Interestingly, the addition of the BtuF+VitB12 complex to BtuCD (called the apo sample) shifted the distance distribution into a narrower peak centered at ~4.5 nm in the envelope (Figure ) (Figure ). Notably, this shift is not observed in the detergent-solubilized sample (see blue vs. orange lines in Figure top panel). The data for vanadate-trapped and ADP-Mg 2+ samples also revealed a similar response. As these distances are nearly the same as the apo sample, it can be concluded that the tested nucleotide states do not cause any additional change in the conformation. The presence of VitB12 may alter the MTSL rotamers in the binding pocket (see Figure and inset in Figure ). Also, the TM5-TM5a helices carrying the MTSL labels may further move apart to accommodate the VitB12 molecule (Figure ). Irrespective of one or both of these aspects leading to the shift of the distance peak, the data sets suggest that VitB12 must be present inside the BtuCD-F complex. In detergent micelles, VitB12 is non-productively released from BtuF upon interaction with BtuCD. This is also evident from our data, where VitB12 failed to induce the conformational change in the micelles (Figure , top panel). Thus, no structure for BtuCD-F with bound VitB12 could be yet solved. Therefore, the envelope environment might stabilize the VitB12-bound BtuCD-F complex. A previous study employing radiolabeled VitB12 in proteoliposomes suggested that AMP-PNP (or ATP) is required for trapping VitB12 inside the transporter. However, subsequent single-molecule fluorescence energy transfer-based studies (in nanodiscs or liposomes) showed that VitB12 could bind to BtuCD-F independent of the nucleotide state, which is in agreement with our observation of VitB12 inducing the shift in the apo, ADP-Mg 2+ and the vanadate trapped states (Figure ). Thus, the release of the Pi following ATP-hydrolysis might be the power stroke for VitB12 translocation. For spectroscopic investigations in the native membranes, the elimination of the background signals is a major challenge. Here, we overcame this problem with an orthogonal labeling strategy employing Gd 3+ and NO spin labels combined with protein overexpression. The level of protein overexpression could be considerably reduced with the ongoing improvements for sensitivity into the nM range. This experimental setup is sensitive exclusively to the Gd 3+ -NO spin pairs and thereby detects the specific interaction between the desired molecules. We show that Gd 3+ labeled BtuF can be introduced into the periplasmic space at sufficient levels. Further, the long TM for the Gd 3+ spins enabled the selective and reliable observation of BtuF-BtuCD interaction under the nanoconfinement in the native envelope. This would be a potential tool to elucidate proteinprotein or protein-ligand/drug interaction for -helical membrane protein complexes, including potential antibiotic targets in the native cellular envelope.