Band 385, Heft 10

Cryoelectron tomography opens a window into the inner space of cells. It combines the potential of three-dimensional imaging with a close-to-life preservation of biological samples. Tomograms with molecular resolution are essentially images of the cellular proteome and, in conjunction with advanced pattern recognition techniques, they can be used to map the molecular landscape inside organelles and cells.

Denitrification represents an important part of the biogeochemical cycle of the essential element nitrogen. It constitutes the predominant pathway of the reductive dissimilation of nitrate in the environment. Via four enzymatic reactions, nitrate is transformed stepwise to nitrite (NO 2 - ), nitric oxide (NO), and nitrous oxide (N 2 O), to finally yield dinitrogen gas (N 2 ). All steps within this metabolic pathway are catalyzed by complex multi-site metalloenzymes with unique spectroscopic and structural features. In recent years, high-resolution crystal structures have become available for these enzymes with the exception of the structure for NO reductase.

Nectins are Ca 2+ -independent immunoglobulin (Ig)-like cell-cell adhesion molecules, which comprise a family consisting of four members. Nectins have five activities: (1) they show Ca 2+ -independent cell-cell adhesion activity by homo- and hetero- trans -interactions through their extracellular regions; (2) they bind afadin, an actin filament (F-actin)-binding protein, through their cytoplasmic tails and are connected to the actin cytoskeleton; (3) they induce activation of Cdc42 and Rac small G proteins through their cytoplasmic tails; (4) they bind Par-3, a cell polarity protein, through their cytoplasmic tails; and (5) they heterophilically trans -interact with Necls, nectin-like molecules, through their extracellular regions. Through these activities, nectins regulate a variety of cellular functions, including adhesion, migration, and polarization. Here we describe these activities and functions of nectins.

Fluorescence methods are now well-established and powerful tools to study biological macromolecules. The canonical amino acid tryptophan (Trp), encoded by a single UGG triplet, is the main reporter of intrinsic fluorescence properties of most natural proteins and peptides and is thus an attractive target for tailoring their spectral properties. Recent advances in research have provided substantial evidence that the natural protein translational machinery can be genetically reprogrammed to introduce a large number of non-coded (i.e. noncanonical) Trp analogues and surrogates into various proteins. Especially attractive targets for such an engineering approach are fluorescent proteins in which the chromophore is formed post-translationally from an amino acid sequence, like the green fluorescent protein from Aequorea victoria . With the currently available translationally active fluoro-, hydroxy-, amino-, halogen-, and chalcogen-containing Trp analogues and surrogates, the traditional methods for protein engineering and design can be supplemented or even fully replaced by these novel approaches. Future research will provide a further increase in the number of Trp-like amino acids that are available for redesign (by engineering of the genetic code) of native Trp residues and enable novel strategies to generate proteins with tailored spectral properties.

Genomic imprinting is an epigenetic phenomenon that results in differential expression of both alleles, depending on their parent of origin. We have recently identified many imprinted small non-coding RNA genes belonging to the C/D RNA and microRNA gene families, both of which are usually known to play key roles in post-transcriptional metabolism of specific genes (e.g. C/D RNAs guide ribose methylation of target RNAs while microRNAs elicit either translational repression or RNA interference). Although the functional and evolutionary significance of this association between C/D RNA genes, microRNA genes and genomic imprinting is still highly elusive, these observations provide a framework for further analysis of the potential role of small non-coding RNAs in epigenetic control.

Recent developments including pulse and multi-frequency techniques make the combination of site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy an attractive approach for the study of protein-protein or protein-oligonucleotide interaction. Analysis of the spin label side chain mobility, its solvent accessibility, the polarity of the spin label micro-environment and distances between spin label side chains allow the modeling of protein domains or protein-protein interaction sites and their conformational changes with a spatial resolution at the level of the backbone fold. Structural changes can be detected with millisecond time resolution. Inter- and intra-molecular distances are accessible in the range from approximately 0.5 to 8 nm by the combination of continuous wave and pulse EPR methods. Recent applications include the study of transmembrane substrate transport, membrane channel gating, gene regulation and signal transfer.

The coupling of ion and water flow through membrane channels is under dispute. Among all human aquaporins only aquaporin-6 exhibits ion channel activity. Whether aquaporin-6 functions also as a water channel cannot yet be determined with confidence. Similarly, a comparison of single-channel water permeabilities from ion channels and aquaporins suggests that ion channels may play a secondary role as water channels. However, the fraction of absorbed fluid that crosses epithelial ion channels still remains to be determined.

Members of the ATP-binding cassette (ABC) superfamily are integral membrane proteins that hydrolyze ATP to drive transport. In the last two decades these proteins have been extensively characterized on a genetic and biochemical level, and in recent years high-resolution crystal structures of several nucleotide-binding domains and full-length transporters have extended our knowledge. Here we discuss the possible mechanisms of transport that have been derived from these crystal structures and the extensive available biochemical data.

