Generation of Fluorogen-Activating Designed Ankyrin Repeat Proteins (FADAs) as Versatile Sensor Tools

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Fluorescent probes are essential for biological imaging.
• Fluorogen-activating proteins (FAPs) are a recent advance, activating dyes only upon binding.
• Existing FAPs are based on antibody single-chain variable fragments.

Purpose of the Study:

• To develop a new class of fluorogen activators, FADAs, based on a designed ankyrin repeat protein scaffold.
• To characterize the FADA's ability to activate fluorogenic dyes.
• To explore FADA applications in cellular imaging and protein interaction detection.

Main Methods:

• Designed ankyrin repeat protein scaffold engineering.
• Ribosome display and yeast surface display for selection.
• Malachite green dye activation assays.
• Structural determination and in vitro measurements.
• Development of FADA biosensors for cell surface and cytosolic imaging.

Main Results:

• A novel FADA was generated, enhancing malachite green fluorescence >11,000-fold.
• The FADA functions as a homodimer for efficient dye activation.
• FADA biosensors demonstrated utility for cell surface and cytosolic protein imaging.
• A prototype biosensor for in situ protein and protein-protein interaction detection was developed.

Conclusions:

• FADAs represent a versatile and easily produced alternative to FAPs.
• The designed ankyrin repeat protein scaffold offers favorable properties for FADA development.
• FADAs significantly extend the capabilities of fluorogen-activating protein technology for diverse biological applications.