Figure 2.

Simulations to explore the consistency and robustness of isothermal analysis. (A) Simulated thermal unfolding curves were generated using a thermodynamic model for unfolding and binding. Parameters were set as follows: T_m = 50 °C, K_d^Tm = 1 µM, ΔH_U^Tm = 120 kcal mol⁻¹, ΔH_b^Tm = −10 kcal mol⁻¹, ΔCp_U^Tm = 4 kcal mol⁻¹ K⁻¹, ΔCp_b^Tm = −0.5 kcal mol⁻¹ K⁻¹, and total protein concentration = 2 µM. By definition, K_U^Tm = 1. Fitting this simulated data using the simpler isothermal approach yields K_U^Tm = 0.99, and K_d^Tm = 0.99 µM. (B) Upon addition of increasing random noise to the simulated unfolding data, the isothermal approach still leads to accurate estimates of K_U^Tm and K_d^Tm, up to values exceeding the noise typically present in real experimental data.