We reasoned that an orthogonal spin pair would enable us to address the above challenges. In this scenario, the IMP in the envelope can be labeled with a nitroxide label. The specific interaction with an orthogonally labeled (using Gd 3+, for example) protein or a ligand and the associated conformational changes can be selectively observed using Gd 3+ -nitroxide PELDOR. Although this approach has been demonstrated for purified membrane proteins, extension into the cellular envelope poses several challenges, including the stability of the spin labels, sufficient expression of the IMP, introduction of the interacting partner into the envelope, and detecting the small extent of complex formation in the complex envelope environment. Here, we report a successful application of this approach to observe protein-protein interaction and substrate-induced conformational shift in the ATP-binding cassette (ABC) transporter BtuCD-F.