Tuning Enzyme Thermostability via Computationally Guided Covalent Stapling and Structural Basis of Enhanced...

Area of Science:

• Protein Engineering
• Biocatalysis
• Structural Biology

Background:

• Enzyme thermostability is crucial for industrial applications but difficult to enhance without compromising activity.
• Previous work introduced genetically encoded thioether staples for enzyme stabilization using O-2-bromoethyl tyrosine (O2beY).

Purpose of the Study:

• To expand the repertoire of electrophilic amino acids for protein stapling.
• To evaluate the efficacy of new stapling strategies for enhancing enzyme thermostability and function.
• To investigate the structural basis of stabilization conferred by protein staples.

Main Methods:

• Utilized a myoglobin-based cyclopropanase as a model enzyme.
• Employed computationally guided installation of genetically encoded electrophilic amino acids (pCaaF, acrylamido, vinylsulfonamido).
• Performed molecular simulations, thermal denaturation assays, and crystallographic analyses.

Main Results:

• p-chloroacetamido-phenylalanine (pCaaF) stapling demonstrated higher efficiency and superior thermodynamic and kinetic stability compared to other variants and the parent protein.
• pCaaF-stapled variants showed enhanced stability against thermal denaturation (ΔTm′ = +27 °C) and heme loss (ΔT50 = +30 °C).
• Catalytic activity and stereoselectivity were maintained, and structural analyses revealed insights into stabilization mechanisms.

Conclusions:

• The pCaaF staple is a promising tool for significantly enhancing enzyme thermostability while preserving function.
• The strategy of using electrophilic amino acids for protein stapling offers a versatile approach for protein engineering.
• Understanding the conformational flexibility and structural interactions of staples provides a basis for rational protein design.