Structure and Function of Redox-Sensitive Superfolder Green Fluorescent Protein Variant

Area of Science:

• Cellular Biology
• Biochemistry
• Microscopy

Background:

• Genetically encoded green fluorescent protein (GFP)-based biosensors are crucial for monitoring cellular redox dynamics.
• Existing biosensors face limitations in small organisms like Plasmodium due to weak fluorescence signals, hindering compartment-specific measurements.
• The malaria parasite Plasmodium presents a significant challenge for precise redox process monitoring.

Purpose of the Study:

• To functionally and structurally characterize an enhanced redox biosensor, superfolder roGFP2 (sfroGFP2).
• To improve fluorescence intensity (FI) for better measurements in small cellular environments.
• To develop a robust biosensor suitable for studying redox changes in Plasmodium.

Main Methods:

• Functional and structural characterization of superfolder roGFP2 (sfroGFP2).
• Crystallography and rigidity theory to understand structural stability.
• Fusion of sfroGFP2 to human glutaredoxin-1 (hGrx1) for enhanced performance in Plasmodium.

Main Results:

• SfroGFP2 exhibits improved fluorescence intensity (FI) in cellulo compared to standard roGFP2.
• SfroGFP2-based biosensors are pH-insensitive within physiological ranges and maintain comparable midpoint potentials.
• Superfolding mutations enhance structural stability, explaining the increased FI in redox-sensitive environments.

Conclusions:

• SfroGFP2 offers improved structural stability and higher FI, making it suitable for small cells where other sensors are limited.
• The sfroGFP2-hGrx1 fusion is the most effective biosensor for measuring oxidizing effects in Plasmodium.
• This enhanced biosensor is vital for studying glutathione redox changes in small cells and subcellular compartments.