Significance: Thymosin-β4 (Tβ4) sequesters actin monomers to help maintain the high concentrations of unpolymerized actin in higher eukaryotic cells. Despite more than two decades of research investigating the Tβ4–actin interaction, the X-ray structure of the full-length Tβ4:actin complex remained unresolved. Here, we report two X-ray structures of Tβ4:actin complexes. The first structure reveals that Tβ4 has two helices that bind at the barbed and pointed faces of actin, whereas the second structure displays a more open actin nucleotide binding cleft and a disruption of the Tβ4 C-terminal helix interaction. These structures, combined with biochemical assays and molecular dynamics simulations, reveal how Tβ4 prevents monomeric actin from joining actin filaments but participates in the exchange of actin with profilin to ensure controlled actin polymerization.

A novel magnetic support was prepared by an oxidization-precipitation method with poly(vinyl alcohol) (PVA) as the entrapment material. Transmission electron microscopy indicated that the magnetic particles had a core-shell structure, containing many nanometer-sized magnetic cores stabilized by the cross-linked PVA. The particles showed a high magnetic responsiveness in magnetic field, and no aggregation of the particles was observed after the particles had been treated in the magnetic field. These facts indicated that the particles were superparamagnetic. Cibacron blue 3GA (CB) was coupled to the particles to prepare a magnetic affinity support (MAS) for protein adsorption. Lysozyme was used as a model protein to test the adsorption properties of the MAS. The adsorption equilibrium of lysozyme to the MAS was described by the Langmuir-type isotherm. The capacity for lysozyme adsorption was more than 70 mg/g MAS (wet weight) at a relatively low CB coupling density (3-5 micromol/g). In addition, 1.0 M NaCl solution could be used to dissociate the adsorbed lysozyme. Finally, the MAS was recycled for the purification of alcohol dehydrogenase (ADH) from clarified yeast homogenates. Under proper conditions, the magnetic separation yielded over 5-fold purification of the enzyme with 60% recovery of the enzyme activity.

A thermostable quorum-quenching lactonase from Geobacillus kaustophilus HTA426 (GI: 56420041) was used as an initial template for in vitro directed evolution experiments. This enzyme belongs to the phosphotriesterase-like lactonase (PLL) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acylhomoserine lactones (AHLs) that are involved in virulence pathways of quorum-sensing pathogenic bacteria. Here we have determined the N-butyryl-L-homoserine lactone-liganded structure of the catalytically inactive D266N mutant of this enzyme to a resolution of 1.6 Å. Using a tunable, bioluminescence-based quorum-quenching molecular circuit, the catalytic efficiency was enhanced, and the AHL substrate range increased through two point mutations on the loops at the C-terminal ends of the third and seventh β-strands. This E101N/R230I mutant had an increased value of k(cat)/K(m) of 72-fold toward 3-oxo-N-dodecanoyl-L-homoserine lactone. The evolved mutant also exhibited lactonase activity toward N-butyryl-L-homoserine lactone, an AHL that was previously not hydrolyzed by the wild-type enzyme. Both the purified wild-type and mutant enzymes contain a mixture of zinc and iron and are colored purple and brown, respectively, at high concentrations. The origin of this coloration is suggested to be because of a charge transfer complex involving the β-cation and Tyr-99 within the enzyme active site. Modulation of the charge transfer complex alters the lactonase activity of the mutant enzymes and is reflected in enzyme coloration changes. We attribute the observed enhancement in catalytic reactivity of the evolved enzyme to favorable modulations of the active site architecture toward productive geometries required for chemical catalysis.

Gelsolin is a severing and capping protein that targets filamentous actin and regulates filament lengths near plasma membranes, contributing to cell movement and plasma membrane morphology. Gelsolin binds to the plasma membrane via phosphatidylinositol 4,5-bisphosphate (PIP₂) in a state that cannot cap F-actin, and gelsolin-capped actin filaments are uncapped by PIP₂ leading to filament elongation. The process by which gelsolin is removed from PIP₂ at the plasma membrane is currently unknown. Gelsolin also binds ATP with unknown function. Here we characterize the role of ATP on PIP₂-gelsolin complex dynamics. Fluorophore-labeled PIP₂ and ATP were used to study their interactions with gelsolin using steady-state fluorescence anisotropy, and Alexa488-labeled gelsolin was utilized to reconstitute the regulation of gelsolin binding to PIP₂-containing phospholipid vesicles by ATP. Under physiological salt conditions ATP competes with PIP₂ for binding to gelsolin, while calcium causes the release of ATP from gelsolin. These data suggest a cycle for gelsolin activity. Firstly, calcium activates ATP-bound gelsolin allowing it to sever and cap F-actin. Secondly, PIP₂-binding removes the gelsolin cap from F-actin at low calcium levels, leading to filament elongation. Finally, ATP competes with PIP₂ to release the calcium-free ATP-bound gelsolin, allowing it to undergo a further round of severing.