Full and Partial Agonism of a Designed Enzyme Switch

Area of Science:

• Protein engineering
• Chemical biology
• Synthetic biology

Background:

• Engineered protein switches are crucial for diagnostics, imaging, and synthetic biology.
• Designing protein switches requires selective and predictable responses tailored to specific applications.
• The 'chemical rescue of structure' method creates allosteric control sites within a protein's functional domain.

Purpose of the Study:

• To understand the principles governing protein switch response to different activating ligands.
• To explore the malleability of designed effector sites in protein switches.
• To guide the design of improved protein switches by tuning ligand-cavity interactions.

Main Methods:

• Disrupting protein structure and activity by creating a buried cavity via mutation.
• Restoring protein structure and activity using an exogenous ligand to replace deleted atoms.
• Utilizing a redesigned β-glycosidase enzyme as a model system to study ligand binding and cavity complementarity.

Main Results:

• The designed effector site is malleable, accommodating ligands of varying sizes.
• Optimal protein activity rescue is achieved with a ligand that precisely replaces the deleted atoms.
• Altering cavity shape through mutations and predicting complementary ligands led to an improved protein switch design.

Conclusions:

• Protein switch response can be tuned by adjusting the ligand-cavity interaction.
• Insights gained will facilitate the design of future systems for controlling protein activity.
• Remolding effector sites offers a pathway to adjust substrate selectivity and downstream signaling, mimicking evolved protein receptors.