Semisynthetic fluorescent sensor proteins based on self-labeling protein tags.
Genetically encoded fluorescent sensor proteins offer the possibility to probe the concentration of key metabolites in living cells. The approaches currently used to generate such fluorescent sensor proteins lack generality, as they require a protein that undergoes a conformational change upon metabolite binding. Here we present an approach that overcomes this limitation. Our biosensors consist of SNAP-tag, a fluorescent protein and a metabolite-binding protein. SNAP-tag is specifically labeled with a synthetic molecule containing a ligand of the metabolite-binding protein and a fluorophore. In the labeled sensor, the metabolite of interest displaces the intramolecular ligand from the binding protein, thereby shifting the sensor protein from a closed to an open conformation. The readout is a concomitant ratiometric change in the fluorescence intensities of the fluorescent protein and the tethered fluorophore. The observed ratiometric changes compare favorably with those achieved in genetically encoded fluorescent sensor proteins. Furthermore, the modular design of our sensors permits the facile generation of ratiometric fluorescent sensors at wavelengths not covered by autofluorescent proteins. These features should allow semisynthetic fluorescent sensor proteins based on SNAP-tag to become important tools for probing previously inaccessible metabolites.
Brun
Matthias A
MA
Institute of Chemical Sciences and Engineering, Ecole Polytechnique Federale de Lausanne (EPFL), CH-1015, Lausanne, Switzerland.
Tan
Kui-Thong
KT
Nakata
Eiji
E
Hinner
Marlon J
MJ
Johnsson
Kai
K
D015374:Biosensing Techniques / Q000379:methods*; D024402:Carbonic Anhydrase II / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006801:Humans; D008024:Ligands; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013449:Sulfonamides / Q000032:analysis; D015032:Zinc / Q000032:analysis
D005456:Fluorescent Dyes; D008024:Ligands; D011506:Proteins; D013449:Sulfonamides; D024402:Carbonic Anhydrase II; D015032:Zinc