Isothermal titration calorimetry (ITC)

ITC experiments were performed for WT‐TTR, the TTR mutants of A97S, V30M, and L55P based on Bulawa et al 2012.14 The detailed experimental conditions were documented in Table S1. Dissociation constants for tafamidis and each TTR protein were determined using a Microcal VP‐ITC isothermal titration calorimeter (Microcal Inc.). The concentrations of proteins and tafamidis used in all ITC experiments are listed in Table S1. The initial injection of 2.5 µL of tafamidis solution was followed by 49 injections of 5 µL each (25°C). The integrated heat was plotted against the molar ratio of tafamidis added to protein in the cell, yielding complete binding isotherms.