The human protein FLJ36880 belongs to the fumarylacetoacetate hydrolase family. The X-ray structure of FLJ36880 has been determined to 2.2 Å resolution employing the semi-automated high-throughput structural genomics approach of the Protein Structure Factory. FLJ36880 adopts a mixed β-sandwich roll fold and forms homodimers in crystals as well as in solution. One Mg 2+ ion is bound to each subunit of the dimeric protein by coordination to three carboxylate oxygens and three water molecules. These metal binding sites are accessible from the same surface of the dimer, partly due to the disorder of the undecapeptide stretch D29 to L39. The overall structure and metal binding site of FLJ36880 bear clear similarities to the C-terminal domain of the bifunctional enzyme HpcE from Escherichia coli C, fumarylacetoacetate hydrolase from Mus musculus and to YcgM (Apc5008) from E. coli 1262. These similarities provide a framework for suggesting biochemical functions and evolutionary relationships of FLJ36880. It appears highly probable that the metal binding sites are involved in an enzymatic activity related to the catabolism of aromatic amino acids. Two point mutations in the active-site of FAH, responsible for the metabolic disease hereditary tyrosinemia type I (HTI) in humans, affect residues that are structurally conserved in FLJ36880 and located in the putative catalytic site.

We present a novel two-step protocol for the differentiation of embryonic stem (ES) cells into the hepatic lineage. Differentiated hepatocyte-like cells express genes and proteins characteristic for endodermal and hepatic cells and acquire a functional hepatic phenotype as demonstrated by albumin secretion and glycogen storage. During differentiation, α-fetoprotein, albumin, transthyretin, α-1-antitrypsin, cytochrome P450 subunits 2b9 and 2b13 and tyrosine aminotransferase transcripts are upregulated. Quantitative RT-PCR data revealed a fetal hepatic phenotype corresponding to day 13–14 of liver development. Terminally differentiated hepatocyte-like cells show a bi-nucleated, cuboidal morphology labeled by albumin, α-1-antitrypsin, liver amylase, dipeptidyl peptidase IV, c-met and cytokeratin 18. ES-derived intermediate cell types transiently and partially co-express nestin with albumin and α-fetoprotein, respectively, but not cytokeratin 19. This finding suggests an ES-derived potential hepatic progenitor cell type, which is partially nestin-, albumin- and α-fetoproteinpositive, but cytokeratin 19-negative.

The surface topography of red blood cells (RBCs) was investigated under near-physiological conditions using atomic force microscopy (AFM). An immobilization protocol was established where RBCs are coupled via molecular bonds of the membrane glycoproteins to wheat germ agglutinin (WGA), which is covalently and flexibly tethered to the support. This results in a tight but non-invasive attachment of the cells. Using tapping-mode AFM, which is known as gentle imaging mode and therefore most appropriate for soft biological samples like erythrocytes, it was possible to resolve membrane skeleton structures without major distortions or deformations of the cell surface. Significant differences in the morphology of RBCs from healthy humans and patients with systemic lupus erythematosus (SLE) were observed on topographical images. The surface of RBCs from SLE patients showed characteristic circular-shaped holes with approx. 200 nm in diameter under physiological conditions, a possible morphological correlate to previously published changes in the SLE erythrocyte membrane.

In eukaryotic cells, double-stranded RNA is degraded to 21mers and triggers RNA interference. Using a pattern description language, we have searched the EMBL database for sequences with the potential to form double strands in cis in Dictyostelium discoideum . No extended inverted repeats were found in mRNAs. However, the antisense direction of some mRNAs encoding regulatory or developmentally regulated proteins showed the ability to form double-stranded regions. In EST archives, we found potential double strands derived from a few genes, but these transcripts are not continuously encoded in the genome. Most likely, they represent hybrid molecules of sense and antisense RNAs.

The technique of segregation of partly melted molecules (SPM) is a convenient and efficient method to isolate DNA fragments associated with CpG islands. The approach is conceptually simple and uses denaturant gradient gel electrophoresis to separate DNA molecules digested with restriction endonucleases. The SPM methodology has successfully been applied to the identification of genes from anonymous, unsequenced DNA fragments and CpG islands methylated in human cancer. In this article the theoretical background and practical application of the SPM method is reviewed.

We have mutated the monomer-monomer interface of the restriction endonuclease EcoRI in order to destabilize the homodimer and to stabilize heterodimers. Mutations of Leu158 to charged amino acid residues result in strong destabilization of the dimer. The largest effect was detected for the L158D mutant which is monomeric even at higher concentrations. It unspecifically degrades DNA by cleaving both single strands independently every 15 nucleotides on the average. Although cleavage is reproducible, it is not determined by nucleotide sequence but by general properties like conformation or deformability as has been found for other unspecific nucleases. Mutations of Ile230, which is in direct contact with Leu158 of the other subunit, cause structural changes with the loss of about ten percent α-helix content, but interfere only marginally with homodimerization and double strand cleavage. Again the mutation to aspartate shows the strongest effects. Mixtures of single mutants, one containing aspartate at one of the two positions and the other lysine at the corresponding position, form heterodimers. These are mainly stabilized compared to the homodimers by re-establishment of the wild-type hydrophobic interaction at the not mutated residues while an interaction of aspartate and lysine seems energetically unfavorable in this structural context.