A water-soluble DsbB variant that catalyzes disulfide-bond formation in vivo.

Escherichia coli DsbB is a transmembrane enzyme that catalyzes the reoxidation of the periplasmic oxidase DsbA by ubiquinone. Here, we sought to convert membrane-bound DsbB into a water-soluble biocatalyst by leveraging a previously described method for in vivo solubilization of integral membrane proteins (IMPs). When solubilized DsbB variants were coexpressed with an export-defective copy of DsbA in the cytoplasm of wild-type E. coli cells, artificial oxidation pathways were created that efficiently catalyzed de novo disulfide-bond formation in a range of substrate proteins, in a manner dependent on both DsbA and quinone. Hence, DsbB solubilization was achieved with preservation of both catalytic activity and substrate specificity. Moreover, given the generality of the solubilization technique, the results presented here should pave the way to unlocking the biocatalytic potential of other membrane-bound enzymes whose utility has been limited by poor stability of IMPs outside of their native lipid-bilayer context.

Isolation of full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of Escherichia coli.

Here we describe a facile and robust genetic selection for isolating full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of redox-engineered Escherichia coli cells. The method is based on the transport of a bifunctional substrate comprised of an antigen fused to chloramphenicol acetyltransferase, which allows positive selection of bacterial cells co-expressing cytoplasmic IgGs called cyclonals that specifically capture the chimeric antigen and sequester the antibiotic resistance marker in the cytoplasm. The utility of this approach is first demonstrated by isolating affinity-matured cyclonal variants that specifically bind their cognate antigen, the leucine zipper domain of a yeast transcriptional activator, with subnanomolar affinities, which represent a ~20-fold improvement over the parental IgG. We then use the genetic assay to discover antigen-specific cyclonals from a naïve human antibody repertoire, leading to the identification of lead IgG candidates with affinity and specificity for an influenza hemagglutinin-derived peptide antigen.

Mechanical heterogeneity in nanoscale films of liquid silica.

Molecular dynamics simulations are carried out for slabs of silica liquid with thicknesses between 1 and 3 nm . A local analysis of the Born contribution to the elastic modulus, CB, shows that the elasticity is not uniform throughout the slabs--CB is identical to that of bulk silica in the slab interior, but CB is larger at the slab edges. The larger CB at the slab edges is due to a distinct atomic level structure characterized by larger density, larger concentration of more highly coordinated ions, and smaller silica rings.

Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria.

Current methods for producing immunoglobulin G (IgG) antibodies in engineered cells often require refolding steps or secretion across one or more biological membranes. Here, we describe a robust expression platform for biosynthesis of full-length IgG antibodies in the Escherichia coli cytoplasm. Synthetic heavy and light chains, both lacking canonical export signals, are expressed in specially engineered E. coli strains that permit formation of stable disulfide bonds within the cytoplasm. IgGs with clinically relevant antigen- and effector-binding activities are readily produced in the E. coli cytoplasm by grafting antigen-specific variable heavy and light domains into a cytoplasmically stable framework and remodelling the fragment crystallizable domain with amino-acid substitutions that promote binding to Fcγ receptors. The resulting cytoplasmic IgGs­named 'cyclonals'­effectively bypass the potentially rate-limiting steps of membrane translocation and glycosylation.

Fold catastrophes and the dependence of free-energy barriers to conformational transitions on applied force.

Applied mechanical force (f) can activate conformational change in molecules by reducing the height of a free-energy barrier (DeltaG(b)). In this paper, molecular dynamics simulations are carried out with umbrella sampling and self-consistent histogram methods to determine free-energy profiles for a coarse-grained model of a protein under an applied force. Applied force is shown to cause fold catastrophes, where free-energy minima are destabilized until they disappear. It is well-known that a fold catastrophe at force f = B implies the scaling DeltaG(b) approximately |B - f|(3/2) in the limit of DeltaG(b) --> 0, but it is not clear whether this scaling is accurate for physically relevant barrier heights. The simulation results show that the fold catastrophe scaling is in fact accurate in the physically relevant regime and that the two-parameter function DeltaG(b) = A(B - f)(3/2) is superior to the two-parameter linear function for parametrizing changes in free-energy barriers with applied force.