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1
LIBONATI, Massimo, and Giovanni GOTTE. "Oligomerization of bovine ribonuclease A: structural and functional features of its multimers." Biochemical Journal 380, no. 2 (June 1, 2004): 311–27. http://dx.doi.org/10.1042/bj20031922.
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Bovine pancreatic RNase A (ribonuclease A) aggregates to form various types of catalytically active oligomers during lyophilization from aqueous acetic acid solutions. Each oligomeric species is present in at least two conformational isomers. The structures of two dimers and one of the two trimers have been solved, while plausible models have been proposed for the structures of a second trimer and two tetrameric conformers. In this review, these structures, as well as the general conditions for RNase A oligomerization, based on the well known 3D (three-dimensional) domain-swapping mechanism, are described and discussed. Attention is also focused on some functional properties of the RNase A oligomers. Their enzymic activities, particularly their ability to degrade double-stranded RNAs and polyadenylate, are summarized and discussed. The same is true for the remarkable antitumour activity of the oligomers, displayed in vitro and in vivo, in contrast with monomeric RNase A, which lacks these activities. The RNase A multimers also show an aspermatogenic action, but lack any detectable embryotoxicity. The fact that both activity against double-stranded RNA and the antitumour action increase with the size of the oligomer suggests that these activities may share a common structural requirement, such as a high number or density of positive charges present on the RNase A oligomers.
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Wootton, Sarah K., and Dongwan Yoo. "Homo-Oligomerization of the Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein and the Role of Disulfide Linkages." Journal of Virology 77, no. 8 (April 15, 2003): 4546–57. http://dx.doi.org/10.1128/jvi.77.8.4546-4557.2003.
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ABSTRACT As a step toward understanding the assembly pathway of the porcine reproductive and respiratory syndrome virus (PRRSV), the oligomeric properties of the nucleocapsid (N) protein were investigated. In this study, we have demonstrated that under nonreducing conditions the N protein forms disulfide-linked homodimers. However, inclusion of an alkylating agent (N-ethylmaleimide [NEM]) prevented disulfide bond formation, suggesting that these intermolecular disulfide linkages were formed as a result of spurious oxidation during cell lysis. In contrast, N protein homodimers isolated from extracellular virions were shown to have formed NEM-resistant intermolecular disulfide linkages, the function of which is probably to impart stability to the virion. Pulse-chase analysis revealed that N protein homodimers become specifically disulfide linked within the virus-infected cell, albeit at the later stages of infection, conceivably when the virus particle buds into the oxidizing environment of the endoplasmic reticulum. Moreover, NEM-resistant disulfide linkages were shown to occur only during productive PRRSV infection, since expression of recombinant N protein did not result in the formation of NEM-resistant disulfide-linked homodimers. Mutational analysis indicated that of the three conserved cysteine residues in the N protein, only the cysteine at position 23 was involved in the formation of disulfide linkages. The N protein dimer was shown to be stable both in the presence and absence of intermolecular disulfide linkages, indicating that noncovalent interactions also play a role in dimerization. Non-disulfide-mediated N protein interactions were subsequently demonstrated both in vitro by the glutathione S-transferase (GST) pull-down assay and in vivo by the mammalian two-hybrid assay. Using a series of N protein deletion mutants fused to GST, amino acids 30 to 37 were shown to be essential for N-N interactions. Furthermore, since RNase A treatment markedly decreased N protein-binding affinity, it appears that at least in vitro, RNA may be involved in bridging N-N interactions. In cross-linking experiments, the N protein was shown to assemble into higher-order structures, including dimers, trimers, tetramers, and pentamers. Together, these findings demonstrate that the N protein possesses self-associative properties, and these likely provide the basis for PRRSV nucleocapsid assembly.
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Kanbara, K., K. Nagai, H. Nakashima, N. Yamamoto, R. J. Suhadolnik, and H. Takaku. "The Relationship between Conformation and Biological Activity of 8-substituted Analogues of 2′,5′-Oligoadenylates." Antiviral Chemistry and Chemotherapy 5, no. 1 (February 1994): 1–5. http://dx.doi.org/10.1177/095632029400500101.
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Analogues of the 2′,5′-linked adenylate trimer 5′-monophosphates, p5′A2′p5′A2′p5′A (pA3) (1a), containing 8-hydroxyadenosine and 8-mercaptoadenosine in the first, second, and third nucleotide positions were tested for their ability to bind to and activate RNase L of mouse L cells. The oligomer, p5′ASH2′p5′ASH2′p5′ASH (pASH3) (1c) had little capacity to bind to RNase L. On the other hand, an analogue of the p5′AOH2′p5′AOH2′p5′AOH (pAOH3) (1b) bound almost as well as the parent 2-5A [pppA(2′p5′A)2] (P3A3) (1d) to RNase L. The 8-substituted analogues of 2-5A were more resistant to degradation by (2′,5′) phosphodiesterase. Finally, the monophosphate, pASH3 (1c) which possessed higher anti-HIV activity than pAg (1a) or pAOH3 (1b).
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LIBONATI, Massimo, Mariarita BERTOLDI, and Salvatore SORRENTINO. "The activity on double-stranded RNA of aggregates of ribonuclease A higher than dimers increases as a function of the size of the aggregates." Biochemical Journal 318, no. 1 (August 15, 1996): 287–90. http://dx.doi.org/10.1042/bj3180287.
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Stable bovine RNase A aggregates larger than dimers (identified as trimers, tetramers, pentamers and hexamers) were obtained by lyophilization of RNase A from 40–50% acetic acid solutions. The RNase activity of these aggregates was compared with that of monomeric RNase A on single- and double-stranded polyribonucleotides. Their activity toward poly(U) and yeast RNA slightly decreases as a function of the size of the aggregates. In contrast, their action on poly(A).poly(U) as substrate progressively increases from a relative activity of 1 for the RNase monomer to 10 for the hexamer. These results are discussed in the light of an already advanced hypothesis about a possible mechanism of RNase attack on double-stranded RNA.
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Filipovic, Dragana, Marija Radojcic, and Bratoljub Milosavljevic. "Determination of the critical molar mass of ovalbumin oligomers degraded by ultrasound." Journal of the Serbian Chemical Society 65, no. 2 (2000): 123–30. http://dx.doi.org/10.2298/jsc0002123f.
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An experimental method has been developed which enables the determination of the critical molar mass (Mmc) of ovalbumin oligomers degraded by ultrasound of known frequency. To test the validity of the Mmc postulate, a series of ovalbumin oligomers was prepared by the radiolytic cross-linking of 1% solutions of ovalbumin monomer dissolved in 50mMNa/K-phosphate buffer pH 7.0 saturated withN2O. Under these conditions, irradiation with 5 kGy from a 60 Co source, yielded ovalbumin dimers, trimers, tetramers, and higher order oligomers. On the basis of the results obtained with the ovalbumin oligomers, it was concluded that for ultrasound of 23 kHz frequency and 5mm amplitude, the Mmc was 274000 - 14000 g/mol. Our results confirmed that the two postulates in the chemistry of polymer degradation by ultrasound are valid when ovalbumin oligomers are used as substrates, i.e., (1) that the higher the molar mass of the original macromolecule, the faster is its degradation rate, and (2) that a lower molar mass limit (LMmL) exists below which the macromolecules are resistent to further degradation.
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Markovic, Ingrid, Helena Pulyaeva, Alexander Sokoloff, and Leonid V. Chernomordik. "Membrane Fusion Mediated by Baculovirus gp64 Involves Assembly of Stable gp64 Trimers into Multiprotein Aggregates." Journal of Cell Biology 143, no. 5 (November 30, 1998): 1155–66. http://dx.doi.org/10.1083/jcb.143.5.1155.
