NMR Investigation of the Binding between Human Profilin I and Inositol 1,4,5-Triphosphate, the Soluble Headgroup of Phosphatidylinositol 4,5-Bisphosphate

Abstract

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) 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 (IP(3)), the headgroup of PI(4,5)P(2). 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 IP(3) 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)P(2). 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.