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A microfluidic device made of polydimethylsiloxane (PDMS) has shown the limitations in single-molecule fluorescence experiments by providing high dye photostability and low sample sticking. Rapid buffer exchange in a laminar sheath flow followed by optical interrogation has minimized the surface-sample contacts and has allowed the in situ addition and combination of other reagents.

The studies have shown that the orthogonal chemistry of a genetically encoded ketone handle together with a single cysteine residue permit efficient and versatile site-specific dual-labeling of proteins for single molecule fluorescence resonance energy transfer (smFRET) studies. This chemistry is not limited to smFRET labeling and is useful for attachment of other biophysical probes and directed protein immobilization.

Cells survive fluctuations in osmolality by accumulating and depleting highly soluble, usually neutral, small organic compounds. Natural selection has converged on a small set of such molecules, called osmolytes. The biophysical characterization of osmolytes, with respect to proteins, has centered on tertiary structure stability. Data about their effect on protein assemblies, whose formation is driven by surface interactions, is lacking. Here, we investigate the effects of osmolytes and related molecules on the stabilities of a protein and a protein complex. The results demonstrate that osmolytes are not differentiated from other cosolutes by their stabilizing influences on protein tertiary structure but by their compatibility with the interactions between protein surfaces that organize the cellular interior. [ABSTRACT FROM AUTHOR]

Abstract: Engineered cytochrome P450s are emerging as powerful synthetic tools due to their ability catalyze non‐native metallocarbenoid and ‐nitrenoid insertion reactions. P450‐mediated cyclopropanation has garnered particular interest due to the high selectivity demonstrated by engineered scaffolds and their application towards the synthesis of therapeutic agents. We previously reported that mutation of a conserved, first‐shell heme‐ligating Cys to Ser led to significant improvements in cyclopropanation activity in a model enzyme, P450BM3h. Here, we demonstrate that mutation of a ubiquitously conserved second‐shell Phe (F393) to His or Ala, provides complementary increases in the P450 heme reduction potential and conversion to cyclopropanation products when compared to first‐shell Cys to Ser mutations. Furthermore, we show that these mutations confer improved non‐natural catalysis in 4 diverse P450 scaffolds. [ABSTRACT FROM AUTHOR]

Whole-cell catalysts for non-natural chemical reactions will open new routes to sustainable production of chemicals. We designed a cytochrome 'P411' with unique serine-heme ligation that catalyzes efficient and selective olefin cyclopropanation in intact Escherichia coli cells. The mutation C400S in cytochrome P450BM3 gives a signature ferrous CO Soret peak at 411 nm, abolishes monooxygenation activity, raises the resting-state FeIII-to-FeII reduction potential and substantially improves NAD(P)H-driven activity. [ABSTRACT FROM AUTHOR]

The article focuses on a study conducted by researcher M.L. Matthews to demonstrate that a syringomycin halogenase SyrB2 can be used to install nitrogenous anions in place of carbon-hydrogen bonds and generate carbon-nitrogen (C-N) bonds. Topics discussed include promiscuous reactions, addition of a glycine mutation and halogenation activities. The study highlights the limitation of C-N bonds such as low product yield.

A correction to the article "A serine-substituted P450 catalyzes highly efficient carbene transfer to olefins in vivo" that was published in the June 23, 2013 issue is presented.

The article presents a study which investigates the development of proteins with the application of a genetically encoded "chemical warheads." Researchers presented that a 21 amino acid code can contribute a selective advantage in a phaged-based system towards the evolution of gp120-binding antibodies. The study also hypothesized that glycan-binding proteins has the possibility to undergo evolution in Boro-X-Escherichia coli since the boronic acid group has an intrinsic reaction for diols.

Thiopeptides are a growing class of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products. Many biosynthetic enzymes for RiPPs, especially thiopeptides, are promiscuous and can accept a wide range of peptide substrates with different amino acid sequences; thus, these enzymes have been used as tools to generate new natural product derivatives. Here, we explore an alternative route to molecular complexity by engineering thiopeptide tailoring enzymes to do new or non-native chemistry. We explore cytochrome P450 enzymes as biocatalysts for cyclopropanation of dehydroalanines, chemical motifs found widely in thiopeptides and other RiPP-based natural products. We find that P450 TbtJ1 and P450 TbtJ2 selectively cyclopropanate dehydroalanines in a number of complex thiopeptide-based substrates and convert them into 1-amino-2-cyclopropane carboxylic acids (ACCAs), which are important pharmacophores. This chemistry takes advantage of the innate affinity of these biosynthetic enzymes for their substrates and enables incorporation of new pharmacophores into thiopeptide architectures. This work also presents a strategy for diversification of natural products through rationally repurposing biosynthetic enzymes as non-natural biocatalysts.