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At least 1,657 records · Page 92
All sourcesNASA NTRSDOE OSTI
DOE OSTI2013

The central role of selenium in the biochemistry and ecology of the harmful pelagophyte, Aureococcus anophagefferens

The trace element selenium (Se) is required for the biosynthesis of selenocysteine (Sec), the 21st amino acid in the genetic code, but its role in the ecology of harmful algal blooms (HABs) is unknown. Here, we examined the role of Se in the biology and ecology of the harmful pelagophyte, Aureococcus anophagefferens, through cell culture, genomic analyses, and ecosystem studies. This organism has the largest and the most diverse selenoproteome identified to date that consists of at least 59 selenoproteins, including known eukaryotic selenoproteins, selenoproteins previously only detected in bacteria, and novel selenoproteins. The A. anophagefferens selenoproteome was dominated by the thioredoxin fold proteins and oxidoreductase functions were assigned to the majority of detected selenoproteins. Insertion of Sec in these proteins was supported by a unique Sec insertion sequence. Se was required for the growth of A. anophagefferens as cultures grew maximally at nanomolar Se concentrations. In a coastal ecosystem, dissolved Se concentrations were elevated before and after A. anophagefferens blooms, but were reduced by >95percent during the peak of blooms to 0.05 nM. Consistent with this pattern, enrichment of seawater with selenite before and after a bloom did not affect the growth of A. anophagefferens, but enrichment during the peak of the bloom significantly increased population growth rates. These findings demonstrate that Se inventories, which can be anthropogenically enriched, can support proliferation of HABs, such as A. anophagefferens through its synthesis of a large arsenal of Se-dependent oxidoreductases that fine-tune cellular redox homeostasis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2011

Crystal Structure of the FimD Usher Bound to its Cognate FimC-FimH Substrate

Type 1 pili are the archetypal representative of a widespread class of adhesive multisubunit fibres in Gram-negative bacteria. During pilus assembly, subunits dock as chaperone-bound complexes to an usher, which catalyses their polymerization and mediates pilus translocation across the outer membrane. Here we report the crystal structure of the full-length FimD usher bound to the FimC-FimH chaperone-adhesin complex and that of the unbound form of the FimD translocation domain. The FimD-FimC-FimH structure shows FimH inserted inside the FimD 24-stranded {beta}-barrel translocation channel. FimC-FimH is held in place through interactions with the two carboxy-terminal periplasmic domains of FimD, a binding mode confirmed in solution by electron paramagnetic resonance spectroscopy. To accommodate FimH, the usher plug domain is displaced from the barrel lumen to the periplasm, concomitant with a marked conformational change in the {beta}-barrel. The amino-terminal domain of FimD is observed in an ideal position to catalyse incorporation of a newly recruited chaperone-subunit complex. The FimD-FimC-FimH structure provides unique insights into the pilus subunit incorporation cycle, and captures the first view of a protein transporter in the act of secreting its cognate substrate.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Crystal structure of a substrate-engaged SecY protein-translocation channel

Hydrophobic signal sequences target secretory polypeptides to a protein-conducting channel formed by a heterotrimeric membrane protein complex, the prokaryotic SecY or eukaryotic Sec61 complex. How signal sequences are recognized is poorly understood, particularly because they are diverse in sequence and length. Structures of the inactive channel show that the largest subunit, SecY or Sec61α, consists of two halves that form an hourglass-shaped pore with a constriction in the middle of the membrane and a lateral gate that faces lipid. The cytoplasmic funnel is empty, while the extracellular funnel is filled with a plug domain. In bacteria, the SecY channel associates with the translating ribosome in co-translational translocation, and with the SecA ATPase in post-translational translocation. How a translocating polypeptide inserts into the channel is uncertain, as cryo-electron microscopy structures of the active channel have a relatively low resolution (~10 Å) or are of insufficient quality. Here we report a crystal structure of the active channel, assembled from SecY complex, the SecA ATPase, and a segment of a secretory protein fused into SecA. The translocating protein segment inserts into the channel as a loop, displacing the plug domain. The hydrophobic core of the signal sequence forms a helix that sits in a groove outside the lateral gate, while the following polypeptide segment intercalates into the gate. The carboxy (C)-terminal section of the polypeptide loop is located in the channel, surrounded by residues of the pore ring. Furthermore, during translocation, the hydrophobic segments of signal sequences, and probably bilayer-spanning domains of nascent membrane proteins, exit the lateral gate and dock at a specific site that faces the lipid phase.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Reversible S-nitrosylation in an engineered azurin

