Entwicklung von "FRET-CAPTURE": Eine Technik die umgebungssensitive Fluorophore als FRET-Donor nutzt, um intermolekulare Protein-Protein-Wechselwirkungen und Proteindynamiken zu untersuchen
Zusammenfassung der Projektergebnisse
Sensors based on Förster Resonance Energy Transfer (FRET) are widely applied for identifying and analyzing macromolecular processes such as protein-protein interactions and conformational dynamics. However, macromolecular structural changes can frequently be too subtle to be detected using traditional FRET-based approaches. With “FRET-CAPTURE” some of the limitation of FRET can be overcome by exploiting solvatochromic fluorophores as donor. They show extremely low fluorescence in polar protic environments, but manifest in greatly enhanced fluorescence upon binding to hydrophobic environments. This approach is applied to both inter- and intramolecular FRET-CAPTURE using the calcium-dependent binding interaction of calmodulin with the M13 peptide as model system. The solvatochromic fluorophore 4-N,N-dimethylamino-1,8-naphthalimide (4DMN) was introduced as the fluorescence donor and a second non-solvatochromic dye served as the acceptor dye. Different donor/acceptor combinations are applied. The binding of the M13 peptide to the calcium-activated calmodulin modified the local environment of 4DMN and revealed a high FRET fluorescence increase of up-to 25-fold using the 4DMN/Alexa Fluor 555 pair. The method has been demonstrated to be useful in simple buffer systems, unfractionated cell lysates and on micro-patterned glass surfaces. The background fluorescence, depending on the FRET acceptor is either negligible or very low, and no washing steps are necessary to remove unbound or non-specific bound probes. The probes provide two readout modes after donor excitation to detect protein-interactions: I) Donor emission at 505 nm and II) FRET-CAPTURE emission depending on the acceptor from 570 – 644 nm, revealing a high apparent Stokes shift of more than 170 nm. The ability to apply acceptor fluorophores with unique emission properties further suggests that the method could be expanded to include the simultaneous application of two suitable FRET-pairs, which would enable multiplexing. Currently, FRET-CAPTURE is applied in sensing cytokines which serve as major signaling molecules in biology. In particular, ligands of the EGF family regulate key cellular processes such as proliferation, differentiation, and apoptosis. Deregulations have been associated with numerous malignancies. It is important to elucidate the cellular dynamics of cytokines in vivo using a non-invasive method. Therefore, a Fibronectin domain type III (FN3) based domain engineered to bind with low nanomolar affinity to human EGF has been labeled selectively with 4DMN. Varying linker length between the protein backbone and 4DMN are used to screen for optimal position that reveal low fluorescence background in the unbound state but a high fluorescence increase after binding to the target protein, respectively, without interfering with binding. A fluorescence increase of up-to 3-fold could be detected after binding to EGF. To guarantee a stable signal in a biological context a fluorescence increase of at least 10-fold is needed. One major issue hereby is the high background fluorescence generated by the interaction of 4DMN with the binder protein FN3 itself. Molecular modeling attempts are used to define suitable positions for the solvatochromic fluorophore with predicted low background fluorescence. This will reduce the need to test all possible linker length and positions and can be used as a general tool for the development of further sensors based on the FN3 system.
Projektbezogene Publikationen (Auswahl)
-
“FRET-CAPTURE: A Sensitive Method for the Detection of Dynamic Protein Interactions” ChemBioChem, 2013 14, 1, 53-57
E. Socher and B. Imperiali
-
“Two-Photon Fluorescence Spectroscopy and Imaging of 4- Dimethylaminonapththalimide Peptide and Protein Conjugates” J. Phys. Chem. B, 2013 117, 15935-15942
Alan M. McLean, Elke Socher, Oleg Varnavski, Travis B. Clark, Barbara Imperiali, and Theodore Goodson III
