Van't Hoff global analyses of variable temperature isothermal titration calorimetry data.

Area of Science:

• Biophysical Chemistry
• Biochemistry
• Enzyme kinetics

Background:

• Isothermal titration calorimetry (ITC) measures binding thermodynamics (equilibrium constants, K_A, and enthalpy changes, ΔH_A).
• Analyzing temperature-dependent ITC data can reveal coupled equilibria, such as protein folding, but requires robust analytical methods.

Purpose of the Study:

• To validate a novel general method for global analysis of variable temperature ITC data.
• To improve the accuracy of extracted thermodynamic parameters from ITC experiments.
• To apply the method to investigate coupled folding and binding in an aminoglycoside acetyltransferase.

Main Methods:

• Global analysis of variable temperature isothermal titration calorimetry (ITC) data.
• Monte Carlo simulations for method validation.
• Application to an aminoglycoside acetyltransferase enzyme system.

Main Results:

• The developed method significantly enhances the accuracy of thermodynamic parameter extraction.
• The analysis requires no prior knowledge of coupled equilibria.
• Validation through Monte Carlo simulations confirmed the method's robustness.

Conclusions:

• The new global analysis method provides accurate thermodynamic insights into biomolecular interactions.
• It is effective for studying complex systems with coupled equilibria, such as enzyme folding and binding.
• This approach advances the utility of variable temperature ITC in biophysical studies.