Enzymatic protein switches built from paralogous input domains

Area of Science:

• Biotechnology
• Protein Engineering
• Molecular Biology

Background:

• Protein switches are crucial for biotechnology and medicine.
• Designing protein switches involves fusing input and output protein domains.
• Predicting successful fusion geometry for protein switch creation is challenging.

Purpose of the Study:

• To identify
• hot spots
• for insertion of output domains within class I periplasmic binding proteins.
• To investigate the transferability of successful switch creation sites between homologous proteins.

Main Methods:

• Directed evolution experiments were employed.
• TEM-1 beta-lactamase (BLA) was inserted into ribose binding protein (RBP), glucose binding protein (GBP), and xylose binding protein (XBP).
• Comparison with previous studies using different output domains (xylanase) was performed.

Main Results:

• Functional protein switches were created by inserting BLA into RBP, GBP, and XBP.
• While some overlapping insertion sites were observed among paralogs, successful switch creation required distinct linkers and BLA circular permutations.
• No correlation in insertion sites was found when comparing BLA insertion with xylanase insertion into XBP.

Conclusions:

• Successful protein switch creation sites are not easily transferable between homologous proteins.
• The specific molecular details of protein fusions, rather than general topological properties, dictate switch functionality.
• Predicting optimal fusion sites for protein switch development remains difficult.