Evolutionary optimization of a nonbiological ATP binding protein for improved folding stability

Area of Science:

• Protein engineering
• Structural biology
• Biophysics

Background:

• Biological proteins sample structural diversity within protein sequence space.
• Previously isolated nonbiological ATP-binding proteins showed poor solubility, hindering characterization.
• Optimization of de novo proteins for stability is crucial for understanding protein evolution.

Purpose of the Study:

• To determine if nonbiological proteins can be optimized for improved folding stability.
• To evolve a protein capable of binding ATP under denaturing conditions.
• To characterize the structure of an evolved, stable, nonbiological protein.

Main Methods:

• mRNA-display selection under increasingly denaturing conditions.
• Evolution of protein variants for ATP binding in 3 M guanidine hydrochloride.
• Biophysical characterization using circular dichroism, tryptophan fluorescence, and NMR spectroscopy.

Main Results:

• Successfully evolved a population of proteins capable of binding ATP in high concentrations of guanidine hydrochloride.
• One evolved protein demonstrated a unique folded structure.
• Biophysical techniques confirmed the protein's stability and unique fold.

Conclusions:

• De novo proteins can be engineered for enhanced folding stability.
• In vitro evolution is a viable strategy for optimizing nonbiological proteins.
• The evolved protein provides insights into the structural diversity accessible from random sequences.