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The baculovirus fusogenic activity depends on the low pH conformation of virally-encoded trimeric glycoprotein, gp64. We used two experimental approaches to investigate whether monomers, trimers, and/or higher order oligomers are functionally involved in gp64 fusion machine. First, dithiothreitol (DTT)- based reduction of intersubunit disulfides was found to reversibly inhibit fusion, as assayed by fluorescent probe redistribution between gp64-expressing and target cells (i.e., erythrocytes or Sf9 cells). This inhibition correlates with disappearance of gp64 trimers and appearance of dimers and monomers in SDS-PAGE. Thus, stable (i.e., with intact intersubunit disulfides) gp64 trimers, rather than independent monomers, drive fusion. Second, we established that merger of membranes is preceded by formation of large (greater than 2 MDa), short-lived gp64 complexes. These complexes were stabilized by cell–surface cross-linking and characterized by glycerol density gradient ultracentrifugation. The basic structural unit of the complexes is stable gp64 trimer. Although DTT-destabilized trimers were still capable of assuming the low pH conformation, they failed to form multimeric complexes. The fact that formation of these complexes correlated with fusion in timing, and was dependent on (a) low pH application, (b) stable gp64 trimers, and (c) cell–cell contacts, suggests that such multimeric complexes represent a fusion machine.
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Salveson, Patrick J., Ryan K. Spencer, and James S. Nowick. "X-ray Crystallographic Structure of Oligomers Formed by a Toxic β-Hairpin Derived from α-Synuclein: Trimers and Higher-Order Oligomers." Journal of the American Chemical Society 138, no. 13 (March 23, 2016): 4458–67. http://dx.doi.org/10.1021/jacs.5b13261.
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Jorba, Núria, Estela Area, and Juan Ortín. "Oligomerization of the influenza virus polymerase complex in vivo." Journal of General Virology 89, no. 2 (February 1, 2008): 520–24. http://dx.doi.org/10.1099/vir.0.83387-0.
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The influenza virus polymerase is a heterotrimer formed by the PB1, PB2 and PA subunits and is responsible for virus transcription and replication. We have expressed the virus polymerase complex by co-transfection of the subunit cDNAs, one of which was tandem affinity purification (TAP)-tagged, into human cells. The intracellular polymerase complexes were purified by the TAP approach, involving two affinity chromatography steps, IgG–Sepharose and calmodulin–agarose. Gel-filtration analysis indicated that, although most of the purified polymerase behaved as a heterotrimer, a significant proportion of the purified material migrated as polymerase dimers, trimers and higher oligomers. Co-purification of polymerase complexes alternatively tagged in the same subunit confirmed that the polymerase complex might form oligomers intracellularly. The implications of this observation for virus infection are discussed.
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Wojciechowska, Daria, Michał Taube, Karolina Rucińska, Joanna Maksim, and Maciej Kozak. "Oligomerization of Human Cystatin C—An Amyloidogenic Protein: An Analysis of Small Oligomeric Subspecies." International Journal of Molecular Sciences 23, no. 21 (November 3, 2022): 13441. http://dx.doi.org/10.3390/ijms232113441.
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Human cystatin C (HCC), an amyloidogenic protein, forms dimers and higher oligomers (trimers, tetramers and donut like large oligomers) via a domain-swapping mechanism. The aim of this study was the characterization of the HCC oligomeric states observed within the pH range from 2.2 to 10.0 and also in conditions promoting oligomerization. The HCC oligomeric forms obtained in different conditions were characterized using size exclusion chromatography, dynamic light scattering and small-angle X-ray scattering. The marked ability of HCC to form tetramers at low pH (2.3 or 3.0) and dimers at pH 4.0–5.0 was observed. HCC remains monomeric at pH levels above 6.0. Based on the SAXS data, the structure of the HCC tetramer was proposed. Changes in the environment (from acid to neutral) induced a breakdown of the HCC tetramers to dimers. The tetrameric forms of human cystatin C are formed by the association of the dimers without a domain-swapping mechanism. These observations were confirmed by their dissociation to dimers at pH 7.4.
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Ganderton, Tim, Jason W. H. Wong, Christina Schroeder, and Philip J. Hogg. "Lateral self-association of VWF involves the Cys2431-Cys2453 disulfide/dithiol in the C2 domain." Blood 118, no. 19 (November 10, 2011): 5312–18. http://dx.doi.org/10.1182/blood-2011-06-360297.
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Abstract VWF is a plasma protein that binds platelets to an injured vascular wall during thrombosis. When exposed to the shear forces found in flowing blood, VWF molecules undergo lateral self-association that results in a meshwork of VWF fibers. Fiber formation has been shown to involve thiol/disulfide exchange between VWF molecules. A C-terminal fragment of VWF was expressed in mammalian cells and examined for unpaired cysteine thiols using tandem mass spectrometry (MS). The VWF C2 domain Cys2431-Cys2453 disulfide bond was shown to be reduced in approximately 75% of the molecules. Fragments containing all 3 C domains or just the C2 domain formed monomers, dimers, and higher-order oligomers when expressed in mammalian cells. Mutagenesis studies showed that both the Cys2431-Cys2453 and nearby Cys2451-Cys2468 disulfide bonds were involved in oligomer formation. Our present findings imply that lateral VWF dimers form when a Cys2431 thiolate anion attacks the Cys2431 sulfur atom of the Cys2431-Cys2453 disulfide bond of another VWF molecule, whereas the Cys2451-Cys2468 disulfide/dithiol mediates formation of trimers and higher-order oligomers. These observations provide the basis for exploring defects in lateral VWF association in patients with unexplained hemorrhage or thrombosis.
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Bock, William, and Enrique Peacock-López. "Chiral Oscillations and Spontaneous Mirror Symmetry Breaking in a Simple Polymerization Model." Symmetry 12, no. 9 (August 20, 2020): 1388. http://dx.doi.org/10.3390/sym12091388.
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The origin of biological homochirality—defined as the preference of biological systems for only one enantiomer—has widespread implications in the study of chemical evolution and the origin of life. The activation—polymerization—epimerization—depolymerization (APED) model is a theoretical model originally proposed to describe chiral symmetry breaking in a simple dimerization system. It is known that the model produces chiral and chemical oscillations for certain system parameters, in particular, the preferential formation of heterochiral polymers. In order to investigate the effect of higher oligomers, our model adds trimers, tetramers, and pentamers. We report sustained oscillations of all chemical species and the enantiomeric excess for a wide range of parameter sets as well as the periodic chiral amplification of a small initial enantiomeric excess to a nearly homochiral state.
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Pangburn, M. K. "Analysis of the natural polymeric forms of human properdin and their functions in complement activation." Journal of Immunology 142, no. 1 (January 1, 1989): 202–7. http://dx.doi.org/10.4049/jimmunol.142.1.202.
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Abstract Many of the anomalies observed in studies or properdin may be explained on the basis of its ability to form a series of multi-subunit polymers and by differences in the functions of these forms of properdin. Dimers (P2), trimers (P3), tetramers (P4), and higher Mr polymers (Pn) of the 46,000-Da subunit were separated by gel filtration or by cation exchange chromatography of purified properdin. The specific activity of each form was measured in two assays. The native properdin activity of P4 was 10 times that of P2 (on a molar basis) with the order: P4 greater than P3 greater than P2 greater than Pn. During C activation P4 was found to be consumed first, P3 second, and P2 last, consistent with their measured specific activities. Assays for activated properdin showed that only Pn caused fluid phase C consumption when incubated in serum at normal concentrations. Pn accumulated during long term storage of purified P and freezing rapidly converted the smaller oligomers to Pn. The isolated oligomers were extremely stable, but did redistribute after denaturation-renaturation cycles by using low pH or guanidine. Renaturation after exposure of any species to denaturing conditions yielded mixtures of 20:54:26 (P4:P3:P2). This distribution was almost identical to that found in fresh normal human serum or plasma, suggesting that a distinct distribution of oligomers exists in blood that provides the C system with an apparently advantageous range of specific activities.
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Kristensen, Line G., James M. Holton, Behzad Rad, Yan Chen, Christopher J. Petzold, Sayan Gupta, and Corie Y. Ralston. "Hydroxyl radical mediated damage of proteins in low oxygen solution investigated using X-ray footprinting mass spectrometry." Journal of Synchrotron Radiation 28, no. 5 (July 20, 2021): 1333–42. http://dx.doi.org/10.1107/s1600577521004744.
