Protein Proximity Observed Using Fluorogen Activating Protein and Dye Activated by Proximal Anchoring (FAP–DAPA) System
| Autor: | Cheryl A. Telmer, Yi Wang, Brigitte F. Schmidt, Marcel P. Bruchez, Zhipeng Yang, M Alexandra Carpenter |
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| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Hydrolases Recombinant Fusion Proteins Ligands 01 natural sciences Biochemistry Article Fluorescence Polyethylene Glycols Immunological synapse Tacrolimus Binding Proteins 03 medical and health sciences Coumarins Hydrocarbons Chlorinated Rosaniline Dyes Hexanes Humans Receptor Fluorescent Dyes 010405 organic chemistry Chemistry TOR Serine-Threonine Kinases HEK 293 cells General Medicine Ligand (biochemistry) Fusion protein 0104 chemical sciences HEK293 Cells 030104 developmental biology FKBP Microscopy Fluorescence Biophysics Molecular Medicine Intracellular Protein Binding |
| Zdroj: | ACS Chem Biol |
| ISSN: | 1554-8937 1554-8929 |
| DOI: | 10.1021/acschembio.0c00419 |
| Popis: | The development and function of tissues, blood, and the immune system is dependent upon proximity for cellular recognition and communication. However, the detection of cell-to-cell contacts is limited due to a lack of reversible, quantitative probes that can function at these dynamic sites of irregular geometry. Described here is a novel chemo-genetic tool developed for fluorescent detection of protein–protein proximity and cell apposition that utilizes the Fluorogen Activating Protein (FAP) in combination with a Dye Activated by Proximal Anchoring (DAPA). The FAP–DAPA system has two protein components, the HaloTag and FAP, expressed on separate protein targets or in separate cells. The proteins function to bind and activate a compound that has the hexyl chloride (HexCl) ligand connected to malachite green (MG), the FAP fluorogen, via a poly(ethylene glycol) spacer spanning up to 28 nm. The dehalogenase protein, HaloTag, covalently binds the HexCl ligand, locally concentrating the attached MG. If the FAP is within range of the anchored fluorogen, it will bind and activate MG specifically when the bath concentration is too low to saturate the FAP receptor. A new FAP variant was isolated with a 1000-fold reduced K(D) of ~10–100 nM so that the fluorogen activation reports proximity without artificially enhancing it. The system was characterized using purified FRB and FKBP fusion proteins and showed a doubling of fluorescence upon rapamycin induced complex formation. In cocultured HEK293 cells (HaloTag and FAP-expressing) fluorescence increased at contact sites across a broad range of labeling conditions, more reliably providing contact-specific fluorescence activation with the lower-affinity FAP variant. When combined with suitable targeting and expression constructs, this labeling system may offer significant improvements in on-demand detection of intercellular contacts, potentially applicable in neurological and immunological synapse measurements and other transient, dynamic biological appositions that can be perturbed using other labeling methods that stabilize these interactions. |
| Databáze: | OpenAIRE |
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