GroEL/ES buffering and compensatory mutations promote protein evolution by stabilizing folding intermediates.

Area of Science:

• Protein Evolution
• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Protein stability is a key constraint in evolution, as most mutations are destabilizing.
• Buffering and compensatory mechanisms are crucial for counteracting destabilization during functional adaptation.
• The interplay between these mechanisms throughout an evolutionary trajectory remains unexplored.

Purpose of the Study:

• To investigate the dynamics of buffering and compensatory mechanisms during protein evolution.
• To understand how these mechanisms influence functional adaptation and protein engineering.
• To explore the role of folding kinetics in cellular protein evolvability.

Main Methods:

• Laboratory evolution of a phosphotriesterase into an arylesterase.
• Utilized a controllable GroEL/ES chaperone co-expression system to modulate selection environments.
• Employed biophysical characterization to analyze protein variants.

Main Results:

• Achieved a >10(4) increase in arylesterase activity by smoothening the evolutionary trajectory.
• Demonstrated that soluble cellular expression did not correlate with in vitro stability.
• Identified that compensatory mutations were linked to the stabilization of folding intermediates.

Conclusions:

• The interplay of buffering and compensatory mechanisms is critical for protein evolution and engineering.
• Cellular folding kinetics, rather than solely in vitro stability, are a key determinant of protein evolvability.
• This study provides novel insights into the molecular mechanisms underlying adaptive protein evolution.