X-ray structure of a soluble Rieske-type ferredoxin from Mus musculus

The X-ray crystal structure of a soluble Rieske ferredoxin from M. musculus was solved at 2.07 Å resolution, revealing an iron–sulfur cluster-binding domain with similar architecture to the Rieske-type domains of bacterial aromatic dioxygenases. The ferredoxin was also shown to be capable of accepting electrons from both eukaryotic and prokaryotic oxidoreductases

The Lantern Vol. 63, No. 2, Spring 1996

• Poet, Lead Me On • St. Patrick\u27s Day • The Last Three Days • The Impressionable • Roundabout • The Bench • Carnivorous • Kyrie • Second Glance • Porch • Cruel Design • A Mime • Flaxen Crown • My Embryonic Ocean of Love • Stone Matrix • Voices from the Past • Skipping the Bullfight: Toreadors and Gaudi • Another Part of My Lacolonialism • Translucent Pane • Linguistics • Treehouse • A Disagreeable Music Piece • Vigil • A Brief History of American Poetry in Englishhttps://digitalcommons.ursinus.edu/lantern/1148/thumbnail.jp

Structural basis for potency differences between GDF8 and GDF11.

BACKGROUND: Growth/differentiation factor 8 (GDF8) and GDF11 are two highly similar members of the transforming growth factor β (TGFβ) family. While GDF8 has been recognized as a negative regulator of muscle growth and differentiation, there are conflicting studies on the function of GDF11 and whether GDF11 has beneficial effects on age-related dysfunction. To address whether GDF8 and GDF11 are functionally identical, we compared their signaling and structural properties. RESULTS: Here we show that, despite their high similarity, GDF11 is a more potent activator of SMAD2/3 and signals more effectively through the type I activin-like receptor kinase receptors ALK4/5/7 than GDF8. Resolution of the GDF11:FS288 complex, apo-GDF8, and apo-GDF11 crystal structures reveals unique properties of both ligands, specifically in the type I receptor binding site. Lastly, substitution of GDF11 residues into GDF8 confers enhanced activity to GDF8. CONCLUSIONS: These studies identify distinctive structural features of GDF11 that enhance its potency, relative to GDF8; however, the biological consequences of these differences remain to be determined

A substrate mimic allows high-throughput assay of the FabA protein and consequently the identification of a novel inhibitor of <i>Pseudomonas aeruginosa</i> FabA

The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 223461, Senior Investigator Award WT100209MA (JHN), Swedish Science Council (GS), Wellcome Trust Strategic grant 100476/Z/12/Z (DWG) and National Institutes of Health R01GM095970 (MB). JHN & ADS are Royal Society Wolfson Merit Award holders.Eukaryotes and prokaryotes possess fatty acid synthase (FAS) biosynthetic pathway(s) that comprise iterative chain elongation, reduction, and dehydration reactions. The bacterial FASII pathway differs significantly from human FAS pathways and is a long-standing target for antibiotic development against Gram-negative bacteria due to differences from the human FAS, and several existing antibacterial agents are known to inhibit FASII enzymes. N-acetylcysteamine (NAC) fatty acid thioesters have been used as mimics of the natural acyl carrier protein (ACP) pathway intermediates to assay FASII enzymes, and we now report an assay of FabV from Pseudomonas aeruginosa using (E)-2-decenoyl-NAC. In addition, we have converted an existing UV absorbance assay for FabA, the bifunctional dehydration/epimerization enzyme and key target in the FAS II pathway, into a high throughput enzyme coupled fluorescence assay that has been employed to screen a library of diverse small molecules. With this approach, N-(4-chlorobenzyl)-3-(2-furyl)-1H-1,2,4-triazol-5-amine (N42FTA) was found to competitively inhibit (pIC50 = 5.7 ± 0.2) the processing of 3-hydroxydecanoyl-NAC by P. aeruginosa FabA. N42FTA was shown to be potent in blocking crosslinking of E. coli ACP and FabA, a direct mimic of the biological process. The co-complex structure of N42FTA with P. aeruginosa FabA protein rationalizes affinity and suggests future design opportunities. Employing NAC fatty acid mimics to developing further high throughput assays for individual enzymes in the FASII pathway should aid in the discovery of new antimicrobials.Publisher PDFPeer reviewe

Small-scale, semi-automated purification of eukaryotic proteins for structure determination

A simple approach that allows cost-effective automated purification of recombinant proteins in levels sufficient for functional characterization or structural studies is described. Studies with four human stem cell proteins, an engineered version of green fluorescent protein, and other proteins are included. The method combines an expression vector (pVP62K) that provides in vivo cleavage of an initial fusion protein, a factorial designed auto-induction medium that improves the performance of small-scale production, and rapid, automated metal affinity purification of His8-tagged proteins. For initial small-scale production screening, single colony transformants were grown overnight in 0.4 ml of auto-induction medium, produced proteins were purified using the Promega Maxwell 16, and purification results were analyzed by Caliper LC90 capillary electrophoresis. The yield of purified [U-15N]-His8-Tcl-1 was 7.5 μg/ml of culture medium, of purified [U-15N]-His8-GFP was 68 μg/ml, and of purified selenomethione-labeled AIA–GFP (His8 removed by treatment with TEV protease) was 172 μg/ml. The yield information obtained from a successful automated purification from 0.4 ml was used to inform the decision to scale-up for a second meso-scale (10–50 ml) cell growth and automated purification. 1H–15N NMR HSQC spectra of His8-Tcl-1 and of His8-GFP prepared from 50 ml cultures showed excellent chemical shift dispersion, consistent with well folded states in solution suitable for structure determination. Moreover, AIA–GFP obtained by proteolytic removal of the His8 tag was subjected to crystallization screening, and yielded crystals under several conditions. Single crystals were subsequently produced and optimized by the hanging drop method. The structure was solved by molecular replacement at a resolution of 1.7 Å. This approach provides an efficient way to carry out several key target screening steps that are essential for successful operation of proteomics pipelines with eukaryotic proteins: examination of total expression, determination of proteolysis of fusion tags, quantification of the yield of purified protein, and suitability for structure determination

