DBD dyes as fluorescence lifetime probes to study conformational changes in proteins

Area of Science:

• Organic chemistry
• Biophysical chemistry
• Spectroscopy

Background:

• Previous [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD)-based fluorophores functioned as sensitive fluorescence lifetime probes for microenvironmental polarity.
• These earlier probes exhibited limitations in aqueous environments.

Purpose of the Study:

• To develop and characterize a new generation of DBD dyes with improved spectroscopic properties, particularly in aqueous surroundings.
• To synthesize functionalized DBD derivatives for biological labeling.
• To investigate the fluorescence quenching mechanism in maleimido derivatives and their application as probes.

Main Methods:

• Synthesis of novel DBD derivatives.
• Spectroscopic characterization including fluorescence lifetime, quantum yield, and Stokes shift determination.
• Investigation of intramolecular fluorescence quenching via photoelectron transfer (PET) mechanism.
• Application of DBD probes in fluorescence lifetime spectroscopy to study protein conformational changes.

Main Results:

• The new DBD dyes exhibit excellent spectroscopic properties in aqueous solutions, including long fluorescence lifetimes (10-20 ns), large Stokes shifts (≈100 nm), high photostability, and high quantum yields (>0.56).
• Thio-reactive maleimido derivatives show fluorescence quenching via PET, which is reversed upon thiol conjugation, confirming successful labeling.
• The probes were successfully utilized to investigate conformational changes in the maltose ATP-binding cassette transporter.

Conclusions:

• The new generation of DBD dyes represents a significant advancement for fluorescence lifetime probes, offering superior performance in aqueous environments.
• The PET-based fluorescence quenching mechanism in maleimido derivatives provides a reliable indicator of thiol conjugation.
• The combined use of both DBD generations offers a versatile toolkit for detailed investigation of protein microenvironments and conformational dynamics.