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384 records · Page 22

The HypA and HypB metallochaperones from Methanococcus maripaludis have unique metal-binding properties and a distinct nickel transfer mechanism

[NiFe] hydrogenases are widespread microbial metalloenzymes that catalyze the reversible conversion of hydrogen (H2) to protons and electrons, playing key roles in energy metabolism. The biosynthesis of the NiFe(CN) 2 CO cofactor involves a suite of maturation proteins, including the HypA and HypB nickel metallochaperones. Here, we define the metal-binding properties, nucleotide-dependent behavior, and functional interplay of HypA and HypB from the hydrogenotrophic methanogenic archaeon, Methanococcus maripaludis . Methanogens have multiple essential nickel-dependent enzymes, so they require efficient systems for nickel delivery that remain largely unexplored. Purified M. maripaludis HypA binds zinc or mononuclear iron at the C-terminal metal binding site, the latter of which has not been reported in other HypA proteins and may serve a unique regulatory role in methanogens. The G-protein metallochaperone HypB binds nickel at the G-domain, which stimulates GTPase activity. Size exclusion chromatography experiments reveal that HypA and HypB form complexes in the presence of nickel, and zinc-bound HypA is optimized for nickel transfer from HypB. The identity of the nucleotide bound to HypB (GDP or GTP) alters the oligomeric state of HypA-HypB complexes, supporting a GTPase-mediated nickel delivery pathway. The HypA-HypB 2 complex configuration is enriched and stable in the presence of GDP and nickel, indicating that this complex delivers nickel to the hydrogenase as opposed to HypA alone. Interestingly, affinity purification-mass spectrometry revealed that HypB interacts with several nickel-dependent proteins, suggesting that HypB may play a broader role in nickel homeostasis in M. maripaludis . Together, this work establishes a biochemical framework for HypAB-mediated nickel trafficking in methanogens.

[NiFe] hydrogenase↗

Purification and immunolocalization of an annexin-like protein in pea seedlings

As part of a study to identify potential targets of calcium action in plant cells, a 35-kDa, annexin-like protein was purified from pea (Pisum sativum L.) plumules by a method used to purify animal annexins. This protein, called p35, binds to a phosphatidylserine affinity column in a calcium-dependent manner and binds 45Ca2+ in a dot-blot assay. Preliminary sequence data confirm a relationship for p35 with the annexin family of proteins. Polyclonal antibodies have been raised which recognize p35 in Western and dot blots. Immunofluorescence and immunogold techniques were used to study the distribution and subcellular localization of p35 in pea plumules and roots. The highest levels of immunostain were found in young developing vascular cells producing wall thickenings and in peripheral root-cap cells releasing slime. This localization in cells which are actively involved in secretion is of interest because one function suggested for the animal annexins is involvement in the mediation of exocytosis.

NASA Discipline Number 40-50↗

Fate of arsenic during the interactions between Mn-substituted goethite and dissolved Fe(II)

Geogenic arsenic has become a globally-distributed groundwater contaminant, liberated from the weathering of arsenic-bearing sulfide minerals and often transported to aquifer sediments adsorbed to iron oxides. Among the iron oxides, goethite (α-FeOOH) is uniquely important for the fate of arsenic because of its widespread abundance, stability, and high affinity for binding arsenic. Goethite is ubiquitous in soils and sediments and often contains substituted elements, including manganese. Structural manganese may affect the surface reactivity and redox capacity of goethite and alter the mechanisms of recrystallization catalyzed by dissolved Fe(II), potentially affecting arsenic adsorption. Here, this study examined the fate of As(V) during the interactions between dissolved Fe(II) and Mn-substituted goethites at pH 4 and 7 as well as associated changes in arsenic speciation. At pH 7, the addition of dissolved Fe(II) initially increases the adsorption of As(V) onto Mn-bearing and Mn-free goethites. For the Mn-substituted goethites, the adsorbed As(V) slowly releases to solution at longer aging times. Fe(II) addition at pH 4 slightly increases As(V) uptake by Mn-substituted goethites, with differences in total sorption correlating with the Mn content in goethite. The addition of Fe(II) releases substantial dissolved manganese but the amount solubilized is higher at pH 4 compared to 7, suggesting that the presence of adsorbed As(V) may substantially promote the Mn release at pH 4. X-ray absorption fine structure spectroscopy shows that arsenic is stabilized as As(V) in all the samples and adsorbed on goethite via a bidentate binuclear mechanism. Fitting results show that the binding distance and coordination numbers are stable in Mn-free goethite and Mn-substituted goethite samples; the effect of substituted Mn on the surface complex structure is minor. High resolution transmission electron microscopy and X-ray diffraction confirm that no secondary ferrous arsenate minerals precipitate under both pH conditions. This study improves our understanding of the Fe(II)-As(V) interactions on iron oxides, and demonstrates that the substituted cations such as manganese may quantitatively alter the geochemical fate of arsenic during the reaction of dissolved Fe(II) with Fe(III) oxides.

