Fluorescence depolarization dynamics of ionic strength sensors using time-resolved anisotropy

Area of Science:

• Biophysics
• Molecular Biology
• Biochemistry

Background:

• Eukaryotic cells utilize dynamic ionic strength for critical biological processes, including enzyme activity and protein interactions.
• Understanding cellular ionic strength is crucial for deciphering complex biological functions.

Purpose of the Study:

• To investigate the fluorescence depolarization dynamics of novel ionic strength biosensors (mCerulean3-linker-mCitrine) in varying Hofmeister salt solutions.
• To establish time-resolved fluorescence depolarization anisotropy as a quantitative method for Förster resonance energy transfer (FRET) analysis.

Main Methods:

• Developed and applied a time-resolved fluorescence depolarization anisotropy approach for FRET analysis.
• Excited the donor (mCerulean3) with 425-nm laser pulses and analyzed acceptor (mCitrine) depolarization in genetically encoded sensors.
• Utilized cleaved sensors and a neutral linker construct (E6G2) as controls for comparative analysis.

Main Results:

• Observed distinct fluorescence depolarization dynamics between intact and cleaved biosensors.
• Demonstrated that increased environmental ionic strength leads to decreased FRET efficiency and increased donor-acceptor distances.
• Chemical equilibrium analyses showed ionic strength-dependent conformational state transitions and thermodynamic parameters (equilibrium constant, Gibbs free energy) for the KE sensor.

Conclusions:

• Time-resolved fluorescence depolarization dynamics of genetically encoded donor-acceptor pairs provide a quantitative FRET analysis method.
• These biosensors and methods offer a valuable complement to traditional techniques for future in vivo studies of ionic strength.
• The sensitivity of FRET parameters to ionic strength highlights the potential of these biosensors for probing cellular environments.