John Tomaszewski

Alkyl Group Incorporation into Nickel Salen during Controlled-Potential Electrolyses in the Presence of Alkyl Halides

During the catalytic reductions of 1-iodooctane, 1-bromooctane, and 1-iodobutane by nickel(I) salen, electrogenerated from nickel(II) salen at a carbon cathode in dimethylformamide containing 0.10 M tetraethylammonium tetrafluoroborate, less than 100% of the alkyl halide is converted into hydrocarbon products (although virtually none of the original alkyl halide remains) and the transition-metal complex loses its activity. For a system originally consisting of nickel(II) salen and either 1-iodooctane or 1-bromooctane, analyses of post-electrolysis solutions by means of high-performance liquid chromatography reveal that the original nickel(II) salen is largely transformed into three new species. Electrospray-ionization mass spectrometry indicates that one species is a monooctylated nickel(II) salen and another species is a dioctylated nickel(II) salen. Nuclear magnetic resonance spectrometry [i.e., correlated spectroscopy (COSY), nuclear Overhauser effect spectroscopy (NOESY), and total correlated spectroscopy (TOCSY) techniques] has been utilized to establish the sites of octylation. For the l-iodobutane-nickel(II) salen system, at least three butylated nickel(II) salen species have been detected, among which are monobutylated and dibutylated complexes.

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. 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.

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. These data demonstrate that the diacylglycerol (DAG) moiety of PIP(2) is not required for high-affinity binding and suggest that profilin uses multivalency as a key means to distinguish between the intact lipid and IP(3). The class of soluble membrane analogues described here is likely to have broad applicability in the study of protein.PI interactions.