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In the method of X-ray footprinting mass spectrometry (XFMS), proteins at micromolar concentration in solution are irradiated with a broadband X-ray source, and the resulting hydroxyl radical modifications are characterized using liquid chromatography mass spectrometry to determine sites of solvent accessibility. These data are used to infer structural changes in proteins upon interaction with other proteins, folding, or ligand binding. XFMS is typically performed under aerobic conditions; dissolved molecular oxygen in solution is necessary in many, if not all, the hydroxyl radical modifications that are generally reported. In this study we investigated the result of X-ray induced modifications to three different proteins under aerobic versus low oxygen conditions, and correlated the extent of damage with dose calculations. We observed a concentration-dependent protecting effect at higher protein concentration for a given X-ray dose. For the typical doses used in XFMS experiments there was minimal X-ray induced aggregation and fragmentation, but for higher doses we observed formation of covalent higher molecular weight oligomers, as well as fragmentation, which was affected by the amount of dissolved oxygen in solution. The higher molecular weight products in the form of dimers, trimers, and tetramers were present in all sample preparations, and, upon X-ray irradiation, these oligomers became non-reducible as seen in SDS-PAGE. The results provide an important contribution to the large body of X-ray radiation damage literature in structural biology research, and will specifically help inform the future planning of XFMS, and well as X-ray crystallography and small-angle X-ray scattering experiments.
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Crepin, Aurélie, Edel Cunill-Semanat, Eliška Kuthanová Trsková, Erica Belgio, and Radek Kaňa. "Antenna Protein Clustering In Vitro Unveiled by Fluorescence Correlation Spectroscopy." International Journal of Molecular Sciences 22, no. 6 (March 15, 2021): 2969. http://dx.doi.org/10.3390/ijms22062969.
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Antenna protein aggregation is one of the principal mechanisms considered effective in protecting phototrophs against high light damage. Commonly, it is induced, in vitro, by decreasing detergent concentration and pH of a solution of purified antennas; the resulting reduction in fluorescence emission is considered to be representative of non-photochemical quenching in vivo. However, little is known about the actual size and organization of antenna particles formed by this means, and hence the physiological relevance of this experimental approach is questionable. Here, a quasi-single molecule method, fluorescence correlation spectroscopy (FCS), was applied during in vitro quenching of LHCII trimers from higher plants for a parallel estimation of particle size, fluorescence, and antenna cluster homogeneity in a single measurement. FCS revealed that, below detergent critical micelle concentration, low pH promoted the formation of large protein oligomers of sizes up to micrometers, and therefore is apparently incompatible with thylakoid membranes. In contrast, LHCII clusters formed at high pH were smaller and homogenous, and yet still capable of efficient quenching. The results altogether set the physiological validity limits of in vitro quenching experiments. Our data also support the idea that the small, moderately quenching LHCII oligomers found at high pH could be relevant with respect to non-photochemical quenching in vivo.
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Grindley, T. Bruce, Rasiah Thangarasa, Pradip K. Bakshi, and T. Stanley Cameron. "The structure of 2,2-dibutyl-1,3,2-dioxastannane in the solid state and in solution." Canadian Journal of Chemistry 70, no. 1 (January 1, 1992): 197–204. http://dx.doi.org/10.1139/v92-031.
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Crystals of 2,2-dibutyl-1,3,2-dioxastannane (1) are orthorhombic, of space group Pnma, with a = 7.663(3), b = 18.437(2), c = 9.277(4) Å, Z = 4, R = 0.0568 (Rw = 0.0551) for 1183 independent reflections with I > 3σ(I). Compound 1 is a polymer in which each monomer unit is joined to the next by a four-membered (SnO)2 ring. The Sn—O bond lengths inside the monomer units average 2.04 Å while those between monomers average 2.57 Å. The mirror plane of the crystal contains the atoms in the four-membered rings and the other oxygen atoms. Two of the three remaining carbon atoms in the six-membered rings of the monomer units are close to the mirror plane. The other carbon atom is disordered above and below the plane. It was shown by 119Sn NMR spectroscopy that solutions of 1 contain mixtures of oligomers that consist mainly of dimers, trimers, and tetramers in chloroform-d. ΔG0 values for dimmer–trimer equilibria and dimmer–tetramer equilibria of −2.5 and −1.5 kcal mol−1 were obtained from integration of low temperature 119Sn NMR spectra. These values favour the higher oligomers slightly less than those for 2,2-dibutyl-1,3,2-dioxastannolane. Keywords: 1,3,2-dioxastannanes, stannylene acetals. X-ray crystallography, 119Sn NMR spectroscopy.
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Patowary, Suparna, Elisa Alvarez-Curto, Tian-Rui Xu, Jessica D. Holz, Julie A. Oliver, Graeme Milligan, and Valerică Raicu. "The muscarinic M3 acetylcholine receptor exists as two differently sized complexes at the plasma membrane." Biochemical Journal 452, no. 2 (May 10, 2013): 303–12. http://dx.doi.org/10.1042/bj20121902.
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The literature on GPCR (G-protein-coupled receptor) homo-oligomerization encompasses conflicting views that range from interpretations that GPCRs must be monomeric, through comparatively newer proposals that they exist as dimers or higher-order oligomers, to suggestions that such quaternary structures are rather ephemeral or merely accidental and may serve no functional purpose. In the present study we use a novel method of FRET (Förster resonance energy transfer) spectrometry and controlled expression of energy donor-tagged species to show that M3Rs (muscarinic M3 acetylcholine receptors) at the plasma membrane exist as stable dimeric complexes, a large fraction of which interact dynamically to form tetramers without the presence of trimers, pentamers, hexamers etc. That M3R dimeric units interact dynamically was also supported by co-immunoprecipitation of receptors synthesized at distinct times. On the basis of all these findings, we propose a conceptual framework that may reconcile the conflicting views on the quaternary structure of GPCRs.
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Casino, Patricia, Roberto Gozalbo-Rovira, Jesús Rodríguez-Díaz, Sreedatta Banerjee, Ariel Boutaud, Vicente Rubio, Billy G. Hudson, Juan Saus, Javier Cervera, and Alberto Marina. "Structures of collagen IV globular domains: insight into associated pathologies, folding and network assembly." IUCrJ 5, no. 6 (October 10, 2018): 765–79. http://dx.doi.org/10.1107/s2052252518012459.
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Basement membranes are extracellular structures of epithelia and endothelia that have collagen IV scaffolds of triple α-chain helical protomers that associate end-to-end, forming networks. The molecular mechanisms by which the noncollagenous C-terminal domains of α-chains direct the selection and assembly of the α1α2α1 and α3α4α5 hetero-oligomers found in vivo remain obscure. Autoantibodies against the noncollagenous domains of the α3α4α5 hexamer or mutations therein cause Goodpasture's or Alport's syndromes, respectively. To gain further insight into oligomer-assembly mechanisms as well as into Goodpasture's and Alport's syndromes, crystal structures of noncollagenous domains produced by recombinant methods were determined. The spontaneous formation of canonical homohexamers (dimers of trimers) of these domains of the α1, α3 and α5 chains was shown and the components of the Goodpasture's disease epitopes were viewed. Crystal structures of the α2 and α4 noncollagenous domains generated by recombinant methods were also determined. These domains spontaneously form homo-oligomers that deviate from the canonical architectures since they have a higher number of subunits (dimers of tetramers and of hexamers, respectively). Six flexible structural motifs largely explain the architectural variations. These findings provide insight into noncollagenous domain folding, while supporting the in vivo operation of extrinsic mechanisms for restricting the self-assembly of noncollagenous domains. Intriguingly, Alport's syndrome missense mutations concentrate within the core that nucleates the folding of the noncollagenous domain, suggesting that this syndrome, when owing to missense changes, is a folding disorder that is potentially amenable to pharmacochaperone therapy.
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Carpenter, Jerome, Yang Wang, Richa Gupta, Yuanli Li, Prashamsha Haridass, Durai B. Subramani, Boris Reidel, et al. "Assembly and organization of the N-terminal region of mucin MUC5AC: Indications for structural and functional distinction from MUC5B." Proceedings of the National Academy of Sciences 118, no. 39 (September 21, 2021): e2104490118. http://dx.doi.org/10.1073/pnas.2104490118.
