Emergence of specific binding and catalysis from a designed generalist binding protein

Area of Science:

• Protein engineering and synthetic biology
• Biochemistry and molecular evolution

Background:

• Natural proteins exhibit promiscuous binding to various molecules, serving as evolutionary starting points.
• This promiscuity is crucial for the stepwise evolution of new specificities and functions.
• Fragment screening is a common method to evaluate protein-ligand interactions, but not previously applied to de novo proteins.

Purpose of the Study:

• To investigate the binding promiscuity of a de novo designed protein, ABLE.
• To assess if promiscuous binding in de novo proteins can serve as a basis for evolving new functions.

Main Methods:

• Applied crystallographic fragment screening to the de novo protein ABLE.
• Evaluated the weak binding interactions identified through screening.
• Evolved new protein functions from these weak binding interactions.

Main Results:

• The de novo protein ABLE demonstrated promiscuous weak binding to small molecule fragments, similar to natural proteins.
• These weak interactions were successfully utilized as starting points for functional evolution.
• Evolved functions included a specific binder for a turn-on fluorophore and a highly efficient Kemp eliminase enzyme.

Conclusions:

• De novo proteins, like natural ones, can exhibit promiscuous binding.
• Promiscuous binding interactions in de novo proteins are valuable for evolving novel, specialized functions.
• This study validates a strategy for protein design and evolution by leveraging weak interactions.