Figure 4: Stepwise enzymatic ATP hydrolysis and subsequent dynamic helix reversals.

CD kinetic analyses of CIAP triggered stereomutation processes of (P)-NDPA-ATP (c=5 × 10⁻⁵ M) self-assembly. (a) Schematic illustration of enzymatic action on (P)-NDPA-ATP supramolecular helix (state-1), which first undergoes helix inversion to form (M)-NDPA-ADP and (M)-NDPA-AMP stacks (state-2) followed by racemization to form Pi-stabilized racemic assemblies, (rac)-NDPA-Pi (state-3). (b–d) The spectral changes during the transformation from state-1→state-2 and (e–g) transition from state-2→state-3. (b,e) Time-dependent changes in the CD intensity at 390 nm, with increasing concentration of CIAP at 35 °C. Time-dependent CD spectral changes, upon addition of 0.84 U ml⁻¹ of CIAP at 20 °C are shown in c (state-1→state-2) and f (state-2→state-3), whereas in g corresponding absorption changes (normalized) from state-1→state-3 is presented. (d) Time-dependent changes in the CD intensity (390 nm) of (P)-NDPA-ATP with 0.28 U ml⁻¹ of CIAP at varying temperature. Lower CD signal with increasing temperature in d is due to weaker ATP binding at elevated temperatures (Supplementary Fig. 15). All experiments were done with one equivalent of AP for better kinetics comparison. In b, kinetics was not probed for prolonged time to see transition from state-2→state-3.