| Summary: | Inositol poly- and pyrophosphates (InsPs and PP-InsPs) are central eukaryotic messengers. These very highly phosphorylated molecules can exist in two distinct conformations, a canonical one with five phosphoryl groups in equatorial positions, and a “flipped” conformation with five axial substituents. Using <sup>13</sup>C-labeled InsPs/PP-InsPs, the behavior of these molecules was investigated by 2D-NMR under solution conditions reminiscent of a cytosolic environment. Remarkably, the most highly phosphorylated messenger 1,5(PP)<sub>2</sub>-InsP<sub>4</sub> (also termed InsP<sub>8</sub>) readily adopts both conformations at physiological conditions. Environmental factors—such as pH, metal cation composition, and temperature—strongly influence the conformational equilibrium. Thermodynamic data revealed that the transition of InsP<sub>8</sub> from the equatorial to the axial conformation is, in fact, an exothermic process. The speciation of InsPs and PP-InsPs also affects their interaction with protein binding partners; addition of Mg<sup>2+</sup> decreased the binding constant K<sub>d</sub> of InsP<sub>8</sub> to an SPX protein domain. The results illustrate that PP-InsP speciation reacts very sensitively to solution conditions, suggesting it might act as an environment-responsive molecular switch.
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