“…First, we reasoned that an increased k off , at a rather stable k on , should be accompanied by an increase in the equilibrium dissociation constant ( K D ), since We, therefore, measured the K D by peptide titration with steady‐state anisotropy 29 and found that the pH did not significantly affect the peptide binding affinity of the complexes (Figure 2c,d ). Second, another readout of peptide binding affinity to MHC‐I is the thermal stability of the peptide‐MHC‐I complexes 20 , 30 , 31 , 32 , 33 , 34 , 35 ; to measure it, we used nanoscale differential scanning fluorimetry (nanoDSF), which quantifies protein unfolding by the change in the fluorescence emission of tryptophan indoles and generates a thermal denaturation curve, the midpoint of which corresponds to the melting temperature ( T m ). 17 We found that there was little influence of the pH on the T m of high‐affinity peptide complexes with wtA2 or wtA24, whereas for medium‐affinity peptides, there was a slight stabilization at low pH (Figure 2e ).…”