Protein Stability─Analysis of Heat and Cold Denaturation without and with Unfolding Models

Area of Science:

• Thermodynamics
• Biophysics
• Protein Chemistry

Background:

• Protein stability is crucial in life sciences, often studied using spectroscopic techniques.
• Differential scanning calorimetry (DSC) directly measures heat capacity (Cp(T)), a key thermodynamic property.
• Current analysis relies on the two-state model, which can lead to inaccurate thermodynamic conclusions.

Purpose of the Study:

• To develop a model-independent method for evaluating DSC heat capacity data.
• To critically assess the standard chemical equilibrium two-state model.
• To propose and validate new models for analyzing protein unfolding thermodynamics.

Main Methods:

• Model-independent evaluation of heat capacity (Cp(T)) data.
• Analysis of protein unfolding enthalpy (ΔH(T)), entropy (ΔS(T)), and free energy (ΔG(T)).
• Comparison of experimental data with predictions from the standard two-state model and two newly proposed models (ΘU(T)-weighted chemical equilibrium and statistical-mechanical two-state models).

Main Results:

• The standard two-state model shows significant divergence from experimental temperature profiles.
• The proposed ΘU(T)-weighted chemical equilibrium and statistical-mechanical two-state models provide excellent fits to experimental data.
• Sigmoidal profiles for enthalpy and entropy, and a trapezoidal profile for free energy were predicted by the new models, validated with lysozyme and β-lactoglobulin denaturation data.

Conclusions:

• The free energy is not the optimal parameter for assessing protein stability; cooperativity and other parameters offer better insights.
• The new models provide a more accurate thermodynamic context for experimental data, suitable for comparison with molecular dynamics calculations.
• This work enables a more robust understanding of protein stability and unfolding mechanisms.