Alpha-helical membrane proteins perform numerous critical functions essential for the survival of living organisms. Traditionally, these proteins are extracted from membranes using detergent solubilization and reconstitution into liposomes or nanodiscs. However, these processes often obscure the effects of nanoconfinement and the native environment on the structure and conformational heterogeneity of the target protein. We demonstrate that pulsed dipolar electron spin resonance spectroscopy, combined with the Gd3+-nitroxide spin pair, enables the selective observation of the vitamin B12 importer BtuCD−F in its native cellular envelope. Despite the high levels of non-specific labeling in the envelope, this orthogonal approach combined with the long phase-memory time for the Gd3+ spin enables the observation of the target protein complex at a few micromolar concentrations with high resolution. In the native envelope, vitamin B12 induces a distinct conformational shift at the BtuCD-BtuF interface, which is not observed in the micelles. This approach offers a general strategy for investigating protein-protein and protein-ligand/drug interactions and conformational changes of the alpha-helical membrane proteins in their native envelope context.