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Elevated levels of MUC5AC, one of the major gel-forming mucins in the lungs, are closely associated with chronic obstructive lung diseases such as chronic bronchitis and asthma. It is not known, however, how the structure and/or gel-making properties of MUC5AC contribute to innate lung defense in health and drive the formation of stagnant mucus in disease. To understand this, here we studied the biophysical properties and macromolecular assembly of MUC5AC compared to MUC5B. To study each native mucin, we used Calu3 monomucin cultures that produced MUC5AC or MUC5B. To understand the macromolecular assembly of MUC5AC through N-terminal oligomerization, we expressed a recombinant whole N-terminal domain (5ACNT). Scanning electron microscopy and atomic force microscopy imaging indicated that the two mucins formed distinct networks on epithelial and experimental surfaces; MUC5B formed linear, infrequently branched multimers, whereas MUC5AC formed tightly organized networks with a high degree of branching. Quartz crystal microbalance-dissipation monitoring experiments indicated that MUC5AC bound significantly more to hydrophobic surfaces and was stiffer and more viscoelastic as compared to MUC5B. Light scattering analysis determined that 5ACNT primarily forms disulfide-linked covalent dimers and higher-order oligomers (i.e., trimers and tetramers). Selective proteolytic digestion of the central glycosylated region of the full-length molecule confirmed that MUC5AC forms dimers and higher-order oligomers through its N terminus. Collectively, the distinct N-terminal organization of MUC5AC may explain the more adhesive and unique viscoelastic properties of branched, highly networked MUC5AC gels. These properties may generate insight into why/how MUC5AC forms a static, “tethered” mucus layer in chronic muco-obstructive lung diseases.
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Signoretto, Caterina, Maria del Mar Lleò, Maria Carla Tafi, and Pietro Canepari. "Cell Wall Chemical Composition ofEnterococcus faecalis in the Viable but Nonculturable State." Applied and Environmental Microbiology 66, no. 5 (May 1, 2000): 1953–59. http://dx.doi.org/10.1128/aem.66.5.1953-1959.2000.
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ABSTRACT The viable but nonculturable (VBNC) state is a survival mechanism adopted by many bacteria (including those of medical interest) when exposed to adverse environmental conditions. In this state bacteria lose the ability to grow in bacteriological media but maintain viability and pathogenicity and sometimes are able to revert to regular division upon restoration of normal growth conditions. The aim of this work was to analyze the biochemical composition of the cell wall ofEnterococcus faecalis in the VBNC state in comparison with exponentially growing and stationary cells. VBNC enterococcal cells appeared as slightly elongated and were endowed with a wall more resistant to mechanical disruption than dividing cells. Analysis of the peptidoglycan chemical composition showed an increase in total cross-linking, which rose from 39% in growing cells to 48% in VBNC cells. This increase was detected in oligomers of a higher order than dimers, such as trimers (24% increase), tetramers (37% increase), pentamers (65% increase), and higher oligomers (95% increase). Changes were also observed in penicillin binding proteins (PBPs), the enzymes involved in the terminal stages of peptidoglycan assembly, with PBPs 5 and 1 being prevalent, and in autolytic enzymes, with a threefold increase in the activity of latent muramidase-1 in E. faecalis in the VBNC state. Accessory wall polymers such as teichoic acid and lipoteichoic acid proved unchanged and doubled in quantity, respectively, in VBNC cells in comparison to dividing cells. It is suggested that all these changes in the cell wall of VBNC enterococci are specific to this particular physiological state. This may provide indirect confirmation of the viability of these cells.
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Jovanovic, Jovan, Michael Spiteller, and Wilhelm Elling. "Indene dimerization products." Journal of the Serbian Chemical Society 67, no. 6 (2002): 393–406. http://dx.doi.org/10.2298/jsc0206393j.
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The reaction of 1H-indene (indene) in the presence of Friedel-Crafts acids was studied. As expected [M. Spiteller, J. Jovanovic, Fuel 78(1999)1263] there were dimers and trimers in the product mixture together with higher oligomers. Among products with double molecular weight relative to the molecular weight of indene, the structure of four compounds was determined: 6-(2?,3?-dihydro-1?H-inden-1?-yl)-1H-indene 2-(2?,3?-dihydro-1?H-inden-1?-yl)-1H-indene 1-(2?,3?-dihydro-1?H-inden-2?-yl)-1H-indene and 2,3,1?,3?-tetrahydro-[1,2?]biindenylidene. It was shown that the first one represents an indene alkylation product and that the others were obtained by bonding of the indan-1-ylium ion and indene at the position 2, followed by acid catalyzed 1,2-hydride rearrangement in the case of the third and fourth one. Considering the indene dimerization products as components of pyrolysis oils and as interesting compounds to be used as model substances for NMR, MS and X-ray analysis, the reaction, separation and isolation parameters were optimized in this study.
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Crepin, Aurélie, Erica Belgio, Barbora Šedivá, Eliška Kuthanová Trsková, Edel Cunill-Semanat, and Radek Kaňa. "Size and Fluorescence Properties of Algal Photosynthetic Antenna Proteins Estimated by Microscopy." International Journal of Molecular Sciences 23, no. 2 (January 11, 2022): 778. http://dx.doi.org/10.3390/ijms23020778.
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Antenna proteins play a major role in the regulation of light-harvesting in photosynthesis. However, less is known about a possible link between their sizes (oligomerization state) and fluorescence intensity (number of photons emitted). Here, we used a microscopy-based method, Fluorescence Correlation Spectroscopy (FCS), to analyze different antenna proteins at the particle level. The direct comparison indicated that Chromera Light Harvesting (CLH) antenna particles (isolated from Chromera velia) behaved as the monomeric Light Harvesting Complex II (LHCII) (from higher plants), in terms of their radius (based on the diffusion time) and fluorescence yields. FCS data thus indicated a monomeric oligomerization state of algal CLH antenna (at our experimental conditions) that was later confirmed also by biochemical experiments. Additionally, our data provide a proof of concept that the FCS method is well suited to measure proteins sizes (oligomerization state) and fluorescence intensities (photon counts) of antenna proteins per single particle (monomers and oligomers). We proved that antenna monomers (CLH and LHCIIm) are more “quenched” than the corresponding trimers. The FCS measurement thus represents a useful experimental approach that allows studying the role of antenna oligomerization in the mechanism of photoprotection.
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Karathanasis, Christos, Juliane Medler, Franziska Fricke, Sonja Smith, Sebastian Malkusch, Darius Widera, Simone Fulda, et al. "Single-molecule imaging reveals the oligomeric state of functional TNFα-induced plasma membrane TNFR1 clusters in cells." Science Signaling 13, no. 614 (January 14, 2020): eaax5647. http://dx.doi.org/10.1126/scisignal.aax5647.
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Ligand-induced tumor necrosis factor receptor 1 (TNFR1) activation controls nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling, cell proliferation, programmed cell death, and survival and is crucially involved in inflammation, autoimmune disorders, and cancer progression. Despite the relevance of TNFR1 clustering for signaling, oligomerization of ligand-free and ligand-activated TNFR1 remains controversial. At present, models range from ligand-independent receptor predimerization to ligand-induced oligomerization. Here, we used quantitative, single-molecule superresolution microscopy to study TNFR1 assembly directly in native cellular settings and at physiological cell surface abundance. In the absence of its ligand TNFα, TNFR1 assembled into monomeric and dimeric receptor units. Upon binding of TNFα, TNFR1 clustered predominantly not only into trimers but also into higher-order oligomers. A functional mutation in the preligand assembly domain of TNFR1 resulted in only monomeric TNFR1, which exhibited impaired ligand binding. In contrast, a form of TNFR1 with a mutation in the ligand-binding CRD2 subdomain retained the monomer-to-dimer ratio of the unliganded wild-type TNFR1 but exhibited no ligand binding. These results underscore the importance of ligand-independent TNFR1 dimerization in NF-κB signaling.
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Nave, R., D. O. Fürst, and K. Weber. "Visualization of the polarity of isolated titin molecules: a single globular head on a long thin rod as the M band anchoring domain?" Journal of Cell Biology 109, no. 5 (November 1, 1989): 2177–87. http://dx.doi.org/10.1083/jcb.109.5.2177.
