Hsc66 substrate specificity is directed toward a discrete region of the iron-sulfur cluster template protein IscU

Hsc66 and Hsc20 comprise a specialized chaperone system important for the assembly of iron-sulfur clusters in Escherchia coli. Only a single substrate, the Fe/S template protein IscU, has been identified for the Hsc66/Hsc20 system, but the mechanism by which Hsc66 selectively binds IscU is unknown. We have investigated Hsc66 substrate specificity using phage display and a peptide array of IscU. Screening of a heptameric peptide phage display library revealed that Hsc66 prefers peptides with a centrally located Pro-Pro motif. Using a cellulose-bound peptide array of IscU we determined that Hsc66 interacts specifically with a region (residues 99-103, LPPVK) that is invariant among all IscU family members. A synthetic peptide (ELPPVKIHC) corresponding to IscU residues 98-106 behaves in a similar manner to native IscU, stimulating the ATPase activity of Hsc66 with similar affinity as IscU, preventing Hsc66 suppression of bovine rhodanese aggregation, and interacting with the peptide-binding domain of Hsc66. Unlike native IscU, however, the synthetic peptide is not bound by Hsc20 and does not synergistically stimulate Hsc66 ATPase activity with Hsc20. Our results indicate that Hsc66 and Hsc20 recognize distinct regions of IscU and further suggest that Hsc66 will not bind LPPVK motifs with high affinity in vivo unless they are in the context of native IscU and can be directed to Hsc66 by Hsc20

Composite Polarons in Ferromagnetic Narrow-band Metallic Manganese Oxides

A new mechanism is proposed to explain the colossal magnetoresistance and related phenomena. Moving electrons accompanied by Jahn-Teller phonon and spin-wave clouds may form composite polarons in ferromagnetic narrow-band manganites. The ground-state and finite-temperature properties of such composite polarons are studied in the present paper. By using a variational method, it is shown that the energy of the system at zero temperature decreases with the formation of composite polaron; the energy spectrum and effective mass of the composite polaron at finite temperature is found to be strongly renormalized by the temperature and the magnetic field. It is suggested that the composite polaron contribute significantly to the transport and the thermodynamic properties in ferromagnetic narrow-band metallic manganese oxides.Comment: Latex, no figur

Three hydrophobic amino acids in Escherichia coli HscB make the greatest contribution to the stability of the HscB-IscU complex

<p>Abstract</p> <p>Background</p> <p>General iron-sulfur cluster biosynthesis proceeds through assembly of a transient cluster on IscU followed by its transfer to a recipient apo-protein. The efficiency of the second step is increased by the presence of HscA and HscB, but the reason behind this is poorly understood. To shed light on the function of HscB, we began a study on the nature of its interaction with IscU. Our work suggested that the binding site of IscU is in the C-terminal domain of HscB, and two different triple alanine substitutions ([L92A, M93A, F153A] and [E97A, E100A, E104A]) involving predicted binding site residues had detrimental effects on this interaction. However, the individual contribution of each substitution to the observed effect remains to be determined as well as the possible involvement of other residues in the proposed binding site.</p> <p>Results</p> <p>In the work reported here, we used isothermal titration calorimetry to characterize the affinity of single alanine HscB mutants for IscU, and subsequently confirmed our results with nuclear magnetic resonance spectroscopy. Alanine substitutions of L92, L96, and F153 severely impaired the ability of HscB to form a complex with IscU; substitutions of R87, R99, and E100 had more modest effects; and substitutions of T89, M93, E97, D103, E104, R152, K156, and S160 had only minor or no detectable effects.</p> <p>Conclusions</p> <p>Our results show that the residues of HscB most important for strong interaction with IscU include three hydrophobic residues (L92, L96, and F153); in addition, we identified a number of other residues whose side chains contribute to a lesser extent to the interaction. Our results suggest that the triple alanine substitution at HscB positions 92, 96, and 153 will destabilize the HscB-IscU complex by ΔΔ<it>G</it>_b≅ 5.7 kcal/mol, equivalent to a ≅ 15000-fold reduction in the affinity of HscB for IscU. We propose that this triple mutant could provide a more definitive test of the functional importance of the HscB-IscU interaction in vivo than those used previously that yielded inconclusive results.</p

Multiple turnover transfer of [2Fe2S] clusters by the iron-sulfur cluster assembly scaffold proteins IscU and IscA

