Structural Characterization of the P1+ Intermediate State of the P-Cluster of Nitrogenase

Nitrogenase is the enzyme that reduces atmospheric dinitrogen (N2) to ammonia (NH3) in biological systems. It catalyzes a series of single-electron transfers from the donor iron protein (Fe protein) to the molybdenum–iron protein (MoFe protein) that contains the iron–molybdenum cofactor (FeMo-co) sites where N2 is reduced to NH3. The P-cluster in the MoFe protein functions in nitrogenase catalysis as an intermediate electron carrier between the external electron donor, the Fe protein, and the FeMo-co sites of the MoFe protein. Previous work has revealed that the P-cluster undergoes redox-dependent structural changes and that the transition from the all-ferrous resting (PN) state to the two-electron oxidized P2+ state is accompanied by protein serine hydroxyl and backbone amide ligation to iron. In this work, the MoFe protein was poised at defined potentials with redox mediators in an electrochemical cell, and the three distinct structural states of the P-cluster (P2+, P1+, and PN) were characterized by X-ray crystallography and confirmed by computational analysis. These analyses revealed that the three oxidation states differ in coordination, implicating that the P1+ state retains the serine hydroxyl coordination but lacks the backbone amide coordination observed in the P2+ states. These results provide a complete picture of the redox-dependent ligand rearrangements of the three P-cluster redox states

SPT-CL J2215−3537: A Massive Starburst at the Center of the Most Distant Relaxed Galaxy Cluster

We present the discovery of the most distant, dynamically relaxed cool core cluster, SPT-CL J2215−3537 (SPT2215), and its central brightest cluster galaxy (BCG) at z = 1.16. Using new X-ray observations, we demonstrate that SPT2215 harbors a strong cool core with a central cooling time of 200 Myr (at 10 kpc) and a maximal intracluster medium cooling rate of 1900 ± 400 M⊙ yr−1. This prodigious cooling may be responsible for fueling the extended, star-forming filaments observed in Hubble Space Telescope imaging. Based on new spectrophotometric data, we detect bright [O ii] emission in the BCG, implying an unobscured star formation rate (SFR) of ${320}_{-140}^{+230}$M⊙ yr−1. The detection of a weak radio source (2.0 ± 0.8 mJy at 0.8 GHz) suggests ongoing feedback from an active galactic nucleus (AGN), though the implied jet power is less than half the cooling luminosity of the hot gas, consistent with cooling overpowering heating. The extreme cooling and SFR of SPT2215 are rare among known cool core clusters, and it is even more remarkable that we observe these at such high redshift, when most clusters are still dynamically disturbed. The high mass of this cluster, coupled with the fact that it is dynamically relaxed with a highly isolated BCG, suggests that it is an exceptionally rare system that must have formed very rapidly in the early universe. Combined with the high SFR, SPT2215 may be a high-z analog of the Phoenix cluster, potentially providing insight into the limits of AGN feedback and star formation in the most massive galaxies

The opposing transcriptional functions of Sin3a and c-Myc are required to maintain tissue homeostasis.

How the proto-oncogene c-Myc balances the processes of stem-cell self-renewal, proliferation and differentiation in adult tissues is largely unknown. We explored c-Myc's transcriptional roles at the epidermal differentiation complex, a locus essential for skin maturation. Binding of c-Myc can simultaneously recruit (Klf4, Ovol-1) and displace (Cebpa, Mxi1 and Sin3a) specific sets of differentiation-specific transcriptional regulators to epidermal differentiation complex genes. We found that Sin3a causes deacetylation of c-Myc protein to directly repress c-Myc activity. In the absence of Sin3a, genomic recruitment of c-Myc to the epidermal differentiation complex is enhanced, and re-activation of c-Myc-target genes drives aberrant epidermal proliferation and differentiation. Simultaneous deletion of c-Myc and Sin3a reverts the skin phenotype to normal. Our results identify how the balance of two transcriptional key regulators can maintain tissue homeostasis through a negative feedback loop

The Photosynthetic Apparatus and Its Regulation in the Aerobic Gammaproteobacterium Congregibacter litoralis gen. nov., sp. nov

