MEL-28/ELYS is required for the recruitment of nucleoporins to chromatin and postmitotic nuclear pore complex assembly

The metazoan nuclear envelope (NE) breaks down and re-forms during each cell cycle. Nuclear pore complexes (NPCs), which allow nucleocytoplasmic transport during interphase, assemble into the re-forming NE at the end of mitosis. Using in vitro NE assembly, we show that the vertebrate homologue of MEL-28 (maternal effect lethal), a recently discovered NE component in Caenorhabditis elegans, functions in postmitotic NPC assembly. MEL-28 interacts with the Nup107-160 complex (Nup for nucleoporin), an important building block of the NPC, and is essential for the recruitment of the Nup107-160 complex to chromatin. We suggest that MEL-28 acts as a seeding point for NPC assembly.     

(NZ)CH...O Contacts assist crystallization of a ParB-like nuclease

Background

The major bottleneck for determination of 3 D structures of proteins using X-rays is the production of diffraction quality crystals. Often proteins are subjected to chemical modification to improve the chances of crystallization

Results

Here, we report the successful crystallization of a nuclease employing a reductive methylation protocol. The key to crystallization was the successful introduction of 44 new cohesive (NZ) CH...O contacts (3.2 – 3.7 Å) by the addition of 2 methyl groups to the side chain amine nitrogen (NZ) of 9 lysine residues of the nuclease. The new contacts dramatically altered the crystallization properties of the protein, resulting in crystals that diffracted to 1.2 Å resolution. Analytical ultracentrifugation analysis and thermodynamics results revealed a more compact protein structure with better solvent exclusion of buried Trp residues in the folded state of the methylated protein, assisting crystallization.

Conclusion

In this study, introduction of novel cohesive (NZ)CH...O contacts by reductive methylation resulted in the crystallization of a protein that had previously resisted crystallization in spite of extensive purification and crystallization space screening. Introduction of (NZ)CH...O contacts could provide a solution to crystallization problems for a broad range of protein targets.     

Interaction of potassium cyanide with the [Ni-4Fe-5S] active site cluster of CO dehydrogenase from Carboxydothermus hydrogenoformans

The Ni-Fe carbon monoxide (CO) dehydrogenase II (CODHII(Ch)) from the anaerobic CO-utilizing hydrogenogenic bacterium Carboxydothermus hydrogenoformans catalyzes the oxidation of CO, presumably at the Ni-(micro(2)S)-Fe1 subsite of the [Ni-4S-5S] cluster in the active site. The CO oxidation mechanism proposed on the basis of several CODHII(Ch) crystal structures involved the apical binding of CO at the nickel ion and the activation of water at the Fe1 ion of the cluster. To understand how CO interacts with the active site, we have studied the reactivity of the cluster with potassium cyanide and analyzed the resulting type of nickel coordination by x-ray absorption spectroscopy. Cyanide acts as a competitive inhibitor of reduced CODHII(Ch) with respect to the substrate CO and is therefore expected to mimic the substrate. It inhibits the enzyme reversibly, forming a nickel cyanide. In this reaction, one of the four square-planar sulfur ligands of nickel is replaced by the carbon atom of cyanide, suggesting removal of the micro(2)S from the Ni-(micro(2)S)-Fe1 subsite. Upon reactivation of the inhibited enzyme, cyanide is released, and the square-planar coordination of nickel by 4S ligands is recovered, which includes the reformation of the Ni-(micro(2)S)-Fe1 bridge. The results are summarized in a model of the CO oxidation mechanism at the [Ni-4Fe-5S] active site cluster of CODHII(Ch) from C. hydrogenoformans.     

High-Level Expression, Refolding and Probing the Natural Fold of the Human Voltage-Dependent Anion Channel Isoforms I and II

The voltage-dependent anion channel (VDAC) is the major protein found in the outer membrane of mitochondria. The channel is responsible for the exchange of ATP/ADP and the translocation of ions and other small metabolites over the membrane. In order to obtain large amounts of pure and suitably folded human VDAC for functional and structural studies, the genes of the human isoforms I and II (HVDAC1 and HVDAC2) were cloned in Escherichia coli. High-level expression led to inclusion body formation. Both proteins could be refolded in vitro by adding denatured protein to a solution of zwitterionic or nonionic detergents. A highly efficient and fast protocol for refolding was developed that yielded more than 50 mg of pure human VDACs per liter of cell culture. The native and functional state of the refolded porins was probed by Fourier transform infrared spectroscopy to determine the secondary structure composition and by electrophysiological measurements, demonstrating the pore-forming activity of HVDAC1. Furthermore, binding of HVDAC1 to immobilized ATP was demonstrated. Limited proteolysis of HVDAC1 protein embedded in detergent micelles in combination with matrix-assisted laser desorption ionization mass spectrometric analysis was applied to identify micelle-exposed regions of the protein and to develop an improved topology model. Our analysis strongly suggests a 16-stranded, antiparallel beta-barrel with one large and seven short loops and turns. Initial crystallization trials of the protein yielded crystals diffracting to 8 Angstrom resolution.     

Composition and hierarchical organisation of a spider silk

Albeit silks are fairly well understood on a molecular level, their hierarchical organisation and the full complexity of constituents in the spun fibre remain poorly defined. Here we link morphological defined structural elements in dragline silk of Nephila clavipes to their biochemical composition and physicochemical properties. Five layers of different make-ups could be distinguished. Of these only the two core layers contained the known silk proteins, but all can vitally contribute to the mechanical performance or properties of the silk fibre. Understanding the composite nature of silk and its supra-molecular organisation will open avenues in the production of high performance fibres based on artificially spun silk material.     