S-Nitrosothiols are known as reagents for NO storage and transportation and as regulators in many physiological processes. Although the S-nitrosylation catalysed by haem proteins is well known, no direct evidence of S-nitrosylation in copper proteins has been reported. Here, we report reversible insertion of NO into a copper–thiolate bond in an engineered copper centre in Pseudomonas aeruginosa azurin by rational design of the primary coordination sphere and tuning its reduction potential by deleting a hydrogen bond in the secondary coordination sphere. The results not only provide the first direct evidence of S-nitrosylation of Cu(II)-bound cysteine in metalloproteins, but also shed light on the reaction mechanism and structural features responsible for stabilizing the elusive Cu(I)–S(Cys)NO species. The fast, efficient and reversible S-nitrosylation reaction is used to demonstrate its ability to prevent NO inhibition of cytochrome bo3 oxidase activity by competing for NO binding with the native enzyme under physiologically relevant conditions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2016

Super-resolution microscopy reveals structural diversity in molecular exchange among peptide amphiphile nanofibres

The dynamic behaviour of supramolecular systems is an important dimension of their potential functions. Here, we report on the use of stochastic optical reconstruction microscopy to study the molecular exchange of peptide amphiphile nanofibres, supramolecular systems known to have important biomedical functions. Solutions of nanofibres labelled with different dyes (Cy3 and Cy5) were mixed, and the distribution of dyes inserting into initially single-colour nanofibres was quantified using correlative image analysis. Our observations are consistent with an exchange mechanism involving monomers or small clusters of molecules inserting randomly into a fibre. Different exchange rates are observed within the same fibre, suggesting that local cohesive structures exist on the basis of beta-sheet discontinuous domains. The results reported here show that peptide amphiphile supramolecular systems can be dynamic and that their intermolecular interactions affect exchange patterns. Lastly, this information can be used to generate useful aggregate morphologies for improved biomedical function.

36 MATERIALS SCIENCE↗
DOE OSTI2016

Mapping polaronic states and lithiation gradients in individual V 2 O 5 nanowires

The rapid insertion and extraction of Li ions from a cathode material is imperative for the functioning of a Li-ion battery. In many cathode materials such as LiCoO 2 , lithiation proceeds through solid-solution formation, whereas in other materials such as LiFePO 4 lithiation/delithiation is accompanied by a phase transition between Li-rich and Li-poor phases. We demonstrate using scanning transmission X-ray microscopy (STXM) that in individual nanowires of layered V 2 O 5 , lithiation gradients observed on Li-ion intercalation arise from electron localization and local structural polarization. Electrons localized on the V 2 O 5 framework couple to local structural distortions, giving rise to small polarons that serves as a bottleneck for further Li-ion insertion. The stabilization of this polaron impedes equilibration of charge density across the nanowire and gives rise to distinctive domains. The enhancement in charge/discharge rates for this material on nanostructuring can be attributed to circumventing challenges with charge transport from polaron formation.