IscU/Isu and IscA/Isa (and related NifU and SufA proteins) have been proposed to serve as molecular scaffolds for preassembly of [FeS] clusters to be used in the biogenesis of iron-sulfur proteins. In vitro studies demonstrating transfer of preformed scaffold-[FeS] complexes to apoprotein acceptors have provided experimental support for this hypothesis, but investigations to date have yielded only single-cluster transfer events. We describe an in vitro assay system that allows for real-time monitoring of [FeS] cluster formation using circular dichroism spectroscopy and use this to investigate de novo [FeS] cluster formation and transfer from Escherichia coli IscU and IscA to apo-ferredoxin. Both IscU and IscA were found to be capable of multiple cycles of [2Fe2S] cluster formation and transfer suggesting that these scaffold proteins are capable of acting "catalytically." Kinetic studies further showed that cluster transfer exhibits Michaelis-Menten behavior indicative of complex formation of holo-IscU and holo-IscA with apoferredoxin and consistent with a direct [FeS] cluster transfer mechanism. Analysis of the dependence of the rate of cluster transfer, however, revealed enhanced efficiency at low ratios of scaffold to acceptor protein suggesting participation of a transient, labile scaffold-[FeS] species in the transfer process

Garden varieties: how attractive are recommended garden plants to butterflies?

One way the public can engage in insect conservation is through wildlife gardening, including the growing of insect-friendly flowers as sources of nectar. However, plant varieties differ in the types of insects they attract. To determine which garden plants attracted which butterflies, we counted butterflies nectaring on 11 varieties of summer-flowering garden plants in a rural garden in East Sussex, UK. These plants were all from a list of 100 varieties considered attractive to British butterflies, and included the five varieties specifically listed by the UK charity Butterfly Conservation as best for summer nectar. A total of 2659 flower visits from 14 butterfly and one moth species were observed. We performed a principal components analysis which showed contrasting patterns between the species attracted to Origanum vulgare and Buddleia davidii. The “butterfly bush” Buddleia attracted many nymphalines, such as the peacock, Inachis io, but very few satyrines such as the gatekeeper, Pyronia tithonus, which mostly visited Origanum. Eupatorium cannibinum had the highest Simpson’s Diversity score of 0.75, while Buddleia and Origanum were lower, scoring 0.66 and 0.50 respectively. No one plant was good at attracting all observed butterfly species, as each attracted only a subset of the butterfly community. We conclude that to create a butterfly-friendly garden, a variety of plant species are required as nectar sources for butterflies. Furthermore, garden plant recommendations can probably benefit from being more precise as to the species of butterfly they attract

Synthesis and characterisation of ruthenium complexes containing a pendent catechol ring

A series of [Ru(bipy)₂L]⁺ and [Ru(phen)₂L]⁺ complexes where L is 2-[5-(3,4-dimethoxyphenyl)-4H-1,2,4-triazol-3-yl]pyridine (HL1) and 4-(5-pyridin-2-yl-4H-1,2,4-triazol-3-yl)benzene-1,2-diol (HL2) are reported. The compounds obtained have been characterised using X-ray crystallography, NMR, UV/Vis and emission spectroscopies. Partial deuteriation is used to determine the nature of the emitting state and to simplify the NMR spectra. The acid-base properties of the compounds are also investigated. The electronic structures of [Ru(bipy)₂L1]⁺ and Ru(bipy)₂HL1]²⁺ are examined using ZINDO. Electro and spectroelectrochemical studies on [Ru(bipy)₂(L2)]⁺ suggest that proton transfer between the catechol and triazole moieties on L2 takes place upon oxidation of the L2 ligand

Exploring rumen microbe-derived fibre-degrading activities for improving feed digestibility

