Labeling of Membrane Receptors in Living Cells

Abstract

The specific labeling of proteins in living cells using a genetically encodable tag and a small synthetic probe targeting the tag has been craved as an alternative to widely used larger fluorescent proteins. We describe a rapid method with a small tag (21 amino acids) for the fluorescence labeling of cell-surface receptors using a high affinity coiled-coil formation without metals or enzymes. The peptide probes K3 (KIAALKE)3 and K4 (KIAALKE)4 labeled with a fluorophore specifically stained the surface-exposed tag sequence E3 (EIAALEK)3 attached to the N-terminus of the mouse-derived prostaglandin EP3 receptor in living cells (Kd = 64 and 6 nM for K3 and K4, respectively). The labeling was quick (<1 min), nontoxic, and available even in culture medium without affecting receptor function. As an application of this tractable method, the agonist-induced internalization of the human-derived 2-adrenergic receptor and epidermal growth factor receptor was successfully visualized.

Induced heterodimerization and purification of two target proteins by a synthetic coiled‐coil tag

“…12 E3/K3 has also recently been used as a fluorescent marker to study surface membrane receptors and their internalization, 24 which suggests that it can also be used in eukaryotic cells. The K3 peptide has a tendency to homodimerize at higher concentrations, as observed in Ref.…”

Fluorescence ratiometric detection of ligand-induced receptor internalization using extracellular coiled-coil tag-probe labeling

“…We recently developed a coiled-coil tag-probe labeling method based on the peptide pair (E3 tag (EIAALKE) 3 and K4 probe (KIA-ALEK) 4 ) that forms a heterodimeric coiled-coil structure ( Fig. 2A) [8]. The tight interaction (apparent K d $ 6 nM) is suitable for long-time observation.…”

Selbstorganisation von Coiled‐Coils in der synthetischen Biologie: Inspiration und Fortschritt

“…Das Sondenpeptid mit vier Heptaden erwies sich als die effektivere Markierung. [156] Abbildung 25. Vesikelaggregation durch Coiled-Coil-Faltung und Wechselwirkung des kationischen Coiled-Coil mit negativ geladenen Membranlipiden.…”