95 research outputs found

    Application of the ramp test from a closed cavity rheometer to obtain the steady-state shear viscosity η ( γ̇ )

    Get PDF
    The steady-state shear viscosity η(˙γ) is required in controlling processing parameters for the extrusion processing of polymer melts. A new method, the so-called ramp test, is investigated in this study to obtain the steady-state shear viscosity with a closed cavity rheometer (CCR). To verify the method and the accuracy of the CCR data, three commercial polyolefin polymers, a low-density polyethylene (LDPE), a linear low-density polyethylene (LLDPE), and a polybutadiene (PBD), were used as model systems. Measurements of the magnitude of the complex viscosity η(ω)∣η^⁎(ω)∣ were compared with the steady-state shear viscosity data obtained by capillary rheometer and CCR. Further, time–temperature superposition master curves of the magnitude of the complex viscosity and steady-state shear viscosity obtained by CCR were developed for LLDPE and PBD. The influence of the cavity sealing on the instrument’s accuracy to obtain the steady-state shear viscosity was investigated using the finite element method simulations. Thus, it was shown that the ramp test performed by CCR is a practical method to determine reliable and reproducible data of the steady-state shear viscosity within a wide range of temperatures (T = 50–180°C) for low and high viscous materials ( η(ω)∣η^⁎(ω)∣ = 1.6–480 kPa s, M w = 144–375 kg mol1^{−1})

    Platinum Group Metal-Doped Tungsten Phosphates for Selective C-H Activation of Lower Alkanes

    Get PDF
    Platinum group metal (PGM)-based catalysts are known to be highly active in the total combustion of lower hydrocarbons. However, through an alternative catalyst design reported in this paper by isolating PGM-based active sites in a tungsten phosphate matrix, we present a class of catalysts for selective oxidation of n-butane, propane, and propylene that do not contain Mo or V as redox-active elements. Two different catalyst concepts have been pursued. Concept A: isolating Ru-based active sites in a tungsten phosphate matrix coming upon as ReO3-type structure. Concept B: dilution of PGM-based active sites through the synthesis of X-ray amorphous Ru tungsten phosphates supported on SiO2. Using a high-throughput screening approach, model catalysts over a wide compositional range were evaluated for C3 and C4 partial oxidation. Bulk crystalline and supported XRD amorphous phases with similar Ru/W/P compositions showed comparable performance. Hence, for these materials, composition is more crucial than the degree of crystallinity. Further studies for optimization on second-generation supported systems revealed even better results. High selectivity for n-butane oxidation to maleic anhydride and propane oxidation to an acrolein/acrylic acid has been achieved

    Deciphering the Catalytic Machinery in 30S Ribosome Assembly GTPase YqeH

    Get PDF
    YqeH, a circularly permuted GTPase (cpGTPase), which is conserved across bacteria and eukaryotes including humans is important for the maturation of small (30S) ribosomal subunit in Bacillus subtilis. Recently, we have shown that it binds 30S in a GTP/GDP dependent fashion. However, the catalytic machinery employed to hydrolyze GTP is not recognized for any of the cpGTPases, including YqeH. This is because they possess a hydrophobic substitution in place of a catalytic glutamine (present in Ras-like GTPases). Such GTPases were categorized as HAS-GTPases and were proposed to follow a catalytic mechanism, different from the Ras-like proteins.MnmE, another HAS-GTPase, but not circularly permuted, utilizes a potassium ion and water mediated interactions to drive GTP hydrolysis. Though the G-domain of MnmE and YqeH share only approximately 25% sequence identity, the conservation of characteristic sequence motifs between them prompted us to probe GTP hydrolysis machinery in YqeH, by employing homology modeling in conjunction with biochemical experiments. Here, we show that YqeH too, uses a potassium ion to drive GTP hydrolysis and stabilize the transition state. However, unlike MnmE, it does not dimerize in the transition state, suggesting alternative ways to stabilize switches I and II. Furthermore, we identify a potential catalytic residue in Asp-57, whose recognition, in the absence of structural information, was non-trivial due to the circular permutation in YqeH. Interestingly, when compared with MnmE, helix alpha2 that presents Asp-57 is relocated towards the N-terminus in YqeH. An analysis of the YqeH homology model, suggests that despite such relocation, Asp-57 may facilitate water mediated catalysis, similarly as the catalytic Glu-282 of MnmE. Indeed, an abolished catalysis by D57I mutant supports this inference.An uncommon means to achieve GTP hydrolysis utilizing a K(+) ion has so far been demonstrated only for MnmE. Here, we show that YqeH also utilizes a similar mechanism. While the catalytic machinery is similar in both, mechanistic differences may arise based on the way they are deployed. It appears that K(+) driven mechanism emerges as an alternative theme to stabilize the transition state and hydrolyze GTP in a subset of GTPases, such as the HAS-GTPases

