*4.3. Aluminium Fluorides (AlFx) as Transition-State Mimic*

AlF<sup>x</sup> is the most frequently used transition-state analogue (Figure 2A), although the pH-dependence of its formation imposes certain limitations to it. At pH ≥ 5 (depending also on the concentration and the anions in the solution), Al3<sup>+</sup> starts to form an aluminium hydroxide complex, Al(OH)3, which is insoluble. However, the presence of an excess of fluoride shifts the pH upon which Al(OH)<sup>3</sup> formation occurs to a higher value. We calculated the concentrations of the different species of aluminium under the conditions used (6 mM of AlCl<sup>3</sup> and 30 mM NH4F) as a function of the pH-value (Figure 8). In our case, the formation of Al(OH)<sup>3</sup> starts at pH 7, and almost all Al3<sup>+</sup> precipitates as Al(OH)<sup>3</sup> at pH ≥ 8. The amount of formed AlF<sup>X</sup> is thus not sufficient to induce the protein:AlF<sup>x</sup> complex formation. At the same time, fluorides present in the solution can form a complex with Mg2<sup>+</sup> generating the transition-state analogue MgF<sup>3</sup> <sup>−</sup>. This effect was followed and confirmed by <sup>19</sup>F NMR for the conversion of a protein:ADP:AlF<sup>4</sup> <sup>−</sup> complex to a protein:ADP:MgF<sup>3</sup> <sup>−</sup> complex by increasing the pH [56]. Moreover, as pointed out above, MgF<sup>3</sup> <sup>−</sup> and AlF<sup>3</sup> are structurally very similar and some structures comprising AlF<sup>3</sup> as transition state mimic are in reality MgF<sup>3</sup> <sup>−</sup> because they were obtained at pH ≥ 8 [56–58].

For DnaB, the DnaB:ADP:AlF<sup>x</sup> complex can easily be prepared at a pH of 6, since the protein is stable at this pH value. In the presence of the transition-state analogue, the 1D CP <sup>31</sup>P spectrum displays two very narrow resonances assigned to the Pα and Pβ of ADP in complex with AlF<sup>x</sup> (Figure 9A, left panel). Note a minor amount of DnaB:ADP in the sample. The 2D <sup>13</sup>C-13C DARR spectrum of DnaB:ADP:AlF<sup>x</sup> displays strong CSPs attributed to conformational changes of the protein (Figure 9A, right panel). While we noticed that the use of vanadate inhibits the binding of DNA, DNA clearly binds to DnaB in the presence of AlFx, as shown by Figure 9B, left panel. Fluorescence anisotropy measurements revealed that the affinity for DNA-binding is even the highest in the presence of ADP:AlF<sup>x</sup> compared to the other ATP-mimics used [82]. The 2D <sup>13</sup>C-13C DARR spectrum of the sample in presence of DNA reveals that several peaks, which belong to the *N*-terminal domain, are again missing, indicating a change in the dynamics of the protein, as was already observed for DnaB:AMPPCP without DNA.

The case of BmrA is more complex, since the optimal pH for sample preparation lies at 8. For optimal use of AlFx, the pH would need to be lowered, but we observed this to result in poor (e.g., strongly broadened) spectra. Nevertheless, we explored this further, and in order to test the pH dependency, BmrA, in the presence of ATP, was incubated with 6 mM of AlCl<sup>3</sup> and 30 mM NH4F at pH 8, 7.5 and 7, and a 1D CP <sup>31</sup>P NMR was recorded for all three conditions (Figure 9C, left panel). The 1D CP <sup>31</sup>P NMR spectrum at pH 8 shows that ATP/ADP is abundantly co-precipitated with Al(OH)<sup>3</sup> which makes the 1D spectrum difficult to analyse due to a broad and rather unstructured resonance of this amorphous species (Figure 9C, left panel). As expected, the fraction of ATP/ADP co-precipitated

with Al(OH)<sup>3</sup> decreases with decreasing pH-values. While at pH 7 the precipitation of Al(OH)<sup>3</sup> is still visible in the <sup>31</sup>P NMR spectrum, one can compare it to BmrA:ADP:Vi, which shows that both spectra overlay with only few minor differences (Figure 9D, right panel). This indicates that the conformation is highly similar to the one observed with vanadate. A 2D <sup>13</sup>C-13C DARR spectrum recorded on the pH 7 sample (Figure 9C, right panel) confirms this, as the resonances largely superimpose. ≥ − − − − − ≥

≥

**Figure 8.** The different species of AlF<sup>x</sup> at different pH. The diagram was generated using ChemEQL [123], which calculates chemical speciation and equilibria. Concentrations used were 6 mM AlCl<sup>3</sup> and 30 mM NH4F.

**Figure 9.** Comparison of the metal fluoride AlF<sup>x</sup> on the systems BmrA and DnaB. 1D CP of BmrA:ADP:AlF<sup>x</sup> at different pH (**A**, right panel) and <sup>13</sup>C-13C-DARR spectra of the alanine region of BmrA:ADP:AlF<sup>x</sup> - (green) and BmrA (**A**, left panel, blue). <sup>31</sup>P 1D CP spectra of BmrA:ADP:AlF<sup>x</sup> at pH 7 overlaid with BmrA:ADP:Vi (**B**, right panel) and <sup>13</sup>C-13C-DARR spectra of the alanine region of BmrA:ADP:AlF<sup>x</sup> and BmrA:ADP:Vi (**B**, left panel). <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 and DnaB:ADP:AlF<sup>x</sup> (**C**), DnaB:DNA and DnaB:DNA:ADP:AlF<sup>x</sup> (**D**). Spectra (**A**) and (**B**) were adapted from Wiegand et al. 2019 [82] (http://creativecommons.org/licenses/by/4.0/.). Panels (**C**) and (**D**) present original data.

≥ ≥ To summarize, the use of AlF<sup>x</sup> as a transition analogue heavily depends on the optimal pH value of the protein. Indeed, biological systems are principally studied at pH 5–9, and it is important to take into account the formation and precipitation of Al(OH)<sup>3</sup> at pH ≥ 7 under our conditions. The spectra of BmrA at pH ≥ 7 well illustrate the consequences of the use of AlF<sup>x</sup> in alkaline conditions. The protein actually adopts a similar conformation as in the presence of vanadate, but high amounts of amorphous species are detected. In contrast, DnaB at pH 6 shows high affinity to AlF<sup>x</sup> which induces substantial conformational changes; and also DNA binding is not affected.
