Structure-function relationships in a molluscan hemocyanin have been investigated by determining the crystal structure of the Rapana thomasiana (gastropod) hemocyanin functional unit RtH2e in deoxygenated form at 3.38 A resolution. This is the first X-ray structure of an unit from the wall of the molluscan hemocyanin cylinder. The crystal structure of RtH2e demonstrates molecular self-assembly of six identical molecules forming a regular hexameric cylinder. This suggests how the functional units are ordered in the wall of the native molluscan hemocyanins. The molecular arrangement is stabilized by specific protomer-to-protomer interactions, which are probably typical for the functional units building the wall of the cylinders. A molecular mechanism for cooperative dioxygen binding in molluscan hemocyanins is proposed on the basis of the molecular interactions between the protomers. In particular, the deoxygenated RtH2e structure reveals a tunnel leading from two opposite sides of the molecule to the active site. The tunnel represents a possible entrance pathway for dioxygen molecules. No such tunnels have been observed in the crystal structure of the oxy-Odg, a functional unit from the Octopus dofleini (cephalopod) hemocyanin in oxygenated form.

Human cofilin possesses the tendency for self-association, as indicated by the rapid formation of dimers and oligomers when reacted with water-soluble carbodiimide, Ellman's reagent, or glutathione disulfide. Intermolecular disulfide bonds involve Cys 39 and probably Cys 147 of two adjacent cofilin units. The disulfide-linked dimers and oligomers exhibit a biological activity distinct from the monomer. While monomeric cofilin decreased viscosity and light-scattering of F-actin solutions, dimers and oligomers caused an increase in viscosity and light scattering. Electron microscopy revealed that cofilin oligomers induce the formation of highly ordered actin bundles with occasionally blunt ends similar to actin-cofilin rods observed in cells under oxidative stress. Bundling activity of the disulfidelinked oligomers could be completely reversed into severing activity by dithiothreitol. Formation of cofilin oligomers occurred also in the presence of actin at pH 8, but not at pH 6.6, and was significantly enhanced in the presence of phosphatidylinositol 4,5-bisphosphate. Our data are consistent with the idea that cofilin exists in two forms in vivo also: as monomers exhibiting the known severing activity and as oligomers exhibiting actin bundling activity. However, stabilization of cofilin oligomers in cytoplasm is probably achieved not by disulfide bonds but by a local increase in cofilin concentration and/or binding of regulatory proteins.The actin cytoskeleton plays an essential role in many cellular processes such as cytokinesis, changing of cell shape, endocytosis, and exocytosis. All of these processes are dependent on the correct spatial and temporal organization of the actin cytoskeleton (i.e. its regulated polymerization and depolymerization). These reorganizations of the actin cytoskeleton are regulated by a variety of actin-binding proteins, which in turn are regulated by external stimuli such as Ca 2ϩ levels, phosphoinositides, pH, or reversible phosphorylation (1-3). The turnover rates of actin filaments in living cells (reviewed in Ref. 4) are 100 -200 times faster than the rate constants determined for pure actin (5). This discrepancy is mainly due to the activity of ADF/cofilins (AC), 1 which are able to increase the filament turnover in vitro up to levels found in living cells (6).The essential role of AC in enhancing the turnover of actin filaments has been demonstrated in Listeria motility assays (6, 7) and in living yeast cells (8).The AC are a class of small (13-19 kDa) actin-binding proteins, which are ubiquitous in eukaryotes (reviewed in Refs. 4 and 9). They typically localize to regions of rapid actin assembly like neural growth cones (10), developing skeletal muscle (11), cell cortex (12, 13), ruffling membranes (14), and cleavage furrow (13,15). AC have been shown to be essential for the viability of Saccharomyces cerevesiae (12, 16), Dictyostelium discoideum (17), Caenorhabtidis elegans (18), Xenopus laevis (13), and Drosophila melanogaster (19). In contrast to other actin-binding proteins...