Aromatic-aromatic interactions and protein stability. Investigation by double-mutant cycles

Area of Science:

• Biochemistry
• Protein structure and stability
• Enzyme kinetics

Background:

• Aromatic-aromatic interactions, particularly involving phenylalanine and tyrosine, are common in proteins.
• These interactions are implicated in protein folding, stability, and tertiary/quaternary structure formation.
• Previous calculations suggest aromatic-aromatic interactions contribute negatively to protein free energy, enhancing stability.

Purpose of the Study:

• To investigate the energetic contribution of a specific aromatic pair (Tyr13-Tyr17) on the barnase alpha-helix to protein stability.
• To compare the stability contributions of tyrosine-tyrosine and phenylalanine-phenylalanine interactions.
• To determine the energetic preference between tyrosine and phenylalanine in this specific protein context.

Main Methods:

• Site-directed mutagenesis of barnase to create single and double alanine mutants at Tyr13 and Tyr17.
• Determination of free energies of unfolding using urea denaturation.
• Application of double-mutant cycle analysis to quantify interaction energies.
• Mutational analysis comparing tyrosine to phenylalanine substitutions.

Main Results:

• The Tyr13-Tyr17 aromatic interaction in barnase contributes -1.3 kcal/mol to protein stability.
• This experimentally determined value aligns with predictions based on changes in solvent-accessible surface area.
• Tyrosine-tyrosine and phenylalanine-phenylalanine interactions provide similar stability contributions.
• Tyrosine is slightly preferred over phenylalanine by 0.3 kcal/mol in this solvent-exposed helical context.

Conclusions:

• Aromatic-aromatic interactions are significant stabilizing forces in proteins.
• The barnase Tyr13-Tyr17 interaction provides a quantifiable measure of aromatic stabilization.
• While similar, tyrosine exhibits a subtle energetic advantage over phenylalanine in certain protein environments.