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TII, the extractable form of titin, was purified from myofibrils and separated by high resolution gel permeation chromatography into two fractions (TIIA and TIIB). Novel specimen orientation methods used before metal shadowing and EM result in striking pictures of the two forms. Molecules layered on mica become uniformly oriented when subjected to centrifugation. TIIB comprises a very homogeneous fraction. All molecules reveal a single globular head at one end on a long and very thin rod of uniform diameter. The lengths of the rods have a very narrow distribution (900 +/- 50 nm). TIIA molecules seem lateral oligomers of TIIB, attached to each other via the head regions. While dimers are the predominant species, trimers and some higher oligomers can also be discerned. Mild proteolysis destroys the heads and converts TIIA and TIIB into TIIB-like rods. Similar molecules also result from titin purified from myofibrils by certain established purification schemes. Headless titin molecules show in gel electrophoresis only the TII band, while head bearing molecules give rise to two additional polypeptides at 165 and 190 kD. Immunoelectron microscopy of myofibrils identifies both titin-associated proteins as M band constituents. We speculate that in the polar images of TII the globular head region corresponds to the M band end of the titin molecules. This hypothesis is supported by immunoelectron micrographs of TIIB molecules using titin antibodies of known epitope location in the half sarcomere. This proposal complements our previous immunoelectron microscopic data on myofibrils. They showed that epitopes present only on the nonextractable TI species locate to the Z line and its immediately adjacent region (Fürst, D. O., M. Osborn, R. Nave, and K. Weber. 1988. J. Cell Biol. 106:1563-1572). Thus, the two distinct ends of the titin molecule attach to Z and M band material respectively.
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Kingsley, David H., Ali Behbahani, Afshin Rashtian, Gary W. Blissard, and Joshua Zimmerberg. "A Discrete Stage of Baculovirus GP64-mediated Membrane Fusion." Molecular Biology of the Cell 10, no. 12 (December 1999): 4191–200. http://dx.doi.org/10.1091/mbc.10.12.4191.
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Viral fusion protein trimers can play a critical role in limiting lipids in membrane fusion. Because the trimeric oligomer of many viral fusion proteins is often stabilized by hydrophobic 4-3 heptad repeats, higher-order oligomers might be stabilized by similar sequences. There is a hydrophobic 4-3 heptad repeat contiguous to a putative oligomerization domain of Autographa californica multicapsid nucleopolyhedrovirus envelope glycoprotein GP64. We performed mutagenesis and peptide inhibition studies to determine if this sequence might play a role in catalysis of membrane fusion. First, leucine-to-alanine mutants within and flanking the amino terminus of the hydrophobic 4-3 heptad repeat motif that oligomerize into trimers and traffic to insect Sf9 cell surfaces were identified. These mutants retained their wild-type conformation at neutral pH and changed conformation in acidic conditions, as judged by the reactivity of a conformationally sensitive mAb. These mutants, however, were defective for membrane fusion. Second, a peptide encoding the portion flanking the GP64 hydrophobic 4-3 heptad repeat was synthesized. Adding peptide led to inhibition of membrane fusion, which occurred only when the peptide was present during low pH application. The presence of peptide during low pH application did not prevent low pH–induced conformational changes, as determined by the loss of a conformationally sensitive epitope. In control experiments, a peptide of identical composition but different sequence, or a peptide encoding a portion of the Ebola GP heptad motif, had no effect on GP64-mediated fusion. Furthermore, when the hemagglutinin (X31 strain) fusion protein of influenza was functionally expressed in Sf9 cells, no effect on hemagglutinin-mediated fusion was observed, suggesting that the peptide does not exert nonspecific effects on other fusion proteins or cell membranes. Collectively, these studies suggest that the specific peptide sequences of GP64 that are adjacent to and include portions of the hydrophobic 4-3 heptad repeat play a dynamic role in membrane fusion at a stage that is downstream of the initiation of protein conformational changes but upstream of lipid mixing.
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Wong, G. William, Sarah A. Krawczyk, Claire Kitidis-Mitrokostas, Tracy Revett, Ruth Gimeno, and Harvey F. Lodish. "Molecular, biochemical and functional characterizations of C1q/TNF family members: adipose-tissue-selective expression patterns, regulation by PPAR-γ agonist, cysteine-mediated oligomerizations, combinatorial associations and metabolic functions." Biochemical Journal 416, no. 2 (November 12, 2008): 161–77. http://dx.doi.org/10.1042/bj20081240.
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The insulin-sensitizing hormone, adiponectin, belongs to the expanding C1q/TNF (tumour necrosis factor) family of proteins. We recently identified a family of adiponectin paralogues designated as CTRP (C1q/TNF-related protein) 1–7, and in the present study describe CTRP10. In the present study, we show that CTRP1, CTRP2, CTRP3, CTRP5 and CTRP7 transcripts are expressed predominantly by adipose tissue. In contrast, placenta and eye expressed the highest levels of CTRP6 and CTRP10 transcripts respectively. Expression levels of CTRP1, CTRP2, CTRP3, CTRP6 and CTRP7 transcripts are up-regulated in 8-week-old obese (ob/ob) mice relative to lean controls. Treatment of mice with a PPAR-γ (peroxisome-proliferator-activated receptor-γ) agonist, rosiglitazone, increased the expression of CTRP1 and decreased CTRP6 transcript levels. All CTRPs are secreted glycoproteins when expressed in mammalian cells. CTRP1, CTRP2, CTRP3, CTRP5 and CTRP6 circulate in the blood and are potential endocrine hormones; their serum levels vary according to the sex and genetic background of mice. Importantly, serum levels of CTRP1 and CTRP6 are increased in adiponectin-null mice. Like adiponectin, all secreted CTRP proteins form trimers as their basic structural units. CTRP3, CTRP5, CTRP6 and CTRP10 trimers are further assembled into higher-order oligomeric complexes via disulfide bonding mediated by their N-terminal cysteine residues. Besides forming homo-oligomers, CTRP1/CTRP6, CTRP2/CTRP7 and adiponectin/CTRP2 are secreted as heterotrimers, thus providing a mechanism to potentially generate functionally distinct ligands. Functional characterization of one such family member, CTRP1, showed that it specifically activates Akt and p44/42-MAPK (mitogen-activated protein kinase) signalling pathways in differentiated mouse myotubes. Moreover, injection of recombinant CTRP1 into mice significantly reduced their serum glucose levels. Thus at least CTRP1 may be considered a novel adipokine. In summary, these molecular, biochemical and functional data provide an important framework to further address the physiological functions and mechanisms of the action of this family of secreted glycoproteins in normal and disease states.
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Kurylowicz, M., H. Paulin, J. Mogyoros, M. Giuliani, and J. R. Dutcher. "The effect of nanoscale surface curvature on the oligomerization of surface-bound proteins." Journal of The Royal Society Interface 11, no. 94 (May 6, 2014): 20130818. http://dx.doi.org/10.1098/rsif.2013.0818.
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The influence of surface topography on protein conformation and association is used routinely in biological cells to orchestrate and coordinate biomolecular events. In the laboratory, controlling the surface curvature at the nanoscale offers new possibilities for manipulating protein–protein interactions and protein function at surfaces. We have studied the effect of surface curvature on the association of two proteins, α-lactalbumin (α-LA) and β-lactoglobulin (β-LG), which perform their function at the oil–water interface in milk emulsions. To control the surface curvature at the nanoscale, we have used a combination of polystyrene (PS) nanoparticles (NPs) and ultrathin PS films to fabricate chemically pure, hydrophobic surfaces that are highly curved and are stable in aqueous buffer. We have used single-molecule force spectroscopy to measure the contour lengths L c for α-LA and β-LG adsorbed on highly curved PS surfaces (NP diameters of 27 and 50 nm, capped with a 10 nm thick PS film), and we have compared these values in situ with those measured for the same proteins adsorbed onto flat PS surfaces in the same samples. The L c distributions for β-LG adsorbed onto a flat PS surface contain monomer and dimer peaks at 60 and 120 nm, respectively, while α-LA contains a large monomer peak near 50 nm and a dimer peak at 100 nm, with a tail extending out to 200 nm, corresponding to higher order oligomers, e.g. trimers and tetramers. When β-LG or α-LA is adsorbed onto the most highly curved surfaces, both monomer peaks are shifted to much smaller values of L c . Furthermore, for β-LG, the dimer peak is strongly suppressed on the highly curved surface, whereas for α-LA the trimer and tetramer tail is suppressed with no significant change in the dimer peak. For both proteins, the number of higher order oligomers is significantly reduced as the curvature of the underlying surface is increased. These results suggest that the surface curvature provides a new method of manipulating protein–protein interactions and controlling the association of adsorbed proteins, with applications to the development of novel biosensors.
