Abstract
Increased efforts are being made for observing proteins in their native environments. Pulsed electron-electron double resonance spectroscopy (PELDOR, also known as DEER) is a powerful tool for this purpose. Conventionally, PELDOR employs an identical spin pair, which limits the output to a single distance for monomeric samples. Here, we show that the Gd(3+)-trityl-nitroxide (NO) three-spin system is a versatile tool to study heterooligomeric membrane protein complexes, even within their native membrane. This allowed for an independent determination of four different distances (Gd(3+)-trityl, Gd(3+)-NO, trityl-NO, and Gd(3+)-Gd(3+)) within the same sample. We demonstrate the feasibility of this approach by observing sequential ligand binding and the dynamics of complex formation in the cobalamin transport system involving four components (cobalamin, BtuB, TonB, and BtuF). Our results reveal that TonB binding alone is sufficient to release cobalamin from BtuB in the native asymmetric bilayers. This approach provides a potential tool for the structural and quantitative analysis of dynamic protein-protein interactions in oligomeric complexes, even within their native surroundings.