*4.4. The Post-Hydrolytic State Induced by ADP*

The last state in the ATP hydrolysis cycle is the post-hydrolytic state, where ADP is still bound to the protein and the inorganic phosphate (previously γ-phosphate) is released from the binding pocket. This state is well mimicked by the addition of ADP. We used the <sup>31</sup>P and <sup>13</sup>C experiments described above to characterize BmrA and DnaB in the presence of ADP. γ

The <sup>31</sup>P CP spectrum for DnaB:ADP is shown in Figure 10A (left panel). The spectrum displays two sharp peaks, which indicates a good homogeneity of the sample. The overlay of the 2D DARR spectra DnaB:ADP and DnaB apo (Figure 10A, right panel) reveals CSPs and also the disappearance of *N*-terminal domain peaks, indicating conformational changes and an increase in the dynamics of the protein.

**Figure 10.** Generation of the post-hydrolytic state using ADP. (**A**) <sup>31</sup>P 1D CP spectrum (left panel) and <sup>13</sup>C-13C-DARR the alanine region spectra overlay (right panel) of DnaB:ADP/DnaB apo; (**B**) <sup>31</sup>P 1D CP spectrum (left panel) and <sup>13</sup>C-13C-DARR the alanine region spectra overlay (right panel) of BmrA:ADP/BmrA. Spectra (**A**) were adapted from Wiegand et al. 2019 [82] (http://creativecommons. org/licenses/by/4.0/.), and spectra in panel (**B**) are original data.

α β α β α β β α α β β α In contrast, the conformational changes of the ABC transporter BmrA are minor between the presence and absence of ADP. First of all, the <sup>31</sup>P CP spectrum shows the presence of two populations of ADP as identified by peak doubling, labeled Pα, Pβ, and Pα', Pβ' (Figure 10B, left panel). These two populations are the result of two different binding modes of ADP to the protein. However, since the intensity of the Pα', Pβ' peaks is 50% lower than Pβ, Pα, there is less Pα', Pβ' bound to the protein than Pβ, Pα. This is reminiscent to the pattern that was observed with vanadate (Figure 6F). Unspecific binding of ADP to the protein can explain this observation. Secondly, the overlay of the 2D DARR spectra of BmrA:ADP and BmrA apo displays few CSPs compared to what we observed in the presence of other ATP analogues. In contrast to DnaB, the binding of ADP does not induce large conformational or dynamics changes in the protein, and binding of ADP to BmrA seems to be very weak.
