Fr Francium

The cover picture shows images of the nucleus in a living U2OS cell. The right-hand image shows continuous fibers of DNA labeled with Picogreen and was acquired using direct stochastic optical reconstruction microscopy (dSTORM). For comparison, the diffraction-limited wide-field image is shown on the left-hand side. Picogreen is a commercially available, DNA-specific fluorophore that is suitable for single-molecule observations. On p. 298 ff., A. Benke and S. Manley describe the use of live-cell dSTORM imaging to visualize nuclear and mitochondrial Picogreen-labeled DNA at super-resolution. As well as chromatin fibers, mitochondrial nucleolids could be imaged at a resolution of 70 nm. They also demonstrate time-lapse dSTORM imaging which will allow the dynamics of chromatin organization to be investigated at an unprecedented level.

Per aspera ad astra: Kinetic isotope effects have indicated a new and comprehensive picture of the central ribosomal enzymatic activity, peptide-bond formation. To this end, isotopes were incorporated in the positions highlighted in red.

Tagging protein AMPylation: A new chemical reporter for AMPylation, recently identified as a key post-translational modification during bacterial infection, is a robust tool for detecting and identifying AMPylated proteins in vitro.

The tools at our disposal define the scientific problems we can solve. Few tools exist for studying and altering the components inside a living animal. The incorporation of unnatural amino acids site-specifically into proteins in Caenorhabditis elegans greatly expands the toolbox currently available to biologists allowing for investigation of inter- and intracellular interactions in an organismal context.

We have implemented the super-resolution method of direct stochastic optical reconstruction microscopy (dSTORM) to image nuclear and mitochondrial DNA in living cells. We also demonstrate time-lapse imaging, all using a dye that associates directly with cellular DNA: the commercially available dye Picogreen (see figure).

The pro form of melB tyrosinase from the melB gene of Aspergillus oryzae was over-produced from E. coli and formed a homodimer that exhibited the spectral features of met-tyrosinase. In the presence of NH₂OH (reductant), the proenzyme bound dioxygen to give a stable (μ-η²:η²-peroxo)dicopper(II) species (oxy form), thus indicating that the pro form tyrosinase can function as an oxygen carrier or storage protein like hemocyanin. The pro form tyrosinase itself showed no catalytic activity toward external substrates, but proteolytic digestion with trypsin activated it to induce tyrosinase activity. Mass spectroscopy analyses, mutagenesis experiments, and colorimetry assays have demonstrated that the tryptic digestion induced cleavage of the C-terminal domain (Glu458–Ala616), although the dimeric structure of the enzyme was retained. The structural changes induced by proteolytic digestion might open the entrance to the enzyme active site for substrate incorporation.

Proteolytic functional change: the melB gene product (melB tyrosinase) from Aspergillus oryzae can function as dioxygen carrier, monooxygenase, and oxidase. The cleavage of the C-terminal domain by proteolytic treatment changes the function of the tyrosinase from dioxygen carrier to catechol oxidase and phenol monooxygenase.

RISC avoidance: By introducing an intercalator such as azobenzene into the 5′-end of the sense strand at a D-threoninol linker, RNAi activity was greatly improved. This enhancement of RNAi activity was attributed to selective loading of the antisense strand into RISC and the suppression of RISC assembly with the modified sense strand.

Thiolysine-mediated chemical ligation has generated fluorescence polarisation assay reagents based on isopeptide-linked ubiquitin-like protein conjugates. These have been used to monitor the activity of ubiquitin(-like) proteases. Thus, it is now possible to generate assay reagents that contain substrate-derived elements around the isopeptide linkage, with no practical limitation.

The genomes of all bacteria with publicly available sequenced genomes have been screened for the presence of sesquiterpene cyclase homologues, resulting in the identification of 55 putative geosmin synthases, 23 homologues of 2-methylisoborneol synthases, and 98 other sesquiterpene cyclase homologues. Most of these enzymes by far were found in actinomycetes. The terpenoid volatiles from 35 strains, including 31 actinomycetes and four strains from other taxa, were collected by using a closed-loop stripping apparatus and identified by GC-MS. All of these bacteria apart from one strain encode sesquiterpene cyclase homologues in their genomes. The identified volatile terpenoids were grouped according to structural similarities and their biosynthetic relationship, and the results of these analyses were correlated to the available genome information, resulting in valuable new insights into bacterial terpene biosynthesis.

The volatiles from 35 bacteria with sequenced genomes, most of them actinomycetes, have been analysed. In the genomes of 34 species, one or several sesquiterpene cyclase homologues were encoded. The diversity of volatile terpenes emitted by these strains was correlated to the available genetic information, giving interesting new insights into bacterial terpene biosynthesis.

The covalent flavoprotein alditol oxidase (AldO) from Streptomyces coelicolor A3(2) was endowed with an extra catalytic functionality by fusing it to a microperoxidase. Purification of the construct resulted in the isolation of a synthetic bifunctional enzyme that was both fully covalently flavinylated and heminylated: an oxiperoxidase. Characterization revealed that both oxidase and peroxidase functionalities were active, with the construct functioning as a single-component xylitol biosensor. In an attempt to reduce the size of the oxidase–peroxidase fusion, we replaced portions of the native AldO sequence with the bacterial cytochrome c CXXCH heme-binding motif. By mutating only three residues of the AldO protein we were able to create a functional oxidase–peroxidase hybrid.

Two in one: We present the merging of two complementary enzyme activities in a single protein backbone to create a new breed of hybrid enzyme: an oxiperoxidase. Such hybrid enzymes have potential for application in biosensors and in high-throughput screening assays.