BACKGROUND: There is accumulating evidence that in some marine environments aerobic bacteriochlorophyll a-producing bacteria represent a significant part of the microbial population. The interaction of photosynthesis and carbon metabolism in these interesting bacteria is still largely unknown and requires further investigation in order to estimate their contribution to the marine carbon cycle. METHODOLOGY/PRINCIPAL FINDINGS: Here, we analyzed the structure, composition and regulation of the photosynthetic apparatus in the obligately aerobic marine gammaproteobacterium KT71(T). Photoheterotrophically grown cells were characterized by a poorly developed lamellar intracytoplasmic membrane system, a type 1 light-harvesting antenna complex and a photosynthetic reaction center associated with a tetraheme cytochrome c. The only photosynthetic pigments produced were bacteriochlorophyll a and spirilloxanthin. Under semiaerobic conditions KT71(T) cells expressing a photosynthetic apparatus showed a light-dependent increase of growth yield in the range of 1.3-2.5 fold. The expression level of the photosynthetic apparatus depended largely on the utilized substrate, the intermediary carbon metabolism and oxygen tension. In addition, pigment synthesis was strongly influenced by light, with blue light exerting the most significant effect, implicating that proteins containing a BLUF domain may be involved in regulation of the photosynthetic apparatus. Several phenotypic traits in KT71(T) could be identified that correlated with the assumed redox state of growing cells and thus could be used to monitor the cellular redox state under various incubation conditions. CONCLUSIONS/SIGNIFICANCE: In a hypothetical model that explains the regulation of the photosynthetic apparatus in strain KT71(T) we propose that the expression of photosynthesis genes depends on the cellular redox state and is maximal under conditions that allow a balanced membrane redox state. So far, bacteria capable of an obligately aerobic, photosynthetic metabolism constitute a unique phenotype within the class Gammaproteobacteria, so that it is justified to propose a new genus and species, Congregibacter litoralis gen. nov, sp. nov., represented by the type strain KT71(T) ( = DSM 17192(T) = NBRC 104960(T))

The Crystal Structure of D212 from Sulfolobus Spindle-Shaped Virus Ragged Hills Reveals a New Member of the PD-(D/E)XK Nuclease Superfamily▿

Structural studies have made significant contributions to our understanding of Sulfolobus spindle-shaped viruses (Fuselloviridae), an important model system for archaeal viruses. Continuing these efforts, we report the structure of D212 from Sulfolobus spindle-shaped virus Ragged Hills. The overall fold and conservation of active site residues place D212 in the PD-(D/E)XK nuclease superfamily. The greatest structural similarity is found to the archaeal Holliday junction cleavage enzymes, strongly suggesting a role in DNA replication, repair, or recombination. Other roles associated with nuclease activity are also considered

Structural and Biochemical Analysis of the Hordeum vulgare L. <i>Hv</i>GR-RBP1 Protein, a Glycine-Rich RNA-Binding Protein Involved in the Regulation of Barley Plant Development and Stress Response

The timing of whole-plant senescence influences important agricultural traits such as yield and grain protein content. Post-transcriptional regulation by plant RNA-binding proteins is essential for proper control of gene expression, development, and stress responses. Here, we report the three-dimensional solution NMR structure and nucleic acid-binding properties of the barley glycine-rich RNA-binding protein <i>Hv</i>GR-RBP1, whose transcript has been identified as being >45-fold up-regulated in earlyas compared to latesenescing near-isogenic barley germplasm. NMR analysis reveals that <i>Hv</i>GR-RBP1 is a multidomain protein comprising a well-folded N-terminal RNA Recognition Motif (RRM) and a structurally disordered C-terminal glycine-rich domain. Chemical shift differences observed in 2D ¹H–¹⁵N correlation (HSQC) NMR spectra of full-length <i>Hv</i>GR-RBP1 and N-<i>Hv</i>GR-RBP1 (RRM domain only) suggest that the two domains can interact both in-trans and intramolecularly, similar to what is observed in the tobacco <i>Nt</i>GR-RBP1 protein. Further, we show that the RRM domain of <i>Hv</i>GR-RBP1 binds single-stranded DNA nucleotide fragments containing the consensus nucleotide sequence 5′-TTCTGX-3′ with low micromolar affinity <i>in vitro</i>. We also demonstrate that the C-terminal glycine-rich (<i>Hv</i>GR) domain of <i>Hv</i>-GR-RBP1 can interact nonspecifically with ssRNA <i>in vitro</i>. Structural similarities with other plant glycine-rich RNA-binding proteins suggest that <i>Hv</i>GR-RBP1 may be multifunctional. Based on gene expression analysis following cold stress in barley and E. coli growth studies following cold shock treatment, we conclude that <i>Hv</i>GR-RBP1 functions in a manner similar to cold-shock proteins and harbors RNA chaperone activity. <i>Hv</i>GR-RBP1 is therefore not only involved in the regulation of barley development including senescence, but also functions in plant responses to environmental stress

Structural Characterization of the P\u3csup\u3e1\u3c/sup\u3e Intermediate State of the P-Cluster Of Nitrogenase

Nitrogenase is the enzyme that reduces atmospheric dinitrogen (N2) to ammonia (NH3) in biological systems. It catalyzes a series of single-electron transfers from the donor iron protein (Fe protein) to the molybdenum–iron protein (MoFe protein) that contains the iron–molybdenum cofactor (FeMo-co) sites where N2 is reduced to NH3. The P-cluster in the MoFe protein functions in nitrogenase catalysis as an intermediate electron carrier between the external electron donor, the Fe protein, and the FeMo-co sites of the MoFe protein. Previous work has revealed that the P-cluster undergoes redox-dependent structural changes and that the transition from the all-ferrous resting (PN) state to the two-electron oxidized P2 state is accompanied by protein serine hydroxyl and backbone amide ligation to iron. In this work, the MoFe protein was poised at defined potentials with redox mediators in an electrochemical cell, and the three distinct structural states of the P-cluster (P2, P1, and PN) were characterized by X-ray crystallography and confirmed by computational analysis. These analyses revealed that the three oxidation states differ in coordination, implicating that the P1 state retains the serine hydroxyl coordination but lacks the backbone amide coordination observed in the P2 states. These results provide a complete picture of the redox-dependent ligand rearrangements of the three P-cluster redox states

