RF and RF catabolites bind MR1. (A) Mechanism of metabolism and photodegradation of RF. (B) Titration curves of the shown ligands binding to MR1 were obtained from the fluorescence polarization–based assay (left). Each data point represents normalized percentage binding from three independent experiments performed in triplicate. Mean values are plotted with SEM represented in error bars. Curve fit for ligands is displayed in the table (right). (C and D) (C) XICs for m/z 243.087 in a lumichrome solvent standard and (D) MS2 fragmentation of the main peak at retention time (RT) 6.82 are depicted. (E) UV-treated MR1 RF XIC for m/z 301.0927. (F) XIC for m/z 243.087 showing lumichrome present at RT 6.82, with an additional peak at RT 7.76. (G) MS2 fragmentation of main peak at RT 6.82, showing aligned fingerprint comparison with lumichrome solvent standard. (H) Thermostability of soluble WT MR1 refolded with the indicated ligands was measured by fluorescence-based thermal shift assay. The graph shows baseline-corrected, normalized emission at 610 nm plotted against temperature (°C). Each point represents the mean of three technical replicates, and error bars represent SD. The Tm50 is indicated by the dotted line at 50%. The table on the right shows the mean Tm50 from three independent experiments, each measured in at least a technical triplicate. XICs, extracted ion chromatograms.