58 GEOSCIENCES↗

A natural mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody

Epitopes that are conserved among SARS-like coronaviruses are attractive targets for design of cross-reactive vaccines and therapeutics. CR3022 is a SARS-CoV neutralizing antibody to a highly conserved epitope on the receptor binding domain (RBD) on the spike protein that is able to cross-react with SARS-CoV-2, but with lower affinity. Using x-ray crystallography, mutagenesis, and binding experiments, we illustrate that of four amino acid differences in the CR3022 epitope between SARS-CoV-2 and SARS-CoV, a single mutation P384A fully determines the affinity difference. CR3022 does not neutralize SARS-CoV-2, but the increased affinity to SARS-CoV-2 P384A mutant now enables neutralization with a similar potency to SARS-CoV. We further investigated CR3022 interaction with the SARS-CoV spike protein by negative-stain EM and cryo-EM. Three CR3022 Fabs bind per trimer with the RBD observed in different up-conformations due to considerable flexibility of the RBD. In one of these conformations, quaternary interactions are made by CR3022 to the N-terminal domain (NTD) of an adjacent subunit. Overall, this study provides insights into antigenic variation and potential cross-neutralizing epitopes on SARS-like viruses.

59 BASIC BIOLOGICAL SCIENCES↗

Discrete Open-Shell Tris(bipyridinium radical cationic) Inclusion Complexes in the Solid State

In the solid state the properties of organic radicals depend heavily on radical-radical interactions that are influenced by the superstructure of the crystalline phase. Here, we report the synthesis and characterization of a substituted tetracationic cyclophane, cyclobis(paraquat-p-1,4- dimethoxyphenylene), and outline how its bisradical dicationic redox state associates with the methyl viologen radical cation (MV •+ ) to give a trisradical tricationic inclusion complex. The (super)structures of the reduced cyclophane and this 1:1 complex in the solid-state deviate from the analogous (super)structures observed for the reduced state of cyclobis(paraquat-p-phenylene) and that of its trisradical tricationic complex. Titration experiments with a range of guests reveal that the methoxy substituents on the para-xylenyl do not influence significantly of the binding affinity of the cyclophane for small neutral guests, whereas the affinity decreases by an order of magnitude for larger radical cationic guests such as MV •+ . X-Ray diffraction reveals that the solid- state structures of the tricationic trisradical complexes to be a discrete entity with weak intermolecular orbital overlap between the neighboring complexes. Transient nutation EPR experiment and DFT calculations confirm that the complex has a doublet spin configuration in the ground state as a result of the strong orbital overlap within the complex, while the quartet-state spin configuration is higher in energy and so is inaccessible at ambient temperature. The superconducting quantum interference device (SQUID) measurement reveals that trisradical tricationic complexes interact antiferromagnetically (J/k = - 2.33 K) and form a 1D Heisenberg antiferromagnetic chain along the a-axis in the crystal. Furthermore, these results offer new insights into the design and synthesis of organic magnetic materials based on host-guest inclusion complexes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

