NMR investigation of the binding between human profilin I and inositol 1,4,5-triphosphate, the soluble headgroup of phosphatidylinositol 4,5-bisphosphate

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) is involved in the regulation of the actin cytoskeleton through interactions with a number of actin-binding proteins. We present here NMR titration experiments that monitor the interaction between the cytoskeletal protein profilin and inositol 1,4,5-triphosphate (IP₃), the headgroup of PI(4,5)P₂. These experiments probe the interaction directly, at equilibrium, and with profilin in its native state. We show the binding between profilin and IP ₃ can readily be observed at high concentrations, even though profilin does not bind to IP₃ under physiological conditions. Moreover, the titration data using wild-type profilin and an R88L mutant support the existence of at least three headgroup binding sites on profilin, consistent with previous experimentation with intact PI(4,5)P₂. This work suggests that various soluble inositol ligands can serve as effective probes to facilitate in Vitro studies of PI-binding proteins that require membrane surfaces for high-affinity binding.