DBD dyes as fluorescence lifetime probes to study conformational changes in proteins.
Previously, [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD)-based fluorophores used as highly sensitive fluorescence lifetime probes reporting on their microenvironmental polarity have been described. Now, a new generation of DBD dyes has been developed. Although they are still sensitive to polarity, in contrast to the 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 photostabilities, and high quantum yields (>0.56). Here, the spectroscopic properties and synthesis of functionalized derivatives for labeling biological targets are described. Furthermore, thio-reactive maleimido derivatives of both DBD generations show strong intramolecular fluorescence quenching. This mechanism has been investigated and is found to undergo a photoelectron transfer (PET) process. After reaction with a thiol group, this fluorescence quenching is prevented, indicating successful bonding. Being sensitive to their environmental polarity, these compounds have been used as powerful fluorescence lifetime probes for the investigation of conformational changes in the maltose ATP-binding cassette transporter through fluorescence lifetime spectroscopy. The differing tendencies of the fluorescence lifetime change for both DBD dye generations promote their combination as a powerful toolkit for studying microenvironments in proteins.
Institut für Chemie, Universität, Potsdam Karl-Liebknecht-Str. 24-25, 14476 Potsdam (Germany), Fax: (+49) 331-977-5065.
Robert
R
Wawrzinek
Joanna
J
Ziomkowska
Johanna
J
Heuveling
Monique
M
Mertens
Andreas
A
Herrmann
Erwin
E
Schneider
Pablo
P
Wessig
D052117:Benzodioxoles / Q000138:chemical synthesis / Q000737:chemistry*; D004579:Electron Transport; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D058619:Maltose-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011789:Quantum Theory
D052117:Benzodioxoles; D005456:Fluorescent Dyes; D058619:Maltose-Binding Proteins
MalF; dyes/pigments; electron transfer; fluorescent probes; maleimides; photoelectron transfer