Copper-containing ceramic precursor synthesis: Solid-state transformations and materials technology

Three copper systems with relevance to materials technology are discussed. In the first, a CuS precursor, Cu4S1O (4-methylpyridine)(sub 4)- (4-MePy), was prepared by three routes: reaction of Cu2S, reaction of CuBr-SMe2, and oxidation of copper powder with excess sulfur in 4-methylpyridine by sulfur. In the second, copper powder was found to react with excess thiourea (H2NC(S)NH2) in 4-methylpyridine to produce thiocyanate (NCS(-)) complexes. Three isolated and characterized compounds are: Cu(NCS)(4-MePy)(sub 2), a polymer, (4-MePy-H)(Cu(NCS)(sub 3)(4-MePy)(sub 2)), a salt, and t-Cu(NCS)(sub 2)(4-MePy)(sub 4). Finally, an attempt to produce a mixed-metal sulfide precursor of Cu and Ga in N-methylimidazole (N-MeIm) resulted in the synthesis of a Cu-containing polymer, Cu(SO4)(N-MeIm). The structures are presented; the chemistry will be briefly discussed in the context of preparation and processing of copper-containing materials for aerospace applications

Expression of a Translationally Fused TAP-Tagged Plasma Membrane Proton Pump in <i>Arabidopsis thaliana</i>

The Arabidopsis thaliana plasma membrane proton ATPase genes, <i>AHA1</i> and <i>AHA2</i>, are the two most highly expressed isoforms of an 11 gene family and are collectively essential for embryo development. We report the translational fusion of a tandem affinity-purification tag to the 5′ end of the AHA1 open reading frame in a genomic clone. Stable expression of TAP-tagged AHA1 in Arabidopsis rescues the embryonic lethal phenotype of endogenous double <i>aha1/aha2</i> knockdowns. Western blots of SDS-PAGE and Blue Native gels show enrichment of AHA1 in plasma membrane fractions and indicate a hexameric quaternary structure. TAP-tagged AHA1 rescue lines exhibited reduced vertical root growth. Analysis of the plasma membrane and soluble proteomes identified several plasma membrane-localized proteins with alterred abundance in TAP-tagged AHA1 rescue lines compared to wild type. Using affinity-purification mass spectrometry, we uniquely identified two additional AHA isoforms, AHA9 and AHA11, which copurified with TAP-tagged AHA1. In conclusion, we have generated transgenic Arabidopsis lines in which a TAP-tagged AHA1 transgene has complemented all essential endogenous AHA1 and AHA2 functions and have shown that these plants can be used to purify AHA1 protein and to identify <i>in planta</i> interacting proteins by mass spectrometry

A RecA protein surface required for activation of DNA polymerase V.

DNA polymerase V (pol V) of Escherichia coli is a translesion DNA polymerase responsible for most of the mutagenesis observed during the SOS response. Pol V is activated by transfer of a RecA subunit from the 3'-proximal end of a RecA nucleoprotein filament to form a functional complex called DNA polymerase V Mutasome (pol V Mut). We identify a RecA surface, defined by residues 112-117, that either directly interacts with or is in very close proximity to amino acid residues on two distinct surfaces of the UmuC subunit of pol V. One of these surfaces is uniquely prominent in the active pol V Mut. Several conformational states are populated in the inactive and active complexes of RecA with pol V. The RecA D112R and RecA D112R N113R double mutant proteins exhibit successively reduced capacity for pol V activation. The double mutant RecA is specifically defective in the ATP binding step of the activation pathway. Unlike the classic non-mutable RecA S117F (recA1730), the RecA D112R N113R variant exhibits no defect in filament formation on DNA and promotes all other RecA activities efficiently. An important pol V activation surface of RecA protein is thus centered in a region encompassing amino acid residues 112, 113, and 117, a surface exposed at the 3'-proximal end of a RecA filament. The same RecA surface is not utilized in the RecA activation of the homologous and highly mutagenic RumA'2B polymerase encoded by the integrating-conjugative element (ICE) R391, indicating a lack of structural conservation between the two systems. The RecA D112R N113R protein represents a new separation of function mutant, proficient in all RecA functions except SOS mutagenesis

Effects of ATP and primer-template binding on RecA-UmuC cross-linking efficiency in pol V Mut.

