Conformational switching is pervasively driven by protein interactions, particularly for intrinsically disordered binding partners. We developed a dually orthogonal fluorescence-based assay to monitor such events, exploiting environmentally sensitive fluorophores. This assay is applied to E3 ligase E6AP, as its AZUL domain induces a disorder-to-order switch in an intrinsically disordered region of the proteasome, the so-named Rpn10 AZUL-binding domain (RAZUL). By testing various fluorophores, we developed an assay appropriate for high-throughput screening of Rpn10:E6AP-disrupting ligands. We found distinct positions in RAZUL for fluorophore labeling with either acrylodan or Atto610, which had disparate spectral responses to E6AP binding. E6AP caused a hypsochromic shift with increased fluorescence of acrylodan-RAZUL while decreasing fluorescence intensity of Atto610-RAZUL. Combining RAZUL labeled with either acrylodan or Atto610 into a common sample achieved robust and orthogonal measurement of the E6AP-induced conformational switch. This approach is generally applicable to disorder-to-order (or vice versa) transitions mediated by molecular interactions.
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