    Final Pre-40S Maturation Depends on the Functional Integrity of the 60S Subunit Ribosomal Protein L3

    Get PDF
    Ribosomal protein L3 is an evolutionarily conserved protein that participates in the assembly of early pre-60S particles. We report that the rpl3[W255C] allele, which affects the affinity and function of translation elongation factors, impairs cytoplasmic maturation of 20S pre-rRNA. This was not seen for other mutations in or depletion of L3 or other 60S ribosomal proteins. Surprisingly, pre-40S particles containing 20S pre-rRNA form translation-competent 80S ribosomes, and translation inhibition partially suppresses 20S pre-rRNA accumulation. The GTP-dependent translation initiation factor Fun12 (yeast eIF5B) shows similar in vivo binding to ribosomal particles from wild-type and rpl3[W255C] cells. However, the GTPase activity of eIF5B failed to stimulate processing of 20S pre-rRNA when assayed with ribosomal particles purified from rpl3[W255C] cells. We conclude that L3 plays an important role in the function of eIF5B in stimulating 3′ end processing of 18S rRNA in the context of 80S ribosomes that have not yet engaged in translation. These findings indicate that the correct conformation of the GTPase activation region is assessed in a quality control step during maturation of cytoplasmic pre-ribosomal particles

    Evolution of protein-coupled RNA dynamics during hierarchical assembly of ribosomal complexes

    Get PDF
    Assembly of 30S ribosomes involves the hierarchical addition of ribosomal proteins that progressively stabilize the folded 16S rRNA. Here, we use three-color single molecule FRET to show how combinations of ribosomal proteins uS4, uS17 and bS20 in the 16S 5' domain enable the recruitment of protein bS16, the next protein to join the complex. Analysis of real-time bS16 binding events shows that bS16 binds both native and non-native forms of the rRNA. The native rRNA conformation is increasingly favored after bS16 binds, explaining how bS16 drives later steps of 30S assembly. Chemical footprinting and molecular dynamics simulations show that each ribosomal protein switches the 16S conformation and dampens fluctuations at the interface between rRNA subdomains where bS16 binds. The results suggest that specific protein-induced changes in the rRNA dynamics underlie the hierarchy of 30S assembly and simplify the search for the native ribosome structure

    Structure of Metaphase Chromosomes: A Role for Effects of Macromolecular Crowding

    Get PDF
    In metaphase chromosomes, chromatin is compacted to a concentration of several hundred mg/ml by mechanisms which remain elusive. Effects mediated by the ionic environment are considered most frequently because mono- and di-valent cations cause polynucleosome chains to form compact ∼30-nm diameter fibres in vitro, but this conformation is not detected in chromosomes in situ. A further unconsidered factor is predicted to influence the compaction of chromosomes, namely the forces which arise from crowding by macromolecules in the surrounding cytoplasm whose measured concentration is 100–200 mg/ml. To mimic these conditions, chromosomes were released from mitotic CHO cells in solutions containing an inert volume-occupying macromolecule (8 kDa polyethylene glycol, 10.5 kDa dextran, or 70 kDa Ficoll) in 100 µM K-Hepes buffer, with contaminating cations at only low micromolar concentrations. Optical and electron microscopy showed that these chromosomes conserved their characteristic structure and compaction, and their volume varied inversely with the concentration of a crowding macromolecule. They showed a canonical nucleosomal structure and contained the characteristic proteins topoisomerase IIα and the condensin subunit SMC2. These observations, together with evidence that the cytoplasm is crowded in vivo, suggest that macromolecular crowding effects should be considered a significant and perhaps major factor in compacting chromosomes. This model may explain why ∼30-nm fibres characteristic of cation-mediated compaction are not seen in chromosomes in situ. Considering that crowding by cytoplasmic macromolecules maintains the compaction of bacterial chromosomes and has been proposed to form the liquid crystalline chromosomes of dinoflagellates, a crowded environment may be an essential characteristic of all genomes