36 MATERIALS SCIENCE↗
DOE OSTI2017

Structural insights into the mycobacteria transcription initiation complex from analysis of X-ray crystal structures

The mycobacteria RNA polymerase (RNAP) is a target for antimicrobials against tuberculosis, motivating structure/function studies. Here we report a 3.2 Å-resolution crystal structure of a Mycobacterium smegmatis (Msm) open promoter complex (RPo), along with structural analysis of the Msm RPo and a previously reported 2.76 Å-resolution crystal structure of an Msm transcription initiation complex with a promoter DNA fragment. We observe the interaction of the Msm RNAP α-subunit C-terminal domain (αCTD) with DNA, and we provide evidence that the a CTD may play a role in Mtb transcription regulation. Here, our results reveal the structure of an Actinobacteria-unique insert of the RNAP β' subunit. Finally, our analysis reveals the disposition of the N-terminal segment of Msm σ A , which may comprise an intrinsically disordered protein domain unique to mycobacteria. The clade-specific features of the mycobacteria RNAP provide clues to the profound instability of mycobacteria RPo compared with E. coli.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Crystal structure of Clostridium difficile toxin A

Clostridium difficile infection is the leading cause of hospital-acquired diarrhoea and pseudomembranous colitis. Disease is mediated by the actions of two toxins, TcdA and TcdB, which cause the diarrhoea, as well as inflammation and necrosis within the colon. The toxins are large (308 and 270 kDa, respectively), homologous (47% amino acid identity) glucosyltransferases that target small GTPases within the host. The multidomain toxins enter cells by receptor-mediated endocytosis and, upon exposure to the low pH of the endosome, insert into and deliver two enzymatic domains across the membrane. Eukaryotic inositol-hexakisphosphate (InsP6) binds an autoprocessing domain to activate a proteolysis event that releases the N-terminal glucosyltransferase domain into the cytosol. Here, we report the crystal structure of a 1,832-amino-acid fragment of TcdA (TcdA 1832 ), which reveals a requirement for zinc in the mechanism of toxin autoprocessing and an extended delivery domain that serves as a scaffold for the hydrophobic α-helices involved in pH-dependent pore formation. A surface loop of the delivery domain whose sequence is strictly conserved among all large clostridial toxins is shown to be functionally important, and is highlighted for future efforts in the development of vaccines and novel therapeutics.

bacterial pathogenesis↗
DOE OSTI2015

Human DNA polymerase θ grasps the primer terminus to mediate DNA repair

DNA polymerase θ protects against genomic instability via an alternative end-joining repair pathway for DNA double-strand breaks. Polymerase θ is overexpressed in breast, lung and oral cancers, and reduction of its activity in mammalian cells increases sensitivity to double-strand break–inducing agents, including ionizing radiation. Reported in this paper are crystal structures of the C-terminal polymerase domain from human polymerase θ, illustrating two potential modes of dimerization. One structure depicts insertion of ddATP opposite an abasic-site analog during translesion DNA synthesis. The second structure describes a cognate ddGTP complex. Polymerase θ uses a specialized thumb subdomain to establish unique upstream contacts to the primer DNA strand, including an interaction with the 3'-terminal phosphate from one of five distinctive insertion loops. Finally, these observations demonstrate how polymerase θ grasps the primer to bypass DNA lesions or extend poorly annealed DNA termini to mediate end-joining.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Cooperative adsorption of carbon disulfide in diamine-appended metal–organic frameworks

Over one million tons of CS 2 are produced annually, and emissions of this volatile and toxic liquid, known to generate acid rain, remain poorly controlled. As such, materials capable of reversibly capturing this commodity chemical in an energy-efficient manner are of interest. Recently, we detailed diamine-appended metal–organic frameworks capable of selectively capturing CO 2 through a cooperative insertion mechanism that promotes efficient adsorption–desorption cycling. We therefore sought to explore the ability of these materials to capture CS 2 through a similar mechanism. Employing crystallography, spectroscopy, and gas adsorption analysis, we demonstrate that CS 2 is indeed cooperatively adsorbed in N,N-dimethylethylenediamine-appended M 2 (dobpdc) (M = Mg, Mn, Zn; dobpdc 4- = 4,4'-dioxidobiphenyl-3,3'-dicarboxylate), via the formation of electrostatically paired ammonium dithiocarbamate chains. In the weakly thiophilic Mg congener, chemisorption is cleanly reversible with mild thermal input. Finally, this work demonstrates that the cooperative insertion mechanism can be generalized to other high-impact target molecules.

