Equilibrium cluster fluids: pair interactions via inverse design.

Inverse methods of statistical mechanics are becoming productive tools in the design of materials with specific microstructures or properties. While initial studies have focused on solid-state design targets (e.g., assembly of colloidal superlattices), one can alternatively design fluid states with desired morphologies. This work addresses the latter and demonstrates how a simple iterative Boltzmann inversion strategy can be used to determine the isotropic pair potential that reproduces the radial distribution function of a fluid of amorphous clusters with prescribed size. The inverse designed pair potential of this "ideal" cluster fluid, with its broad attractive well and narrow repulsive barrier at larger separations, is qualitatively different from the so-called SALR form most commonly associated with equilibrium cluster formation in colloids, which features short-range attractive (SA) and long-range repulsive (LR) contributions. These differences reflect alternative mechanisms for promoting cluster formation with an isotropic pair potential, and they in turn produce structured fluids with qualitatively different static and dynamic properties. Specifically, equilibrium simulations show that the amorphous clusters resulting from the inverse designed potentials display more uniformity in size and shape, and they also show greater spatial and temporal resolution than those resulting from SALR interactions.

Construction, overexpression, and purification of Arthrobacter globiformis amine oxidase-Strep-tag II fusion protein.

The copper-containing amine oxidase from Arthrobacter globiformis has been expressed and purified as a fusion protein with a C-terminal Strep-tag II peptide. This tag facilitates the rapid purification of the enzyme on a large scale using the StrepTactin POROS medium. For example, we have demonstrated that 50 mg of protein can be obtained in 2 days from 2 L of Escherichia coli. The purified fusion protein displays turnover and spectroscopic properties that are essentially identical to those of the wild-type enzyme. Given the location of the C-terminus in four amine oxidase crystal structures, this strategy should be quite general for the rapid purification of amine oxidases from multiple sources.

Expression, purification, and characterization of NosL, a novel Cu(I) protein of the nitrous oxide reductase (nos) gene cluster.

NosL, one of the accessory proteins of the nos (nitrous oxide reductase) gene cluster, has been heterologously expressed, purified, and characterized. NosL is a monomeric protein of 18,540 MW that specifically and stoichiometrically binds Cu(I). The copper ion in NosL is ligated by a Cys residue, and one Met and one His are thought to serve as the other ligands. While it is possible to oxidize Cu(I)-NosL with ferricyanide, the Cu(II) ion thus formed appears to dissociate from the protein. The function of Cu(I)NosL is not yet known, but the data indicate that NosL does not act as an electron transfer partner to nitrous oxide reductase. NosL is encoded on the same transcript as three other gene products (NosD, NosF, and NosY). These have been shown to be required for assembly of the active site in nitrous oxide reductase, which is thought to be a copper cluster. Accordingly, it is possible that NosL is a copper chaperone involved in metallocenter assembly.

The nos (nitrous oxide reductase) gene cluster from the soil bacterium Achromobacter cycloclastes: cloning, sequence analysis, and expression.

The nitrous oxide (N2O) reductase (nos) gene cluster from Achromobacter cycloclastes has been cloned and sequenced. Seven protein coding regions corresponding to nosR, nosZ (structural N2O reductase gene), nosD, nosF, nosY, nosL, and nosX are detected, indicating a genetic organization similar to that of Rhizobium meliloti. To aid homology studies, nosR from R. meliloti has also been sequenced. Comparison of the deduced amino acid sequences with corresponding sequences from other organisms has also allowed structural and functional inferences to be made. The heterologous expression of NosD, NosZ (N2O reductase), and NosL is also reported. A model of the CuA site in N2O reductase, based on the crystal structure of this site in bovine heart cytochrome c oxidase, is presented. The model suggests that a His residue of the CuA domain may be a ligand to the catalytic CuZ site. In addition, the origin of the spectroscopically-observed Cys coordination to CuZ is discussed in terms of the sequence alignment of seven N2O reductases.