Solution Structure and Molecular Determinants of Hemoglobin Binding of the First NEAT Domain of IsdB in Staphylococcus aureus

The human pathogen Staphylococcus aureus acquires heme iron from hemoglobin (Hb) via the action of a series of iron-regulated surface determinant (Isd) proteins. The cell wall anchored IsdB protein is recognized as the predominant Hb receptor, and is comprised of two NEAr transporter (NEAT) domains that act in concert to bind, extract, and transfer heme from Hb to downstream Isd proteins. Structural details of the NEAT 2 domain of IsdB have been investigated, but the molecular coordination between NEAT 2 and NEAT 1 to extract heme from hemoglobin has yet to be characterized. To obtain a more complete understanding of IsdB structure and function, we have solved the 3D solution structure of the NEAT 1 domain of IsdB (IsdB^N1) spanning residues 125–272 of the full-length protein by NMR. The structure reveals a canonical NEAT domain fold and has particular structural similarity to the NEAT 1 and NEAT 2 domains of IsdH, which also interact with Hb. IsdB^N1 is also comprised of a short N-terminal helix, which has not been previously observed in other NEAT domain structures. Interestingly, the Hb binding region (loop 2 of IsdB^N1) is disordered in solution. Analysis of Hb binding demonstrates that IsdB^N1 can bind metHb weakly and the affinity of this interaction is further increased by the presence of IsdB linker domain. IsdB^N1 loop 2 variants reveal that phenylalanine 164 (F164) of IsdB is necessary for Hb binding and rapid heme transfer from metHb to IsdB. Together, these findings provide a structural role for IsdB^N1 in enhancing the rate of extraction of metHb heme by the IsdB NEAT 2 domain

Structural Characterization of the P\u3csup\u3e1\u3c/sup\u3e Intermediate State of the P-Cluster Of Nitrogenase

Nitrogenase is the enzyme that reduces atmospheric dinitrogen (N2) to ammonia (NH3) in biological systems. It catalyzes a series of single-electron transfers from the donor iron protein (Fe protein) to the molybdenum–iron protein (MoFe protein) that contains the iron–molybdenum cofactor (FeMo-co) sites where N2 is reduced to NH3. The P-cluster in the MoFe protein functions in nitrogenase catalysis as an intermediate electron carrier between the external electron donor, the Fe protein, and the FeMo-co sites of the MoFe protein. Previous work has revealed that the P-cluster undergoes redox-dependent structural changes and that the transition from the all-ferrous resting (PN) state to the two-electron oxidized P2 state is accompanied by protein serine hydroxyl and backbone amide ligation to iron. In this work, the MoFe protein was poised at defined potentials with redox mediators in an electrochemical cell, and the three distinct structural states of the P-cluster (P2, P1, and PN) were characterized by X-ray crystallography and confirmed by computational analysis. These analyses revealed that the three oxidation states differ in coordination, implicating that the P1 state retains the serine hydroxyl coordination but lacks the backbone amide coordination observed in the P2 states. These results provide a complete picture of the redox-dependent ligand rearrangements of the three P-cluster redox states

Columnar water vapor retrievals from multifilter rotating shadowband radiometer data

[1] The multifilter rotating shadowband radiometer (MFRSR) measures direct and diffuse irradiances in the visible and near-infrared spectral range. In addition to characteristics of atmospheric aerosols, MFRSR data also allow retrieval of precipitable water vapor (PWV) column amounts, which are determined from the direct normal irradiances in the 940-nm spectral channel. The HITRAN 2004 spectral database was used in our retrievals to model the water vapor absorption. We present a detailed error analysis describing the influence of uncertainties in instrument calibration and spectral response, as well as those in available spectral databases, on the retrieval results. The results of our PWV retrievals from the Southern Great Plains (SGP) site operated by the Department of Energy (DOE) Atmospheric Radiation Measurement (ARM) Program were compared with correlative standard measurements by microwave radiometers (MWRs) and a global positioning system (GPS) water vapor sensor, as well as with retrievals from other solar radiometers (AERONET&apos;s CIMEL, AATS-6). Some of these data are routinely available at the SGP&apos;s Central Facility; however, we also used measurements from a wider array of instrumentations deployed at this site during the water vapor intensive observation period (WVIOP2000) in September-October 2000. The WVIOP data show better agreement between different solar radiometers or between different microwave radiometers (both groups showing relative biases within 4%) than between these two groups of instruments, with MWR values being consistently higher (up to 14%) than those from solar instruments (especially in the large PWV column amount range). We also demonstrate the feasibility of using MFRSR network data for creation of 2D data sets comparable with that of the MODIS satellite water vapor product