GMMA Can Stabilize Proteins Across Different Functional Constraints

Area of Science:

• Protein engineering and design
• Molecular biology
• Biotechnology

Background:

• Stabilizing proteins while preserving function is a key challenge in protein engineering.
• PYR1, a plant hormone receptor, is engineered for diverse small molecule ligand binding.

Purpose of the Study:

• To identify generalized mutations for stabilizing PYR1 variants without compromising function.
• To find mutations that confer stability across different PYR1 functionalities.

Main Methods:

• Global multi-mutant analysis (GMMA) was employed to identify stabilizing substitutions.
• FACS and deep sequencing were used on PYR1 variant libraries with distinct functionalities.
• GMMA was applied to identify mutations conferring enhanced thermal stability.

Main Results:

• Five specific substitutions were identified that enhance protein stability.
• These substitutions increased the thermal inactivation temperature of PYR1 variants by 2-6°C.
• No decrease in functionality was observed in the engineered PYR1 variants.

Conclusions:

• Generalized mutations can effectively stabilize PYR1 variants across different ligand-binding capabilities.
• The identified substitutions offer a promising strategy for engineering stable and functional proteins.
• This work advances protein engineering for applications requiring enhanced protein stability.