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. 2023 Mar 23;145(15):8344–8354. doi: 10.1021/jacs.2c08636

Figure 3.

Figure 3

Kinetic parameters of ATP-synthesis activity. (A) Initial velocity of ATP synthesis as a function of ADP concentration. 4 μM prototypes (N-half, black curve and N-αβα, pink curve) were titrated with varying concentrations of ADP (50 to 5000 μM). Reactions were incubated for 1 h at 37 °C in tricine buffer (pH 7.6) with 0.5 mM MgCl2. Shown here are the initial velocities (expressed as μM ATP synthesized per second) for N-half and N-αβα prototypes at varying ADP concentrations. Data were fit to the standard Michaelis-Menton equation in GraphPad Prism 8.3.0. Error bars represent the SEM from four to eight independent measurements. (B) Time course analysis of ATP-synthesis activity. The N-half prototype (4 μM; black circles and 1 μM; pink circles) was incubated in the presence of saturating ADP concentrations (1 mM) in tricine buffer (pH 7.6) with 0.5 mM MgCl2. Luminescence was measured at varying time points, and ATP synthesized in the reaction was calculated using standard curves (see the “Materials and Methods” section). Error bars represent the SEM from three independent measurements. (C) ATP-synthesis activity of prototypes and P-loop mutants. Shown here are steady-state (1 h) values of ATP synthesis at varying concentrations of “wild-type” (N-half and N-αβα) and mutant prototypes. Reactions were carried out in the presence of 1 mM ADP and 0.5 mM MgCl2. Samples were incubated at 37 °C for 1 h. The “0 μM” (purple) bar indicates background luminescence from the ADP control sample (1 mM ADP + 0.5 mM MgCl2). The structural models of the N-half [topology: β1-(P-loop)-α1−β2-α2] and N-αβα prototypes [topology: α2−β1-(P-loop)-α1] are shown next to the bar diagram. The P-loop (G1xxG2xG3KT) connecting the β1 strand to α1 helix is colored red, and the mutated regions are shown as yellow sticks. Error bars represent the SEM from three to six independent measurements. The models of P-loop prototypes are adapted using PyMOL (pymol.org) with permission from ref (26). Copyright 2021, Proceedings of the National Academy of Sciences of the United States of America.