Figure 3
From: Isothermal Analysis of ThermoFluor Data can readily provide Quantitative Binding Affinities
Determination of maltose/MBP binding affinity using isothermal analysis of thermal unfolding data. (A) Thermal unfolding of MBP is monitored using SYPRO Orange. Data were collected using 12 increasing maltose concentrations, each in triplicate; 4 representative unfolding curves are shown, after normalization using the Boltzmann equation. (B) The fraction of unfolded protein is calculated at 53 °C for each maltose concentration. Fitting using Equations 6 and 8 yields a Kd value of 2.7 µM and a Ku value of 1.3. (C) Extracting instead the fraction of unfolded protein at 56 °C yields a Kd value of 3.2 µM. (D) The thermal unfolding transition was instead monitored using MBP’s intrinsic tryptophan fluorescence, and the fraction of unfolded protein was calculated at 56 °C for each maltose concentration. Two replicates were carried out for each maltose concentration. Fitting this complementary experimental data using Equations 6 and 8 yields a Kd value of 2.3 µM.
