Statistical Mechanical Model for pH-Induced Protein Folding: Application to Apomyoglobin.

Area of Science:

• Biochemistry
• Physical Chemistry
• Protein Dynamics

Background:

• The pH dependence of protein folding and stability is crucial but lacks quantitative mechanistic models.
• Existing models (Monod-Wyman-Changeux, Linderstrøm-Lang) focus on specific structures or states, limiting analysis of overall folding kinetics.
• A gap exists in models that capture the complete kinetic events of pH-induced folding/unfolding independent of molecular species properties.

Purpose of the Study:

• To develop and apply a novel statistical mechanical model for quantitatively analyzing pH-induced protein folding mechanisms.
• To investigate the folding kinetics and intermediates of horse apomyoglobin across a wide pH range.
• To elucidate the roles of protonation, specific binding, and Coulombic interactions in protein folding.

Main Methods:

• Construction of a statistical mechanical model incorporating protonation mechanisms.
• Combined manual search and least-squares fitting procedures for model parameterization.
• Continuous- and stopped-flow fluorescence measurements of horse apomyoglobin folding kinetics (40 μs to 100 s) at pH 2.2–6.7 and 8 °C.
• Analysis using a five-state sequential kinetic scheme.

Main Results:

• The model successfully quantifies pH-induced folding/unfolding, driven by specific binding and Coulombic interactions.
• Kinetic folding/unfolding intermediates were found to share mechanisms with the equilibrium intermediate, suggesting their equivalence.
• Progressive acquisition of native-like properties by intermediates and transition states during the folding process was observed.

Conclusions:

• The developed statistical mechanical model provides a quantitative framework for understanding pH-modulated protein folding.
• The findings highlight the interplay of protonation, specific binding, and electrostatic forces in governing protein conformational changes.
• The model's applicability extends beyond protein folding to other association/dissociation processes.