36 MATERIALS SCIENCE↗
DOE OSTI2021

High-fidelity DNA ligation enforces accurate Okazaki fragment maturation during DNA replication

DNA ligase 1 (LIG1, Cdc9 in yeast) finalizes eukaryotic nuclear DNA replication by sealing Okazaki fragments using DNA end-joining reactions that strongly discriminate against incorrectly paired DNA substrates. Whether intrinsic ligation fidelity contributes to the accuracy of replication of the nuclear genome is unknown. Here, we show that an engineered low-fidelity LIG1 Cdc9 variant confers a novel mutator phenotype in yeast typified by the accumulation of single base insertion mutations in homonucleotide runs. The rate at which these additions are generated increases upon concomitant inactivation of DNA mismatch repair, or by inactivation of the Fen1 Rad27 Okazaki fragment maturation (OFM) nuclease. Biochemical and structural data establish that LIG1 Cdc9 normally avoids erroneous ligation of DNA polymerase slippage products, and this protection is compromised by mutation of a LIG1 Cdc9 high-fidelity metal binding site. Collectively, our data indicate that high-fidelity DNA ligation is required to prevent insertion mutations, and that this may be particularly critical following strand displacement synthesis during the completion of OFM.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Tuning local chemistry of P2 layered-oxide cathode for high energy and long cycles of sodium-ion battery

Layered transition-metal oxides have attracted intensive interest for cathode materials of sodium-ion batteries. However, they are hindered by the limited capacity and inferior phase transition due to the gliding of transition-metal layers upon Na + extraction and insertion in the cathode materials. Here, we report that the large-sized K + is riveted in the prismatic Na + sites of P2-Na 0.612 K 0.056 MnO 2 to enable more thermodynamically favorable Na + vacancies. The Mn-O bonds are reinforced to reduce phase transition during charge and discharge. 0.901 Na + per formula are reversibly extracted and inserted, in which only the two-phase transition of P2 ↔ P’2 occurs at low voltages. It exhibits the highest specific capacity of 240.5 mAh g -1 and energy density of 654 Wh kg -1 based on the redox of Mn 3+ /Mn 4+ , and a capacity retention of 98.2% after 100 cycles. This investigation will shed lights on the tuneable chemical environments of transition-metal oxides for advanced cathode materials and promote the development of sodium-ion batteries.

25 ENERGY STORAGE↗
DOE OSTI2021

Structural basis of LAIR1 targeting by polymorphic Plasmodium RIFINs

RIFIN, a large family of Plasmodium variant surface antigens, plays a crucial role in malaria pathogenesis by mediating immune suppression through activation of inhibitory receptors such as LAIR1, and antibodies with LAIR1 inserts have been identified that bind infected erythrocytes through RIFIN. However, details of RIFIN-mediated LAIR1 recognition and receptor activation have been unclear. Here, we use negative-stain EM to define the architecture of LAIR1-inserted antibodies and determine crystal structures of RIFIN-variable 2 (V2) domain in complex with a LAIR1 domain. These structures reveal the LAIR1-binding region of RIFIN to be hydrophobic and membrane-distal, to exhibit extensive structural diversity, and to interact with RIFIN-V2 in a one-to-one fashion. Through structural and sequence analysis of various LAIR1 constructs, we identify essential elements of RIFIN-binding on LAIR1. Furthermore, a structure-derived LAIR1-binding sequence signature ascertained >20 LAIR1-binding RIFINs, including some from P. falciparum field strains and Plasmodium species infecting gorillas and chimpanzees.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structural basis of DNA synthesis opposite 8-oxoguanine by human PrimPol primase-polymerase