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Kwon, Bum Gun, Jea-Jun Ko, and Jeong-Hun Park. "Qualitative Evaluation of Factors Inducing Environmental Pollution of the Sandy Beaches of Jeju Island Using Styrene Oligomers." Journal of Korean Society of Environmental Engineers 43, no. 12 (December 31, 2021): 700–708. http://dx.doi.org/10.4491/ksee.2021.43.12.700.
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Objectives : Plastic pollution is a very important environmental issue in Korea as well as abroad. The objective of this study is to evaluate the internal and external factors that cause pollution of the coastal environment of Jeju Island using styrene oligomers (SOs) originated from polystyrene (PS) plastic.Methods : In order to achieve the above objective, this study is conducted to quantitatively measure the concentration of 12 individual SOs chemicals, through gas chromatography/mass spectroscopy (GC/MS) analyzing seawater and beach sand samples around sandy beaches in Jeju Island. This study evaluates the degree of environmental pollution according to internal or external factors of the sandy beach by using the physicochemical characteristic that SOs species are adsorbed on the surface of sand particles.Results and Discussion : The average concentration of SOs in the beach sand of Jeju Island ranges from a minimum of 9.80 ng/g to a maximum of 13.62 ng/g, and the average concentration of SOs in seawater is relatively low with a constant 0.05 to 0.11 µg/L. Although the concentration distribution of SOs species differs considerably depending on the sample collected, the concentration of SOs decreases in the order of styrene trimers (7 isomers) > styrene dimers (4 isomers) > styrene monomer. As a result of monitoring, the concentration of SOs at the sandy beaches of Jeju Island is much higher in the beach sand than in the seawater. This result means that the major beaches of Jeju Island can be polluted mainly by internal factors (e.g. population density, number of travelers according to population movement, and so on), because SOs species are adsorbed on the surface of the sand particles and their mobility is limited.Conclusions : This study shows that the sandy beaches of Jeju Island are mainly polluted by internal factors. It is thought that the pollution degree of the sandy beaches is the highest in the order of Gwakji Beach < Samyang Beach, Hamdeok Beach, Pyoseon Beach < Ihoteho Beach, Sagye Beach < Seopjikoji Beach, Gimnyeong Beach, and Hyeopjae Beach. This study is expected to contribute to the evaluation of the causes of plastic pollution in the coastal environment of Jeju Island.
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Purde, Vedud, Florian Busch, Elena Kudryashova, Vicki H. Wysocki, and Dmitri S. Kudryashov. "Oligomerization Affects the Ability of Human Cyclase-Associated Proteins 1 and 2 to Promote Actin Severing by Cofilins." International Journal of Molecular Sciences 20, no. 22 (November 12, 2019): 5647. http://dx.doi.org/10.3390/ijms20225647.
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Actin-depolymerizing factor (ADF)/cofilins accelerate actin turnover by severing aged actin filaments and promoting the dissociation of actin subunits. In the cell, ADF/cofilins are assisted by other proteins, among which cyclase-associated proteins 1 and 2 (CAP1,2) are particularly important. The N-terminal half of CAP has been shown to promote actin filament dynamics by enhancing ADF-/cofilin-mediated actin severing, while the central and C-terminal domains are involved in recharging the depolymerized ADP–G-actin/cofilin complexes with ATP and profilin. We analyzed the ability of the N-terminal fragments of human CAP1 and CAP2 to assist human isoforms of “muscle” (CFL2) and “non-muscle” (CFL1) cofilins in accelerating actin dynamics. By conducting bulk actin depolymerization assays and monitoring single-filament severing by total internal reflection fluorescence (TIRF) microscopy, we found that the N-terminal domains of both isoforms enhanced cofilin-mediated severing and depolymerization at similar rates. According to our analytical sedimentation and native mass spectrometry data, the N-terminal recombinant fragments of both human CAP isoforms form tetramers. Replacement of the original oligomerization domain of CAPs with artificial coiled-coil sequences of known oligomerization patterns showed that the activity of the proteins is directly proportional to the stoichiometry of their oligomerization; i.e., tetramers and trimers are more potent than dimers, which are more effective than monomers. Along with higher binding affinities of the higher-order oligomers to actin, this observation suggests that the mechanism of actin severing and depolymerization involves simultaneous or consequent and coordinated binding of more than one N-CAP domain to F-actin/cofilin complexes.
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Huamán-Castilla, Nils Leander, David Campos, Diego García-Ríos, Javier Parada, Maximiliano Martínez-Cifuentes, María Salomé Mariotti-Celis, and José Ricardo Pérez-Correa. "Chemical Properties of Vitis Vinifera Carménère Pomace Extracts Obtained by Hot Pressurized Liquid Extraction, and Their Inhibitory Effect on Type 2 Diabetes Mellitus Related Enzymes." Antioxidants 10, no. 3 (March 17, 2021): 472. http://dx.doi.org/10.3390/antiox10030472.
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Grape pomace polyphenols inhibit Type 2 Diabetes Mellitus (T2DM)-related enzymes, reinforcing their sustainable recovery to be used as an alternative to the synthetic drug acarbose. Protic co-solvents (ethanol 15% and glycerol 15%) were evaluated in the hot pressurized liquid extraction (HPLE) of Carménère pomace at 90, 120, and 150 °C in order to obtain extracts rich in monomers and oligomers of procyanidins with high antioxidant capacities and inhibitory effects on α-amylase and α-glucosidase. The higher the HPLE temperature (from 90 °C to 150 °C) the higher the total polyphenol content (~79%, ~83%, and ~143% for water-ethanol, water-glycerol and pure water, respectively) and antioxidant capacity of the extracts (Oxygen Radical Absorbance Capacity, ORAC), increased by ~26%, 27% and 13%, while the half maximal inhibitory concentration (IC50) decreased by ~65%, 67%, and 59% for water-ethanol, water-glycerol, and pure water extracts, respectively). Water-glycerol HPLE at 150 and 120 °C recovered the highest amounts of monomers (99, 421, and 112 µg/g dw of phenolic acids, flavanols, and flavonols, respectively) and dimers of procyanidins (65 and 87 µg/g dw of B1 and B2, respectively). At 90 °C, the water-ethanol mixture extracted the highest amounts of procyanidin trimers (13 and 49 µg/g dw of C1 and B2, respectively) and procyanidin tetramers of B2 di-O-gallate (13 µg/g dw). Among the Carménère pomace extracts analyzed in this study, 1000 µg/mL of the water-ethanol extract obtained, at 90 °C, reduced differentially the α-amylase (56%) and α-glucosidase (98%) activities. At the same concentration, acarbose inhibited 56% of α-amylase and 73% of α-glucosidase activities; thus, our grape HPLE extracts can be considered a good inhibitor compared to the synthetic drug.
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Aldhayan, Dhaifallah, and Ahmed Aouissi. "Gas Phase Oligomerization of Isobutene over Acid Treated Kaolinite Clay Catalyst." Bulletin of Chemical Reaction Engineering & Catalysis 12, no. 1 (April 30, 2017): 119. http://dx.doi.org/10.9767/bcrec.12.1.758.119-126.
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<p>Natural Kaolin Clay was calcined and treated by sulfuric acid. The resulting solid acid catalyst was characterized by FTIR, TGA, and X-ray powder diffraction (XRD) and tested for isobutene oligomerization in a gas phase. The characterization results showed that the acid treated clay underwent chemical and structural transformations. After acid treatment, the Si/Al ratio was increased, and the crystalline raw clay became amorphous. The effects of various parameters such as reaction temperature, reaction time and contact time on isobutene oligomerization were investigated. Catalytic tests showed that isobutene oligomerization led to dimers and trimers as major products. Tetramers were obtained as by- products. At relatively high reaction temperatures and long contact times, the conversion was enhanced while the selectivity of dimers was decreased in favor of higher oligomers. Copyright © 2017 BCREC GROUP. All rights reserved</p><p><em>Received: 27<sup>th</sup> October 2016; Revised: 21<sup>st</sup> December 2016; Accepted: 22<sup>nd</sup> December 2016</em></p><p><strong>How to Cite:</strong> Aldhayan, D., Aouissi, A. (2017). Gas Phase Oligomerization of Isobutene over Acid Treated Kaolinite Clay Catalyst. <em>Bulletin of Chemical Reaction Engineering & Catalysis</em>, 12 (1): 119-126 (doi:10.9767/bcrec.12.1.758.119-126)</p><p><strong>Permalink/DOI</strong>: http://dx.doi.org/10.9767/bcrec.12.1.758.119-126</p><p> </p>
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Navarro-Hoyos, Mirtha, Elizabeth Arnáez-Serrano, Silvia Quesada-Mora, Gabriela Azofeifa-Cordero, Krissia Wilhelm-Romero, Maria Isabel Quirós-Fallas, Diego Alvarado-Corella, Felipe Vargas-Huertas, and Andrés Sánchez-Kopper. "HRMS Characterization, Antioxidant and Cytotoxic Activities of Polyphenols in Malus domestica Cultivars from Costa Rica." Molecules 26, no. 23 (December 4, 2021): 7367. http://dx.doi.org/10.3390/molecules26237367.
