In the case of the cell envelope, labeling of reactive cysteines in other -helical proteins will lead to undesired background labeling (Figure ). In agreement, we did not observe any difference in the spectral shape and intensity between the cysteine variant and the wild-type protein (Figure ). MTSL is stable in the envelope environment (Figure ), and the contribution from endogenous Mn 2+ appears negligible (Figure ). Therefore, we adopted an orthogonal labeling strategy by introducing a Gd 3+ spin label at position S138C in BtuF (Figure and). This BtuF variant efficiently binds to a spinlabeled VitB12, thereby revealing a good labeling efficiency (L.E., ~100%, Figure ). The combination of Gd 3+ with NO provides great advantages, in particular for the envelope sample. By observing BtuF-Gd 3+ (and pumping NO), distances come exclusively through the interaction with BtuCD, and the effect of non-specific NO labeling is selectively eliminated (which can also verified with a control sample in which BtuCD is not overexpressed; please see the following sections). Owing to its narrow central transition and favorable relaxation times, Gd 3+ offers higher sensitivity, especially when combined with the nitroxide spin, which has a narrower spectrum. Further, this approach provides structural information for BtuF-BtuCD interaction as well as for the TM5-TM5a helices on which the NO labels are located. Relaxation measurements revealed a short phase memory time (TM, ~800 ns) for the nitroxide-labeled envelope sample (Figure ). Remarkably, Gd 3+ -BtuF introduced into the envelope gave a fivefold larger TM (~4 s), thereby facilitating the observation of long dipolar evolution time windows. A NO-NO PELDOR using the envelope gave a broad distribution, which makes it difficult to make any meaningful conclusion (Figure ). We also tested another position, D131C, located away from the membranes on the NBDs, to test whether it is less affected by the non-specific labeling. However, these positions also gave a similarly broad distribution (Figure ). Overall, due to the complexity of the native envelope, a direct distance measurement using cysteinebased labeling, as demonstrated for the outer membrane proteins, would be difficult.