High affinity binding to profilin by a covalently constrained, soluble mimic of phosphatidylinositol-4,5-bisphosphate micelles.

Phosphoinositide (PI) lipids are essential regulators of a wide variety of cellular functions. We present here the preparation of a multivalent analogue of a phosphatidylinositol-4,5-bisphosphate (PIP(2)) micelle containing only the polar headgroup portion of this lipid. We show that this dendrimer binds to the cytoskeletal protein profilin with an affinity indistinguishable from that of PIP(2), despite the fact that profilin discriminates between PIP(2) and its monomeric hydrolysis product inositol-1,4,5-triphosphate (IP(3)) under physiological conditions.

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)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.

Synthesis and characterization of covalent mimics of phosphatidylinositol-4,5-bisphosphate micelles.

There is increasing evidence that multivalency plays an important role in protein-lipid recognition and membrane targeting in biological systems. We describe here the preparation and characterization of multivalent analogues of the signaling lipid phosphatidylinositol-4,5-bisphosphate (PIP2). Tetherable analogues of the PIP2 headgroup were appended to polyamidoamine dendrimers via a squarate linker to afford polymers displaying four or eight headgroup moieties.

A low-barrier hydrogen bond between histidine of secreted phospholipase A2 and a transition state analog inhibitor.

This work describes in-depth NMR characterization of a unique low-barrier hydrogen bond (LBHB) between an active site residue from the enzyme and a bound inhibitor: the complex between secreted phospholipase A(2) (sPLA(2), from bee venom and bovine pancreas) and a transition-state analog inhibitor HK32. A downfield proton NMR resonance, at 17-18 ppm, was observed in the complex but not in the free enzyme. On the basis of site-specific mutagenesis and specific 15N-decoupling, this downfield resonance was assigned to the active site H48, which is part of the catalytic dyad D99-H48.