Publikation

R. Wawrzinek, J. Ziomkowska, J. Heuveling, M. Mertens, A. Herrmann, E. Schneider, P. Wessig
DBD Dyes as Fluorescence Lifetime Probes to Study Conformational Changes in Proteins
Chemistry Eur. J. 2013, 19, 17349-17357
DOI: 10.1002/chem.201302368
Previously described [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD)-based fluorophores have been used as highly sensitive fluorescence lifetime probes reporting on their microenvironmental polarity. Now, a new generation of DBD dyes has been developed. Still being sensitive to the polarity, contrary to former DBD dyes, they have extraordinary spectroscopic properties, even in aqueous surroundings. They are characterized by long fluorescence lifetimes (10-20 ns), large Stokes shifts (~100 nm), high photostability and high quantum yields (>0.56). Here, spectroscopic properties and the synthesis of functionalized derivates to label biological targets are described. Furthermore thio-reactive maleimido derivates of both DBD generations show strong intramolecular fluorescence quenching. This mechanism has been investigated and found to undergo a photo electron transfer (PET). Once reacted with a thiol group, this fluorescence quenching is prevented, indicating successful bonding. Sensitive to their environmental polarity, these compounds were used as powerful fluorescence lifetime probes to investigate conformational changes in the maltose ATP-binding cassette transporter via fluorescence lifetime spectroscopy. The differing tendencies of fluorescence lifetime change for both DBD dye generations promote their combination as a powerful toolkit to study microenvironments in proteins.
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