Computational modeling of a new fluorescent biosensor for caspase proteolytic activity improves dynamic range

Area of Science:

• Biochemistry and Molecular Biology
• Biophysics
• Biotechnology

Background:

• Fluorescence resonance energy transfer (FRET) protein biosensors, typically comprising yellow fluorescent protein (YFP), a cleavage sequence, and cyan fluorescent protein (CFP), are used to measure protease activity.
• The dynamic range of existing FRET biosensors is often limited, posing a challenge for applications like high-throughput drug screening that necessitate a greater dynamic range.

Purpose of the Study:

• To computationally improve the FRET dynamic range of protease activity biosensors.
• To validate the computational strategy through experimental verification for enhanced biosensor performance.

Main Methods:

• A computational approach was developed utilizing the atomic structure of caspase-3 bound to its inhibitor.
• The strategy was applied to a FRET biosensor designed for caspase-3 protease activity.
• Experimental validation was performed in both in vitro and in vivo settings.

Main Results:

• The computational approach successfully improved the FRET dynamic range of the caspase-3 biosensor.
• Experimental results demonstrated an average FRET dynamic range improvement of at least 60%.

Conclusions:

• A computational strategy based on atomic structures can significantly enhance the FRET dynamic range of protease biosensors.
• This method offers a viable approach to improve FRET biosensors for protease activity, benefiting applications like drug discovery.