    Proofreading of pre-40S ribosome maturation by a translation initiation factor and 60S subunits

    Get PDF
    In the final steps of yeast ribosome synthesis, immature translation-incompetent pre-40S particles that contain 20S pre-rRNA are converted to the mature translation-competent subunits containing the 18S rRNA. An assay for 20S pre-rRNA cleavage in purified pre-40S particles showed that cleavage by the PIN domain endonuclease Nob1 was strongly stimulated by the GTPase activity of the cytoplasmic translation initiation factor eIF5b/Fun12. Cleavage of the 20S pre-rRNA was also inhibited in vivo and in vitro by blocking binding of Fun12 to the 25S rRNA through specific methylation of its binding site. Cleavage competent pre-40S particles stably associate with Fun12 and form 80S complexes with 60S ribosomal subunits. We propose that recruitment of 60S subunits promotes GTP-hydrolysis by Fun12, leading to structural rearrangements within the pre-40S particle that bring Nob1 and the pre-rRNA cleavage site together

    Structure of the pre-60S ribosomal subunit with nuclear export factor Arx1 bound at the exit tunnel

    Get PDF
    Pre-ribosomal particles evolve in the nucleus through transient interaction with biogenesis factors, before export to the cytoplasm. Here, we report the architecture of the late pre-60S particle purified from Saccharomyces cerevisiae through Arx1, a nuclear export factor with structural homology to methionine aminopeptidases, or its binding partner Alb1. Cryo-electron microscopy reconstruction of the Arx1-particle at 11.9 Å resolution reveals regions of extra densities on the pre-60S particle attributed to associated biogenesis factors, confirming the immature state of the nascent subunit. One of these densities could be unambiguously assigned to Arx1. Immuno-electron microscopy and UV cross-linking localize Arx1 close to the ribosomal exit tunnel in direct contact with ES27, a highly dynamic eukaryotic rRNA expansion segment. The binding of Arx1 at the exit tunnel may position this export factor to prevent premature recruitment of ribosome-associated factors active during translation

    Heterozygous Yeast Deletion Collection Screens Reveal Essential Targets of Hsp90

    Get PDF
    Hsp90 is an essential eukaryotic chaperone with a role in folding specific “client” proteins such as kinases and hormone receptors. Previously performed homozygous diploid yeast deletion collection screens uncovered broad requirements for Hsp90 in cellular transport and cell cycle progression. These screens also revealed that the requisite cellular functions of Hsp90 change with growth temperature. We present here for the first time the results of heterozygous deletion collection screens conducted at the hypothermic stress temperature of 15°C. Extensive bioinformatic analyses were performed on the resulting data in combination with data from homozygous and heterozygous screens previously conducted at normal (30°C) and hyperthermic stress (37°C) growth temperatures. Our resulting meta-analysis uncovered extensive connections between Hsp90 and (1) general transcription, (2) ribosome biogenesis and (3) GTP binding proteins. Predictions from bioinformatic analyses were tested experimentally, supporting a role for Hsp90 in ribosome stability. Importantly, the integrated analysis of the 15°C heterozygous deletion pool screen with previously conducted 30°C and 37°C screens allows for essential genetic targets of Hsp90 to emerge. Altogether, these novel contributions enable a more complete picture of essential Hsp90 functions
    corecore