<p>Western blot using an antibody to UmuC demonstrating increased UmuC-RecA cross-linking with increasing ATPγS concentration and decreasing UmuC-RecA cross-linking with increasing primer-template concentration in the presence of 500 μM ATPγS. pol V Mut was generated using RecA N113Bpa as described in Methods, with ATPγS and primer-template hairpin DNA added where indicated. ATPγS concentration ranges from 0.8 to 500 μM and primer-template DNA concentration ranges from 0.01–5 μM.</p

RumA′₂B can be activated by all RecA variants.

<p>Transactivation reaction was carried out on 3-nt overhang hairpin for UmuD′₂C (A) and RumA′₂B (B). RecA* (400 nM) was preformed on 30 nt ssDNA then mixed with UmuD′₂C or RumA′₂B (400 nM each) and incubated for 30 mins at 37°C. Unlike UmuD′₂C (A), RumA′₂B can be activated by RecA variants D112R, D112R N113R, and S117F (B).</p

RecA D112R and D112R N113R exhibit wild-type RecA binding affinities for pol V.

<p>(A) Location of investigated residues on the RecA protein surface. The D112 and N113 residues compose an acidic knob on the RecA surface. The RecA monomer represented in electrostatic coloring scheme (red = negative charged residues, blue = positive charged residues) was generated in Pymol (PDB 3CMU [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005066#pgen.1005066.ref067" target="_blank">67</a>]). In this illustration, the monomer shown is located at the 3' end of the ssDNA (the 3'-proximal RecA monomer), which is the monomer removed by pol V during the activation cycle. (B) Altering this acidic surface to basic residues does not affect the binding affinity for pol V. Equilibrium binding isotherms of wild-type RecA, RecA D112R, and RecA D112R titrated with pol V as monitored by fluorescence depolarization. All data are the average of at least three experiments. Error bars are one standard deviation from the mean.</p

Analysis of cross-linked RecA-UmuC product #1.

<p>The first of the two identified cross-linked products appeared in samples generated via both active and inactive pol V protocols, although it seemed to be somewhat more prominent in the inactive samples. The UmuC peptide involved in the cross-linking encompasses residues 361–376. MS/MS spectra for each of the three peaks seen in the extracted ion chromatogram (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005066#pgen.1005066.g012" target="_blank">Fig. 12</a>) for the RecA-UmuC cross-linked product are shown at the top. The predicted crosslinking locations for peaks A,B, and C are shown at the bottom. The lettering corresponds to the unique elution profile for the same precursor ion.</p

The RecA 3'-surface interacts with UmuC in pol V Mut.

<p>The photo-reactive unnatural amino acid Bpa was incorporated into RecA at position N113 (RecA N113Bpa) and used to probe RecA/pol V Mut interactions. The protocol for the generation of pol V Mut was followed as described in Methods, with ATP, ATPγS, and template-primer added where indicated. Samples were exposed to UV light to covalently crosslink the RecA N113Bpa to nearby interacting partners (A) Coomassie stained SDS-PAGE of samples. UmuC, RecA, and UmuD' bands are indicated. Higher molecular weight bands appear upon cross-linking via UV light. The major cross-linked species runs at ~100 kDa. (B) Western blot of reactions presented in (A) using antibodies to the RecA protein. (C) UmuC western blot of reactions presented in (A). UmuC is present in the major cross-linked band at ~100 kDa. (D) UmuD' western blot of reactions presented in (A). A non-specific band is present at ~49 kDa which is not UV crosslinking dependent. A weak band observed at ~60 kDa was UV-dependent, but was not observed via SDS-PAGE. Therefore, it could not be pursued via mass spectrometry analysis.</p