Underlying thermodynamics of pH-dependent allostery

Area of Science:

• Biophysics
• Protein Engineering
• Computational Biology

Background:

• Understanding protein protonation and conformational changes is vital for developing pH sensors and switches.
• Nitrophorin 4 (NP4) exhibits pH-dependent activity regulated by conformational shifts.
• The interplay between protonation and conformation is crucial for protein function.

Purpose of the Study:

• To comprehensively study the pH-regulated switch between closed and open conformations in NP4.
• To elucidate the role of specific amino acids, like D30, in pH-dependent behavior.
• To analyze the free-energy landscape and thermodynamic model of NP4's conformational change.

Main Methods:

• Computational analysis of protein protonation and conformational states.
• Free-energy landscape calculations as a function of pH.
• Estimation of free-energy barriers and equilibrium constants.

Main Results:

• Aspartic acid 30 (D30) was identified as the sole amino acid with distinct pKas in open and closed NP4 conformations.
• Accurate estimations of free-energy barriers and equilibrium constants were obtained.
• The study demonstrated that conformational equilibria can tune observed pKa, as shown with the K125L mutant.

Conclusions:

• Coupling between protonation and conformational equilibria enables efficient pH sensing near physiological pH.
• Specific combinations of equilibrium constants constrain protein evolution and function.
• The findings offer insights into pH-regulated allostery, valuable for drug design and protein engineering.