wiskostatin and pyrene

The Wiskott-Aldrich syndrome protein (WASP) is an effector of the Rho GTPase Cdc42 and a key component of signaling pathways that regulate the actin cytoskeleton. WASP is regulated by a number of ligands, and the mechanisms by which these act are beginning to be understood through detailed biochemical analyses. Here we describe the protocols we use to study WASP proteins, including the methods we use to purify signaling components and the assays we use to quantitatively characterize the biochemical and biophysical properties of WASP, its activation by Cdc42, and its inhibition by the small molecule wiskostatin. These methods have broad use within the WASP-related cytoskeletal-signaling pathway but are also applicable to investigations of other intramolecular and intermolecular interactions.

Topics: Actin-Related Protein 2-3 Complex; Actins; Carbazoles; cdc42 GTP-Binding Protein; Fluorescence Resonance Energy Transfer; Guanidine; Humans; Nuclear Magnetic Resonance, Biomolecular; Propanolamines; Protein Denaturation; Protein Structure, Quaternary; Protein Structure, Tertiary; Pyrenes; Urea; Wiskott-Aldrich Syndrome Protein, Neuronal