Ruminal fibre degradation is mediated by a complex community of rumen microbes, and its efficiency is crucial for optimal dairy productivity. Enzymes produced by rumen microbes are primarily responsible for degrading the complex structural polysaccharides that comprise fibre in the plant cell walls of feed materials. Because rumen microbes have evolved with their ruminant hosts over millions of years to perform this task, their enzymes are hypothesised to be optimally suited for activity at the temperature, pH range, and anaerobic environment of the rumen. However, fibre-rich diets are not fully digested, which represents a loss in potential animal productivity. Thus, there is opportunity to improve fibre utilisation through treating feeds with rumen microbe-derived fibrolytic enzymes and associated activities that enhance fibre degradation. This research aims to gain a better understanding of the key rumen microbes involved in fibre degradation and the mechanisms they employ to degrade fibre, by applying cultivation-based and culture-independent genomics approaches to rumen microbial communities of New Zealand dairy cattle. Using this knowledge, we aim to identify new opportunities for improving fibre degradation to enhance dairy productivity. Rumen content samples were taken over the course of a year from a Waikato dairy production herd. Over 1,000 rumen bacterial cultures were obtained from the plant-adherent fraction of the rumen contents. Among these cultures, two, 59 and 103 potentially new families, genera and species of rumen bacteria were identified, respectively. Many of the novel strains are being genome sequenced within the Hungate 1000 rumen microbial reference genome programme, which is providing deeper insights into the range of mechanisms used by the individual strains for fibre degradation. This information has been used to guide the selection of rumen bacterial strains with considerable potential as fibrolytic enzyme producers in vitro, with the intent of developing the strains so that their enzymes may be used as feed pre-treatments for use on farm. Culture-independent metagenomic approaches were also used to explore the activities involved in fibre degradation from the rumen microbial communities. Functional screening has revealed a range of novel enzymes and a novel fibre disrupting activity. Enrichment for the cell-secreted proteins from the community revealed evidence of a diverse range of cellulosomes, which are cell-surface associated multi-enzyme complexes that efficiently degrade plant cell wall polysaccharides. Biochemical and structural characterisation of these proteins has been conducted. In conclusion, cultivation and culture-independent genomic approaches have been applied to New Zealand bovine rumen microbial communities, and have provided considerable new insights into ruminal fibre degradation processes. Novel activities and bacterial species that display desirable activities on fibrous substrates in vitro are now being explored for their potential to improve ruminal fibre degradation, to allow the development of new technologies that will enhance dairy productivity

The major barriers to evidence-informed conservation policy and possible solutions

Conservation policy decisions can suffer from a lack of evidence, hindering effective decision-making. In nature conservation, studies investigating why policy is often not evidence-informed have tended to focus on Western democracies, with relatively small samples. To understand global variation and challenges better, we established a global survey aimed at identifying top barriers and solutions to the use of conservation science in policy. This obtained the views of 758 people in policy, practice, and research positions from 68 countries across six languages. Here we show that, contrary to popular belief, there is agreement between groups about how to incorporate conservation science into policy, and there is thus room for optimism. Barriers related to the low priority of conservation were considered to be important, while mainstreaming conservation was proposed as a key solution. Therefore, priorities should focus on convincing the public of the importance of conservation as an issue, which will then influence policy-makers to adopt pro-environmental long-term policies.NERC (1653183) Grantham Foundation for the Protection of the Environment Kenneth Miller Trust (unknown) NERC (1653183) NERC (NE/L002507/1) European Commission (308454

Solution Structure of the Iron−Sulfur Cluster Cochaperone HscB and Its Binding Surface for the Iron−Sulfur Assembly Scaffold Protein IscU†‡

ABSTRACT: The interaction between IscU and HscB is critical for successful assembly of iron-sulfur clusters. NMR experiments were performed on HscB to investigate which of its residues might be part of the IscU binding surface. Residual dipolar couplings ( 1 DHN and 1 DCRHR) indicated that the crystal structure of HscB [Cupp-Vickery, J. R., and Vickery, L. E. (2000) Crystal structure of Hsc20, a J-type cochaperone from Escherichia coli, J. Mol. Biol. 304, 835-845] faithfully represents its solution state. NMR relaxation rates ( 15 N R1, R2) and 1 H- 15 N heteronuclear NOE values indicated that HscB is rigid along its entire backbone except for three short regions which exhibit flexibility on a fast time scale. Changes in the NMR spectrum of HscB upon addition of IscU mapped to the J-domain/C-domain interface, the interdomain linker, and the C-domain. Sequence conservation is low in the interface and in the linker, and NMR changes observed for these residues likely result from indirect effects of IscU binding. NMR changes observed in the conserved patch of residues in the C-domain (L92, M93, L96, E97, E100, E104, and F153) were suggestive of a direct interaction with IscU. To test this, we replaced several of these residues with alanine and assayed for the ability of HscB to interact with IscU and to stimulate HscA ATPase activity. HscB(L92A,M93A,F153A) and HscB(E97A,E100A,E104A) both showed decreased binding affinity for IscU; the (L92A,M93A,F153A) substitution also strongly perturbed the allosteric interactio