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There is increasing interest in research into fruits as sources of secondary metabolites because of their potential bioactivities. In this study, the phenolic profiles of Malus domestica Anna and Jonagold cultivars from Costa Rica were determined by Ultra Performance Liquid Chromatography coupled with High Resolution Mass Spectrometry (HRMS) using a quadrupole-time-of-flight analyzer (UPLC-QTOF-ESI MS), on enriched-phenolic extracts from skins and flesh, obtained through Pressurized Liquid Extraction (PLE). In total, 48 different phenolic compounds were identified in the skin and flesh extracts, comprising 17 flavan-3-ols, 12 flavonoids, 4 chalcones, 1 glycosylated isoprenoid and 14 hydroxycinnamic acids and derivatives. Among extracts, the flesh of Jonagold exhibits a larger number of polyphenols and is especially rich in procyanidin trimers, tetramers and pentamers. Evaluating total phenolic content (TPC) and antioxidant activities using ORAC and DPPH procedures yields higher values for this extract (608.8 mg GAE/g extract; 14.80 mmol TE/g extract and IC50 = 3.96 µg/mL, respectively). In addition, cytotoxicity evaluated against SW620 colon cancer cell lines and AGS gastric cancer cell lines also delivered better effects for Jonagold flesh (IC50 = 62.4 and 60.0 µg/mL, respectively). In addition, a significant negative correlation (p < 0.05) was found between TPC and cytotoxicity values against SW620 and AGS adenocarcinoma (r = −0.908, and −0.902, respectively). Furthermore, a significant negative correlation (p < 0.05) was also found between the number of procyanidins and both antioxidant activities and cytotoxicity towards SW620 (r = −0.978) and AGS (r = −0.894) cell lines. These results align with Jonagold flesh exhibiting the highest abundance in procyanidin oligomers and yielding better cytotoxic and antioxidant results. In sum, our findings suggest the need for further studies on these Costa Rican apple extracts—and particularly on the extracts from Jonagold flesh—to increase the knowledge on their potential benefits for health.
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Medina, Rafael, Deisy Perdomo, Carolina Möller, and José Bubis. "Cross-linking of bovine rhodopsin with sulfosuccinimidyl 4-(N maleimidomethyl)cyclohexane-1-carboxylate affects its functionality." Biochemical Journal 477, no. 12 (June 24, 2020): 2295–312. http://dx.doi.org/10.1042/bcj20200376.
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Rhodopsin is the photoreceptor protein involved in visual excitation in retinal rods. The functionality of bovine rhodopsin was determined following treatment with sulfosuccinimidyl 4-(N maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), a bifunctional reagent capable of forming covalent cross-links between suitable placed lysines and cysteines. Denaturing polyacrylamide gel electrophoresis showed that rhodopsin incubated with sulfo-SMCC generated intermolecular dimers, trimers, and higher oligomers, although most of the sulfo-SMCC-treated protein remained as a monomer. Minor alterations on the absorption spectrum of light-activated sulfo-SMCC-treated rhodopsin were observed. However, only ∼2% stimulation of the guanine nucleotide binding activity of transducin was measured in the presence of sulfo-SMCC-cross-linked photolyzed rhodopsin. Moreover, rhodopsin kinase was not able of phosphorylating sulfo-SMCC-cross-linked rhodopsin after illumination. Rhodopsin was purified in the presence of either 0.1% or 1% n-dodecyl β-d-maltoside, to obtain dimeric and monomeric forms of the protein, respectively. Interestingly, no generation of the regular F1 and F2 thermolytic fragments was perceived with sulfo-SMCC-cross-linked rhodopsin either in the dimeric or monomeric state, implying the formation of intramolecular connections in the protein that might thwart the light-induced conformational changes required for interaction with transducin and rhodopsin kinase. Structural analysis of the rhodopsin three-dimensional structure suggested that the following lysine and cysteine pairs: Lys66/Lys67 and Cys316, Cys140 and Lys141, Cys140 and Lys248, Lys311 and Cys316, and/or Cys316 and Lys325 are potential candidates to generate intramolecular cross-links in the protein. Yet, the lack of fragmentation of sulfo-SMCC-treated Rho with thermolysin is consistent with the formation of cross-linking bridges between Lys66/Lys67 and Cys316, and/or Cys140 and Lys248.
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Matsuyama, Shutoku, Sue Ellen Delos, and Judith M. White. "Sequential Roles of Receptor Binding and Low pH in Forming Prehairpin and Hairpin Conformations of a Retroviral Envelope Glycoprotein." Journal of Virology 78, no. 15 (August 1, 2004): 8201–9. http://dx.doi.org/10.1128/jvi.78.15.8201-8209.2004.
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ABSTRACT A general model has been proposed for the fusion mechanisms of class I viral fusion proteins. According to this model a metastable trimer, anchored in the viral membrane through its transmembrane domain, transits to a trimeric prehairpin intermediate, anchored at its opposite end in the target membrane through its fusion peptide. A subsequent refolding event creates a trimer of hairpins (often termed a six-helix bundle) in which the previously well-separated transmembrane domain and fusion peptide (and their attached membranes) are brought together, thereby driving membrane fusion. While there is ample biochemical and structural information on the trimer-of-hairpins conformation of class I viral fusion proteins, less is known about intermediate states between native metastable trimers and the final trimer of hairpins. In this study we analyzed conformational states of the transmembrane subunit (TM), the fusion subunit, of the Env glycoprotein of the subtype A avian sarcoma and leukosis virus (ASLV-A). By analyzing forms of EnvA TM on mildly denaturing sodium dodecyl sulfate gels we identified five conformational states of EnvA TM. Following interaction of virions with a soluble form of the ASLV-A receptor at 37°C, the metastable form of EnvA TM (which migrates at 37 kDa) transits to a 70-kDa and then to a 150-kDa species. Following subsequent exposure to a low pH (or an elevated temperature or the fusion promoting agent chlorpromazine), an additional set of bands at >150 kDa, and then a final band at 100 kDa, forms. Both an EnvA C-helix peptide (which inhibits virus fusion and infectivity) and the fusion-inhibitory agent lysophosphatidylcholine inhibit the formation of the >150- and 100-kDa bands. Our data are consistent with the 70- and 150-kDa bands representing precursor and fully formed prehairpin conformations of EnvA TM. Our data are also consistent with the >150-kDa bands representing higher-order oligomers of EnvA TM and with the 100-kDa band representing the fully formed six-helix bundle. In addition to resolving fusion-relevant conformational intermediates of EnvA TM, our data are compatible with a model in which the EnvA protein is activated by its receptor (at neutral pH and a temperature greater than or equal to room temperature) to form prehairpin conformations of EnvA TM, and in which subsequent exposure to a low pH is required to stabilize the final six-helix bundle, which drives a later stage of fusion.
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Meyer, D. F., M. O. Mayans, P. H. E. Groot, K. E. Suckling, K. R. Bruckdorfer, and S. J. Perkins. "Time-course studies by neutron solution scattering and biochemical assays of the aggregation of human low-density lipoprotein during Cu2+-induced oxidation." Biochemical Journal 310, no. 2 (September 1, 1995): 417–26. http://dx.doi.org/10.1042/bj3100417.
