Figure 1.

P-loop prototypes and luciferase assay to detect ATP synthesis. (A)Representative P-loop prototypes used for this study. The “intact” prototype contains two copies of the β-(P-loop)-α motif connected by scaffolding β-(loop)-α elements [topology: β1-(P-loop)-α1−β2-(loop)-α2−β3-(P-loop)-α3−β4-(loop)-α4.²⁶ Short prototypes “N-half” and “N-αβα” were constructed from the “intact” prototype by truncation and circular permutation.²⁶ The structural models depict the ancestrally inferred β1 strand and α1 helix in green and the connecting P-loop in red. The scaffolding β strands, α helices, and connecting loops are shown in cyan. The descriptor below each prototype indicates the strand topology and the order of secondary structural elements. The prototypes have a propensity to oligomerize;²⁶ therefore, the monomeric models shown here are only schematic descriptions. 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. Sequences of prototypes are listed in Supporting Information Table S2. (B) ATP-synthesis activity of P-loop prototypes. Luciferase assay showing a linear increase in ATP synthesis (expressed as μM of ATP synthesized per second) with the increasing concentration of prototypes. N-half with ADP (turquoise triangles); N-αβα with ADP (black circles); N-half with ADP and PolyP (violet triangles); and N-αβα with ADP and PolyP (pink squares). The black dotted line indicates the background luminescence from 1 mM ADP (generally equivalent to 0.2–0.3 μM ATP). The red circle on the x-axis indicates background luminescence from 5 μM prototypes. Reactions were carried out in the presence of 1 mM ADP and 0.5 mM MgCl₂ with and without 0.5 mM PolyP at 37 °C for 1 h (see the “Materials and Methods” section). Error bars represent the standard error of mean (SEM) from four to eight independent experiments.