Fluorescent Proteins Detect Host Structural Rearrangements via Electrostatic Mechanism

Area of Science:

• Biophysics
• Molecular Biology
• Computational Chemistry

Background:

• Genetically encoded fluorescent biosensors are crucial for detecting protein rearrangements via allostery.
• Understanding the photophysics of these sensors, particularly enhanced Green Fluorescent Protein (eGFP), is essential for rational design but currently limited.
• The precise mechanisms by which mechanical forces modulate fluorescence in eGFP remain unclear.

Purpose of the Study:

• To elucidate the photophysical mechanisms underlying fluorescence modulation by mechanical perturbation in eGFP.
• To develop a transferable model for predicting fluorescence intensity changes in response to mechanical forces.
• To propose a mechanistic hypothesis for the operation of the genetically encoded voltage probe ArcLight.

Main Methods:

• Employed molecular dynamics (MD) simulations combined with quantum chemistry calculations.
• Developed a 2D electric field map of fluorescence quantum yield based on protein environment interactions.
• Utilized steered MD simulations to investigate mechanical perturbations and their effect on fluorescence.

Main Results:

• Constructed a transferable map of fluorescence quantum yield as a function of the electric field for Tsien's Class 2 GFP variants.
• Demonstrated that this map allows direct estimation of fluorescence intensity shifts from MD simulations.
• Generated a mechanistic hypothesis for the operation of the ArcLight voltage probe.

Conclusions:

• The developed photophysical model provides a framework for understanding and predicting fluorescence changes in eGFP due to mechanical forces.
• This approach facilitates the rational design of advanced genetically encoded fluorescent biosensors.
• The proposed mechanism for ArcLight offers new insights into the function of genetically encoded voltage probes.