Increasing protein stability by engineering the n → π* interaction at the β-turn

Area of Science:

• Biochemistry
• Structural Biology
• Protein Science

Background:

• Non-covalent n → π* interactions between carbonyl groups are abundant in protein structures.
• These interactions are predicted to be crucial for protein structure determination.
• Experimental validation of these weak interactions in proteins has been difficult.

Purpose of the Study:

• To experimentally investigate the role of n → π* interactions in protein structural stability.
• To amplify the strength of n → π* interactions for experimental testing.
• To determine the influence of amino acid side-chain identity on n → π* interaction strength.

Main Methods:

• Utilized amino acid substitution and thioamide incorporation to enhance n → π* interactions.
• Focused on a solvent-exposed β-turn in GB1 proteins and the Pin 1 WW domain.
• Employed statistical analysis of protein structures to identify n → π* interactions.

Main Results:

• Demonstrated that amplified n → π* interactions increase protein structural stability.
• Showed that these interactions restrict the ϕ torsion angle, contributing to stability.
• Identified amino acid side-chain identity and rotameric conformation as key factors in interaction strength.

Conclusions:

• n → π* interactions are experimentally confirmed to enhance protein structural stability.
• The strength and impact of n → π* interactions are significantly influenced by amino acid side-chain properties.
• This work provides a foundation for understanding and manipulating protein structure through non-covalent interactions.