Structural basis for RNA recognition by NusB and NusE in the initiation of transcription antitermination

Processive transcription antitermination requires the assembly of the complete antitermination complex, which is initiated by the formation of the ternary NusB–NusE–BoxA RNA complex. We have elucidated the crystal structure of this complex, demonstrating that the BoxA RNA is composed of 8 nt that are recognized by the NusB–NusE heterodimer. Functional biologic and biophysical data support the structural observations and establish the relative significance of key protein–protein and protein–RNA interactions. Further crystallographic investigation of a NusB–NusE–dsRNA complex reveals a heretofore unobserved dsRNA binding site contiguous with the BoxA binding site. We propose that the observed dsRNA represents BoxB RNA, as both single-stranded BoxA and double-stranded BoxB components are present in the classical lambda antitermination site. Combining these data with known interactions amongst antitermination factors suggests a specific model for the assembly of the complete antitermination complex

African tropical rainforest net carbon dioxide fluxes in the twentieth century

The African humid tropical biome constitutes the second largest rainforest region, significantly impacts global carbon cycling and climate, and has undergone major changes in functioning owing to climate and land-use change over the past century. We assess changes and trends in CO2 fluxes from 1901 to 2010 using nine land surface models forced with common driving data, and depict the inter-model variability as the uncertainty in fluxes. The biome is estimated to be a natural (no disturbance) net carbon sink (−0.02 kg C m−2 yr−1 or −0.04 Pg C yr−1, p < 0.05) with increasing strength fourfold in the second half of the century. The models were in close agreement on net CO2 flux at the beginning of the century (σ1901 = 0.02 kg C m−2 yr−1), but diverged exponentially throughout the century (σ2010 = 0.03 kg C m−2 yr−1). The increasing uncertainty is due to differences in sensitivity to increasing atmospheric CO2, but not increasing water stress, despite a decrease in precipitation and increase in air temperature. However, the largest uncertainties were associated with the most extreme drought events of the century. These results highlight the need to constrain modelled CO2 fluxes with increasing atmospheric CO2 concentrations and extreme climatic events, as the uncertainties will only amplify in the next century

A Novel, “Double-Clamp” Binding Mode for Human Heme Oxygenase-1 Inhibition

The development of heme oxygenase (HO) inhibitors is critical in dissecting and understanding the HO system and for potential therapeutic applications. We have established a program to design and optimize HO inhibitors using structure-activity relationships in conjunction with X-ray crystallographic analyses. One of our previous complex crystal structures revealed a putative secondary hydrophobic binding pocket which could be exploited for a new design strategy by introducing a functional group that would fit into this potential site. To test this hypothesis and gain further insights into the structural basis of inhibitor binding, we have synthesized and characterized 1-(1H-imidazol-1-yl)-4,4-diphenyl-2-butanone (QC-308). Using a carbon monoxide (CO) formation assay on rat spleen microsomes, the compound was found to be ∼15 times more potent (IC50 = 0.27±0.07 µM) than its monophenyl analogue, which is already a potent compound in its own right (QC-65; IC50 = 4.0±1.8 µM). The crystal structure of hHO-1 with QC-308 revealed that the second phenyl group in the western region of the compound is indeed accommodated by a definitive secondary proximal hydrophobic pocket. Thus, the two phenyl moieties are each stabilized by distinct hydrophobic pockets. This “double-clamp” binding offers additional inhibitor stabilization and provides a new route for improvement of human heme oxygenase inhibitors

Structure-Based Design of Non-Natural Amino Acid Inhibitors of Amyloid Fibrillation

Many globular and natively disordered proteins can convert into amyloid fibers. These fibers are associated with numerous pathologies1 as well as with normal cellular functions2,3, and frequently form during protein denaturation4,5. Inhibitors of pathological amyloid fibers could serve as leads for therapeutics, provided the inhibitors were specific enough to avoid interfering with normal processes. Here we show that computer-aided, structure-based design can yield highly specific peptide inhibitors of amyloid formation. Using known atomic structures of segments of amyloid fibers as templates, we have designed and characterized an all D-amino acid inhibitor of fibrillation of the tau protein found in Alzheimer’s disease, and a non-natural L-amino acid inhibitor of an amyloid fiber that enhances sexual transmission of HIV. Our results indicate that peptides from structure-based designs can disrupt the fibrillation of full-length proteins, including those like tau that lack fully ordered native structures.We thank M.I. Ivanova, J. Corn, T. Kortemme, D. Anderson, M.R. Sawaya, M. Phillips, S. Sambashivan, J. Park, M. Landau, Q. Zhang, R. Clubb, F. Guo, T. Yeates, J. Nowick, J. Zheng, and M.J. Thompson for discussions, HHMI, NIH, NSF, the GATES foundation, and the Joint Center for Translational Medicine for support, R. Peterson for help with NMR experiments, E. Mandelkow for providing tau constructs, R. Riek for providing amyloid beta, J. Stroud for amyloid beta preparation. Support for JK was from the Damon Runyon Cancer Research Foundation, for HWC by the Ruth L. Kirschstein National Research Service Award, for JM from the programme for junior-professors by the ministry of science, Baden-Württemberg, and for SAS by a UCLA-IGERT bioinformatics traineeship