PrimPol is a human DNA polymerase-primase that localizes to mitochondria and nucleus and bypasses the major oxidative lesion 7,8-dihydro-8-oxoguanine (oxoG) via translesion synthesis, in mostly error-free manner. We present structures of PrimPol insertion complexes with a DNA template-primer and correct dCTP or erroneous dATP opposite the lesion, as well as extension complexes with C or A as a 3'–terminal primer base. We show that during the insertion of C and extension from it, the active site is unperturbed, reflecting the readiness of PrimPol to accommodate oxoG(anti). The misinsertion of A opposite oxoG(syn) also does not alter the active site, and is likely less favorable due to lower thermodynamic stability of the oxoG(syn)•A base-pair. During the extension step, oxoG(syn) induces an opening of its base-pair with A or misalignment of the 3'-A primer terminus. Together, the structures show how PrimPol accurately synthesizes DNA opposite oxidatively damaged DNA in human cells.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Au-activated N motifs in non-coherent cupric porphyrin metal organic frameworks for promoting and stabilizing ethylene production

Direct implementation of metal-organic frameworks as the catalyst for CO 2 electroreduction has been challenging due to issues such as poor conductivity, stability, and limited > 2e - products. In this study, Au nanoneedles are impregnated into a cupric porphyrin-based metal-organic framework by exploiting ligand carboxylates as the Au 3+ -reducing agent, simultaneously cleaving the ligand-node linkage. Surprisingly, despite the lack of a coherent structure, the Au-inserted framework affords a superb ethylene selectivity up to 52.5% in Faradaic efficiency, ranking among the best for metal-organic frameworks reported in the literature. Through operando X-ray, infrared spectroscopies and density functional theory calculations, the enhanced ethylene selectivity is attributed to Au-activated nitrogen motifs in coordination with the Cu centers for C-C coupling at the metalloporphyrin sites. Furthermore, the Au-inserted catalyst demonstrates both improved structural and catalytic stability, ascribed to the altered charge conduction path that bypasses the incoherent framework. This study underlines the modulation of reticular metalloporphyrin structure by metal impregnation for steering the CO 2 reduction reaction pathway.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Biosensor Guided Polyketide Synthases Engineering for Optimization of Domain Exchange Boundaries

Type I modular polyketide synthases (PKSs) are multi-domain enzymes functioning like assembly lines. Many engineering attempts have been made for the last three decades to replace, delete and insert new functional domains into PKSs to produce novel molecules. However, inserting heterologous domains often destabilize PKSs, causing loss of activity and protein misfolding. To address this challenge, here we develop a fluorescence-based solubility biosensor that can quickly identify engineered PKSs variants with minimal structural disruptions. Using this biosensor, we screen a library of acyltransferase (AT)-exchanged PKS hybrids with randomly assigned domain boundaries, and we identify variants that maintain wild type production levels. We then probe each position in the AT linker region to determine how domain boundaries influence structural integrity and identify a set of optimized domain boundaries. Overall, we have successfully developed an experimentally validated, high-throughput method for making hybrid PKSs that produce novel molecules.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Correlative operando microscopy of oxygen evolution electrocatalysts

Transition metal (oxy)hydroxides are promising electrocatalysts for the oxygen evolution reaction.1–3 The properties of these materials evolve dynamically and heterogeneously4 with applied voltage through ion insertion redox reactions, converting inactive materials at open-circuit into active electrocatalysts during operation.5 Thus, the catalytic state is inherently far-from-equilibrium, complicating its direct observation. Here, we establish the link between the oxygen evolution activity and the local operational chemical, physical, and electronic nanoscale structure of single-crystalline β-Co(OH)2 platelet particles through a suite of newly developed correlative operando scanning probe and X-ray microscopy techniques. At pre-catalytic voltages, the particles swell to form an α-CoO2H1.5·0.5H2O-like structure with associated Co2.5+ oxidation state produced through hydroxide intercalation. With increasing voltage to drive oxygen evolution, interlayer water and protons de-intercalate to form contracted β-CoOOH particles with Co3+ species. While these transformations manifest heterogeneously through the bulk of the particles, the electrochemical current is primarily restricted to the particle edge facets. The observed Tafel behavior is correlated to the local concentration of Co3+ at these reactive edge sites, demonstrating the link between bulk ion-insertion and surface catalytic activity.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