Structural prediction of a rhodamine-based biosensor and comparison with biophysical data

Area of Science:

• Biochemistry
• Structural Biology
• Computational Chemistry

Background:

• A previously developed protein-based biosensor utilizes the phosphate binding protein from Escherichia coli.
• The biosensor is engineered by labeling the protein with two rhodamine fluorophores.
• Understanding the structural dynamics of this biosensor is crucial for its application in phosphate detection.

Purpose of the Study:

• To calculate the predicted molecular structure of the protein-based inorganic phosphate (Pi) biosensor.
• To provide molecular models of the biosensor in both the presence and absence of Pi.
• To investigate the conformational changes and fluorophore interactions within the biosensor.

Main Methods:

• Utilized classical molecular dynamics simulations.
• Employed hybrid Car-Parrinello/molecular mechanics simulations.
• Validated computational models by comparing with experimental absorption and circular dichroism spectra.

Main Results:

• Developed molecular models for the biosensor with and without inorganic phosphate (Pi).
• Observed a distinct 'face A to face B' stacked conformation of rhodamine fluorophores in the absence of Pi, differing from free fluorophores.
• Determined that Pi binding induces a protein conformational change, disrupting significant rhodamine stacking and showing hydrophobic contact with LEU291.

Conclusions:

• The study provides accurate molecular models of the Pi biosensor, validated by experimental data.
• Phosphate binding significantly alters the biosensor's structure, specifically the conformation and stacking of the rhodamine fluorophores.
• These findings enhance the understanding of the biosensor's mechanism and can guide further optimization.