Estimating the contribution of engineered surface electrostatic interactions to protein stability by using...

Area of Science:

• Protein engineering
• Biophysical chemistry
• Structural biology

Background:

• Coulombic interactions between surface protein charges significantly influence protein stability.
• Estimating the precise energetic contribution of individual charged pairs is challenging using traditional protein engineering due to pleiotropic effects of mutations.

Purpose of the Study:

• To quantify the electrostatic interaction energy between Asp-12 and Arg-16 in a barnase mutant using a double-mutant cycle approach.
• To investigate the contribution of this specific ion pair to the overall protein stability.

Main Methods:

• Employed a double-mutant cycle strategy involving wild-type barnase and its single and double mutants (Asp-12, Thr-16).
• Analyzed changes in free energy of unfolding under varying salt concentrations to isolate electrostatic contributions.
• Utilized structural information from homologous binase to infer ion pair geometry.

Main Results:

• Free energy changes of unfolding were non-additive in single mutants, indicating electrostatic interactions.
• Additivity of free energy changes was restored at high salt concentrations, confirming the shielding of electrostatic effects.
• The Asp-12/Arg-16 ion pair in barnase mutant increased stability by 0.5 kcal/mol, with only a portion attributed to electrostatics.

Conclusions:

• Double-mutant cycles provide a robust method for measuring pairwise interaction energetics in proteins.
• The study quantifies the energetic impact of a specific ion pair, contributing to a deeper understanding of protein stability determinants.