The dependence of the chemical shift on pH is then fit to the Henderson-Hasselbalch equation, and the pK a is resolved from the point of inflection. NMR can estimate the pK a with an accuracy of 0.1 pK unit ; however, this strongly depends on the nuclei considered (i.e., C vs N), and the fit to the Henderson-Hasselbalch curve, which can be difficult due to conformational changes , titration coupling , or if the chemical shift simply reports a different titration event . Even with the above caveats, NMR remains the experimental method of choice to resolve pK a values in proteins and is, in general, very reliable. There are alternative approaches for measuring pK a values including fluorometry, kinetic assays, and isothermal titration calorimetry . However, they have their own challenges and generally obtain pK a values with higher uncertainty compared to the NMR-based approach.