Effect of oculopharyngeal muscular dystrophy-associated extension of seven alanines on the fibrillation properties of the N-terminal domain of PABPN1

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease that usually manifests itself within the fifth decade. The most prominent symptoms are progressive ptosis, dysphagia, and proximal limb muscle weakness. The disorder is caused by trinucleotide (GCG) expansions in the N-terminal part of the poly(A)-binding protein 1 (PABPN1) that result in the extension of a 10-alanine segment by up to seven more alanines. In patients, biopsy material displays intranuclear inclusions consisting primarily of PABPN1. Poly l-alanine-dependent fibril formation was studied using the recombinant N-terminal domain of PABPN1. In the case of the protein fragment with the expanded poly l-alanine sequence [N-(+7)Ala], fibril formation could be induced by low amounts of fragmented fibrils serving as seeds. Besides homologous seeds, seeds derived from fibrils of the wild-type fragment (N-WT) also accelerated fibril formation of N-(+7)Ala in a concentration-dependent manner. Seed-induced fibrillation of N-WT was considerably slower than that of N-(+7)Ala. Using atomic force microscopy, differences in fibril morphologies between N-WT and N-(+7)Ala were detected. Furthermore, fibrils of N-WT showed a lower resistance against solubilization with the chaotropic agent guanidinium thiocyanate than those from N-(+7)Ala. Our data clearly reveal biophysical differences between fibrils of the two variants that are likely caused by divergent fibril structures.     

Consistency analysis of metabolic correlation networks

Background  

Metabolic correlation networks are derived from the covariance of metabolites in replicates of metabolomics experiments. They constitute an interesting intermediate between topology (i.e. the system's architecture defined by the set of reactions between metabolites) and dynamics (i.e. the metabolic concentrations observed as fluctuations around steady-state values in the metabolic network).     

Enabling coupled quantitative genomics and proteomics analyses from rat spinal cord samples

Translational research is progressing toward combined genomics and proteomics analyses of small and precious samples. In our analyses of spinal cord material, we systematically evaluated disruption and extraction techniques to determine an optimum process for the coupled analysis of RNA and protein from a single 5-mm segment of tissue. Analyses of these distinct molecular species were performed using microarrays and high resolution two-dimensional gels, respectively. Comparison of standard homogenization with automated frozen disruption (AFD) identified negligible differences in the relative abundance of genes (44) with all genes identified by either process. Analysis on either the Affymetrix or Applied Biosystems Inc. gene array platforms provided good correlations between the extraction techniques. In contrast, the AFD technique enabled identification of more unique proteins from spinal cord tissue than did standard homogenization. Furthermore use of an optimized CHAPS/urea extraction provided better protein recovery, as shown by quantitative two-dimensional gel analyses, than did solvent precipitation during TRIzol-based RNA extraction. Thus, AFD of tissue samples followed by protein and RNA isolation from separate aliquots of the frozen powdered sample is the most effective route to ensure full, quantitative analyses of both molecular entities.     

Structural and chemical profiling of the human cytosolic sulfotransferases

The human cytosolic sulfotransfases (hSULTs) comprise a family of 12 phase II enzymes involved in the metabolism of drugs and hormones, the bioactivation of carcinogens, and the detoxification of xenobiotics. Knowledge of the structural and mechanistic basis of substrate specificity and activity is crucial for understanding steroid and hormone metabolism, drug sensitivity, pharmacogenomics, and response to environmental toxins. We have determined the crystal structures of five hSULTs for which structural information was lacking, and screened nine of the 12 hSULTs for binding and activity toward a panel of potential substrates and inhibitors, revealing unique “chemical fingerprints” for each protein. The family-wide analysis of the screening and structural data provides a comprehensive, high-level view of the determinants of substrate binding, the mechanisms of inhibition by substrates and environmental toxins, and the functions of the orphan family members SULT1C3 and SULT4A1. Evidence is provided for structural “priming” of the enzyme active site by cofactor binding, which influences the spectrum of small molecules that can bind to each enzyme. The data help explain substrate promiscuity in this family and, at the same time, reveal new similarities between hSULT family members that were previously unrecognized by sequence or structure comparison alone.     

Plastic and genetic responses of a common sedge to warming have contrasting effects on carbon cycle processes

Climate warming affects plant physiology through genetic adaptation and phenotypic plasticity, but little is known about how these mechanisms influence ecosystem processes. We used three elevation gradients and a reciprocal transplant experiment to show that temperature causes genetic change in the sedge Eriophorum vaginatum. We demonstrate that plants originating from warmer climate produce fewer secondary compounds, grow faster and accelerate carbon dioxide (CO₂) release to the atmosphere. However, warmer climate also caused plasticity in E. vaginatum, inhibiting nitrogen metabolism, photosynthesis and growth and slowing CO₂ release into the atmosphere. Genetic differentiation and plasticity in E. vaginatum thus had opposing effects on CO₂ fluxes, suggesting that warming over many generations may buffer, or reverse, the short‐term influence of this species over carbon cycle processes. Our findings demonstrate the capacity for plant evolution to impact ecosystem processes, and reveal a further mechanism through which plants will shape ecosystem responses to climate change.