Destabilization of a protein helix by electrostatic interactions

Area of Science:

• Protein Engineering
• Biochemistry
• Structural Biology

Background:

• Proteins contain alpha-helices with inherent dipoles.
• Charged residues near helix termini can interact with this dipole.
• Understanding these interactions is key to protein stability.

Purpose of the Study:

• To investigate electrostatic interactions between charged residues and the helix dipole in ribonuclease T1.
• To quantify the impact of these interactions on protein stability.
• To explore the role of these interactions in protein unfolding.

Main Methods:

• Protein engineering: specific mutations of charged residues (Glu28, Asp29) to uncharged residues (Gln28, Asn29) in ribonuclease T1.
• Stability measurements of wild-type and variant proteins across a range of pH values.
• Determination of activation energies for protein unfolding.

Main Results:

• Mutations of Glu28 and Asp29 to uncharged residues had additive effects on protein stability.
• Individual mutations destabilized the protein by 0.7 kJ/mol at pH 2 and stabilized it by 3.2 kJ/mol at pH 7.
• Deprotonation of Glu28 and Asp29 reduces stabilization free energy by approximately 4 kJ/mol each, likely due to unfavorable helix dipole interactions.

Conclusions:

• Unfavorable electrostatic interactions between acidic residues and the helix dipole significantly impact protein stability.
• These interactions are lost early in the unfolding process, as indicated by parallel changes in thermodynamic stability and activation energy.
• Protein engineering is a valuable tool for dissecting electrostatic contributions to protein stability.