Profilin is the prototype of small proteins that sequester actin monomers. Recently a host of demonstrated or putative new functions for profilin have emerged. For example, genetic experiments from flies and yeast support a role for profilin in the function of the actin cytoskeleton. Also, the assembly of actin filaments to form new filopodia and pseudopodia is controlled by growth factors. Biochemical studies suggest a dual role for profilin as a component of the signal transduction machinery and as a messenger from the membrane to actin and other ligands, and interactions with the lipids phosphoinositiol bisphosphate (PIP2) and inositol triphosphate (IP3) have been implicated. In addition, the ability to bind poly-L-proline is a common feature among profilins. To probe these and other functions the following experiments (among other) are proposed. * Refinement of the NMR ad x-ray structures of profilin I from Acanthamoeba and a detailed comparison of the refined structures; determination of the profilin II structure by molecular replacement. * Polyproline interactions: by NMR determine the residues of profilin I that interact with poly-L-proline. Using phage display libraries, search for polyproline-containing peptides that bind strongly to profilin; determine the structure of the complex by NMR or crystallography. * Lipid interactions: analysis of the binding of PIP2 to profilin II by contrast-matching low angle scattering; attempt to determine which residues of profilin II bind IP3 (a challenging experiment). Acanthamoeba actophorin is a member of a family of actin monomer binding/actin filament severing proteins that includes actin depolymerizing factor, destrin, gelsolin, and many more distantly related molecules. Their physiological role is far from clear. Our experiments here include: * Completion and refinement of the actophorin crystal structure by the MAD phasing method; crystallization of the complex actophorin with ADP- actin (it has recently emerged that actophorin binds much more strongly to ADP actin that to ATP actin). * Exploration of the mechanism of actin-filament severing. Attempts will also be made to crystallize a number of other cytoskeletal molecules. These include: * New components of the cytoskeleton: the lipid-binding tail of myosin I; the acting-binding head of alpha actinin; the regulatory tailpiece of myosin II; one or more of the recently discovered unconventional actins. * The C-terminus of myosin Ic, which contains an SH3 domain plus mysterious segments rich in the sequence GPA.
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