A New Type of Foreign Clast in A Polymict Ureilite: A CAI or AL-Rich Chondrule

Introduction: Polymict ureilites are breccias interpreted to represent regolith formed on a ureilitic asteroid [1-3]. They consist of approximately 90-95% clasts of various ureilite types (olivine-pyroxene rocks with Fo 75-95), a few % indigenous feldspathic clasts, and a few % foreign clasts [4-20]. The foreign clasts are diverse, including fragments of H, L, LL and R chondrites, angrites, other achondrites, and dark clasts similar to CC [6,7,9-19]. We report a new type of foreign clast in polymict ureilite DaG 999. Methods: Clast 8 in Dar al Gani (DaG) 999/1 (Museum fur Naturkunde) was discovered during a survey of feldspathic clasts in polymict ureilites [19,20]. It was studied by BEI, EMPA, and X-ray mapping on the JEOL 8530F electron microprobe at ARES, JSC. Petrography and Mineral Compositions: Clast 8 is sub-rounded to irregular in shape, approximately 85 micrometers in diameter, and consists of approximately 68% pyroxene and 32% mesostasis (by area). Part of the pyroxene (top half of clast in Fig. 1a and 2) shows a coarse dendritic morphology; the rest appears massive. Mesostasis may be glassy and contains fine needles/grains of pyroxene. The pyroxene has very high CaO (23.5 wt.%) and Al2O3 (19.7 wt.%), with the formula: (Ca(0.91)Mg(0.63)Fe(0.01)Al(sup VI) (0.38)Cr(0.01)Ti(0.05)1.99 Si2O6. The bulk mesostasis also has very high Al2O3 (approximately 26 wt.%). A bulk composition for the clast was obtained by combining modal abundances with phase compositions (Table 1, Fig. 3). Discussion: The pyroxene in clast 8 has a Ca-Al-(Ti)- rich (fassaitic) composition that is clearly distinct from compositions of pyroxenes in main group ureilites [22] or indigenous feldspathic clasts in polymict ureilites [4-8]. It also has significantly higher Al than fassaite in angrites (up to approximately 12 wt.% [23]), which occur as xenoliths in polymict ureilites. Ca-Al-Ti rich pyroxenes are most commonly found in CAIs, Al-rich chondrules and other types of refractory inclusions in chondrites [21,24-31]. However, the clast 8 pyroxene matches only the most Al-Ca-rich of these, e.g., pyroxenes in type B CAIs in CV3 chondrites [25,30,31], a pyroxene-hibonite spherule and a pyroxene-anorthitespinel fragment from unique CC Acfer 094 [29], and one Al-rich chondrule from Chainpur (LL3.4) [21]. The mineralogy of clast 8 is not consistent with the mineral assemblages of any of these objects (since it lacks hibonite, spinel and/or anorthite), which suggests that it is unrepresentatively sectioned or is a fragment of a more mineralogically diverse object. Its bulk composition (Table 1; Fig. 3) is similar to bulk compositions of some Al-rich chondrules, as well as those of Type C CAIs (which plot in the sp+An+L field in Fig. 3), although it is enriched in silica relative to type C CAIs [e.g., 31]. This suggests a more likely affinity to Al-rich chondrules, although most Al-rich chondrules have less Al-Ca-rich pyroxene [21,26,27]. These bulk compositional comparisons may not be definitive, however, if the clast is unrepresentatively sampled. One of eleven Al-rich chondrules from UOCs described by [21] has textural and compositional characteristics that make it a possible progenitor type for clast 8. This chondrule (Chainpur 1251-14-2) is anorthiteporphyritic, with an interstitial dendritic intergrowth of pyroxene (similar in composition to that in clast 8) and plagioclase [21]. Clast 8 is conceivably a fragment from the interstitial area of such an object. The occurrence of glassy mesostasis (in clast 8) rather than plagioclase may not be a significant difference; it could result from a difference only in cooling rate. Al-rich chondrules with glassy mesostasis are rare, and known occurrences are Ca-poor [26], unlike clast 8. Polymict ureilites are known to contain xenoliths of various chondrites (including OC, R and CC) as well as individual ferromagnesian and silica-pyroxene chondrules probably derived from OC or RC [6,9,15,16,18]. This is the first report of an individual chondritic refractory inclusion as a xenolith in a polymict ureilite. An RC-like sample from anomalous polymict ureilite Almahata Sitta contains CAIs, but they are spinel-rich and not similar to clast 8 [13,14]. Further studies of this clast (which, unfortunately, may not be possible), or the discovery of additional (more representative?) materials of this type would be needed to determine the exact nature of this xenolith and the type of chondrite from which it is derived.

Goodrich, C. A.↗