Disentangling\nthe Complexity in Protein Complexes\nUsing Complementary Isotope-Labeling and Multiple-Receiver NMR Spectroscopy

Intrinsically\ndisordered proteins are abundant in eukaryotic systems,\nbut they remain largely elusive pharmacological targets. NMR spectroscopy\nproved to be a suitable method to study these proteins and their interaction\nwith one another or with drug candidates. Although NMR can give atomistic\ninformation about these interplays, molecular complexity due to severe\nspectral overlap, limited sample stability, and quantity remain an\nissue and hamper widespread applications. Here, we propose an approach\nto simultaneously map protein–protein binding sites onto two\ninteracting partners by employing a complementary isotope-labeling\nstrategy and a multiple receiver NMR detection scheme. With one partner\nbeing <sup>15</sup>N,<sup>2</sup>H labeled and the interacting one\nbeing <sup>13</sup>C,<sup>1</sup>H-labeled, we exploited proton and\ncarbon detection to obtain clean and easily readable information.\nThe method is illustrated with an application to the 50 kDa ternary\nprotein complex formed between the prominent oncogenic transcription\nfactor complex Myc/MAX and the tumor suppressor BRCA1.