Supramolecular Assembly of an Evolved Miniprotein Host and Fluorogenic Guest Pair

Area of Science:

• Biochemistry
• Materials Science
• Synthetic Biology

Background:

• Designing host-guest systems for small-molecule-induced assembly in biological contexts is challenging.
• Fibronectin type III domain (Fn3) based miniproteins offer a scaffold for protein engineering.

Purpose of the Study:

• To engineer Fn3-based miniprotein hosts with high affinity for a specific dye.
• To achieve small-molecule-induced supramolecular assembly of these engineered proteins.
• To demonstrate the utility of this system for visualization in living cells.

Main Methods:

• Directed evolution was used to evolve Fn3 miniprotein binders.
• Analytical ultracentrifugation confirmed higher-order assembly.
• Fluorescence microscopy was employed for visualization.

Main Results:

• A consensus binder (6.2.18) showed high affinity for the merocyanine dye sI-Pht.
• Addition of sI-Pht induced supramolecular assembly of the 6.2.18 protein.
• This assembly led to a 570-fold increase in fluorescence.
• The assembly was visualized on the surface of living yeast cells.

Conclusions:

• A novel host-guest system was developed using engineered Fn3 miniproteins and a merocyanine dye.
• This system enables small-molecule-induced protein assembly and fluorescence enhancement.
• The findings present a new paradigm for designing functional host-guest systems in living cells for materials and pathway engineering.