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The oxidative modification of low-density lipoproteins (LDL) is recognized to be a key event in the development of atherosclerotic plaques on artery walls. The characteristics of LDL oxidized by cells of the artery wall can be imitated by the addition of Cu2+ ions to initiate lipid peroxidation in LDL. Neutron scattering of LDL in 2H2O buffers enables the time course of changes in the gross structure of LDL during oxidation to be continuously monitored under conditions close to physiological. Oxidation of LDL [2 mg of apolipoprotein B (apoB) protein/ml] was studied in the presence of 6.4, 25.6 and 51.2 mumol of Cu2+/g of apoB by incubation at 37 degrees C for up to 70 h. Neutron Guinier analyses showed that the radius of gyration RG (indicative of size) and the forward-scattered intensity at zero angle I(0) (indicative of M(r)) continuously increased during oxidation, indicating that LDL had aggregated. Both the rate of aggregation and the change in RG and I(0) values after 10 and 50 h increased with Cu2+ concentration. Distance-distribution functions P(r) showed that, within 4 h, the maximum dimension of LDL increased from 23 to 55 nm. The P(r) curves of oxidatively modified LDL exhibited two peaks at 10-12 nm and 26 nm. The 10-12 nm peak corresponds to native LDL, and the 26 nm peak is assigned to the initial formation of LDL dimers and trimers and their progression to form higher oligomers. The growth of the 26 nm peak depended on Cu2+ concentration. Particle-size-distribution functions Dv(r) suggested that the polydisperse spherical structure of LDL ceased to exist after 30 h, at which point the LDL samples underwent a phase separation. Related, but not identical, changes in the I(Q) and P(r) curves were observed when native LDL was self-aggregated by brief vortexing. Parallel assessment of LDL protein modification by SDS/PAGE showed increased aggregation and degradation of apoB with increased Cu2+ concentrations, and that the main apoB protein band had diminished after 2-8 h, depending on the amount of Cu2+ added. The uptake and degradation of oxidized 125I-labelled LDL by mouse peritoneal macrophages occurred maximally within the first 10 h, and increased in proportion to the Cu2+ concentration. ApoB protein broke down within the first 10 h of oxidation, and this is the period when scavenger receptors on macrophages can recognize and internalize oxidized LDL. Within 10 h, the protein-lipid interactions responsible for the spherical LDL structure became destabilized by protein fragmentation.(ABSTRACT TRUNCATED AT 400 WORDS)
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Belyy, Vladislav, Iratxe Zuazo-Gaztelu, Andrew Alamban, Avi Ashkenazi, and Peter Walter. "Endoplasmic reticulum stress activates human IRE1α through reversible assembly of inactive dimers into small oligomers." eLife 11 (June 22, 2022). http://dx.doi.org/10.7554/elife.74342.
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Protein folding homeostasis in the endoplasmic reticulum (ER) is regulated by a signaling network, termed the unfolded protein response (UPR). Inositol-requiring enzyme 1 (IRE1) is an ER membrane-resident kinase/RNase that mediates signal transmission in the most evolutionarily conserved branch of the UPR. Dimerization and/or higher-order oligomerization of IRE1 are thought to be important for its activation mechanism, yet the actual oligomeric states of inactive, active, and attenuated mammalian IRE1 complexes remain unknown. We developed an automated two-color single-molecule tracking approach to dissect the oligomerization of tagged endogenous human IRE1 in live cells. In contrast to previous models, our data indicate that IRE1 exists as a constitutive homodimer at baseline and assembles into small oligomers upon ER stress. We demonstrate that the formation of inactive dimers and stress-dependent oligomers is fully governed by IRE1’s lumenal domain. Phosphorylation of IRE1’s kinase domain occurs more slowly than oligomerization and is retained after oligomers disassemble back into dimers. Our findings suggest that assembly of IRE1 dimers into larger oligomers specifically enables trans-autophosphorylation, which in turn drives IRE1’s RNase activity.
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Min, Yuan-Qin, Xu-Chu Duan, Yi-Dan Zhou, Anna Kulinich, Wang Meng, Zhi-Peng Cai, Hong-Yu Ma, Li Liu, Xiao-Lian Zhang, and Josef Voglmeir. "Effects of microvirin monomers and oligomers on hepatitis C virus." Bioscience Reports 37, no. 3 (June 30, 2017). http://dx.doi.org/10.1042/bsr20170015.
Full textAbstract:
Microvirin (MVN) is a carbohydrate-binding protein which shows high specificity for high-mannose type N-glycan structures. In the present study, we tried to identify whether MVN could bind to high-mannose containing hepatitis C virus (HCV) envelope glycoproteins, which are heavily decorated high-mannose glycans. In addition, recombinantly expressed MVN oligomers in di-, tri- and tetrameric form were evaluated for their viral inhibition. MVN oligomers bound more efficiently to HCV virions, and displayed in comparison with the MVN monomer a higher neutralization potency against HCV infection. The antiviral effect was furthermore affected by the peptide linker sequence connecting the MVN monomers. The results indicate that MVN oligomers such as trimers and tetramers may be used as future neutralization agents against HCV infections.
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Moore, Sara R., Smrithi S. Menon, Neeti S. Galwankar, Sadik A. Khuder, Michael K. Pangburn, and Viviana P. Ferreira. "A novel assay that characterizes properdin function shows neutrophil-derived properdin has a distinct oligomeric distribution." Frontiers in Immunology 13 (January 12, 2023). http://dx.doi.org/10.3389/fimmu.2022.918856.
Full textAbstract:
Properdin acts as an essential positive regulator of the alternative pathway of complement by stabilizing enzymatic convertases. Identical properdin monomers form head-to-tail associations of oligomers in a reported 20:54:26 ratio (most often described as an approximate 1:2:1 ratio) of tetramers (P4), trimers (P3), and dimers (P2), in blood, under normal physiological conditions. Oligomeric size is proportional to properdin function with tetramers being more active, followed by trimers and dimers. Neutrophils are the most abundant granulocyte, are recruited to inflammatory microenvironments, and are a significant source of properdin, yet the ratio of properdin oligomers released from neutrophils is unknown. The oligomer ratio of neutrophil-derived properdin could have functional consequences in local microenvironments where neutrophils are abundant and complement drives inflammation. We investigated the oligomer properties of neutrophil-derived properdin, as compared to that of normal human sera, using a novel ELISA-based method that detects function of properdin in a way that was proportional to the oligomeric size of properdin (i.e., the larger the oligomer, the higher the detected function). Unexpectedly, neutrophil-derived properdin had 5-fold lower function than donor-matched serum-derived properdin. The lower function was due to a lower percentage of tetramers/trimers and more dimers, indicating a significantly different P4:P3:P2 ratio in neutrophil-derived properdin (18:34:48) as compared to donor-matched serum (29:43:29). Release of lower-order oligomers by neutrophils may constitute a novel regulatory mechanism to control the rate of complement activation in cellular microenvironments. Further studies to determine the factors that affect properdin oligomerization and whether, or how, the predominant dimers in neutrophil-derived properdin, assimilate to the ~1:2:1 ratio found in serum are warranted.
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Sica, Mauricio P., and Cristian R. Smulski. "Coarse Grained Molecular Dynamic Simulations for the Study of TNF Receptor Family Members' Transmembrane Organization." Frontiers in Cell and Developmental Biology 8 (January 21, 2021). http://dx.doi.org/10.3389/fcell.2020.577278.
Full textAbstract:
The Tumor Necrosis Factor (TNF) and the TNF receptor (TNFR) superfamilies are composed of 19 ligands and 30 receptors, respectively. The oligomeric properties of ligands, both membrane bound and soluble, has been studied most. However, less is known about the oligomeric properties of TNFRs. Earlier reports identified the extracellular, membrane-distal, cysteine-rich domain as a pre-ligand assembly domain which stabilizes receptor dimers and/or trimers in the absence of ligand. Nevertheless, recent reports based on structural nuclear magnetic resonance (NMR) highlight the intrinsic role of the transmembrane domains to form dimers (p75NTR), trimers (Fas), or dimers of trimers (DR5). Thus, understanding the structural basis of transmembrane oligomerization may shed light on the mechanism for signal transduction and the impact of disease-associated mutations in this region. To this end, here we used an in silico coarse grained molecular dynamics approach with Martini force field to study TNFR transmembrane homotypic interactions. We have first validated this approach studying the three TNFR described by NMR (p75NTR, Fas, and DR5). We have simulated membrane patches composed of 36 helices of the same receptor equidistantly distributed in order to get unbiassed information on spontaneous proteins assemblies. Good agreement was found in the specific residues involved in homotypic interactions and we were able to observe dimers, trimers, and higher-order oligomers corresponding to those reported in NMR experiments. We have, applied this approach to study the assembly of disease-related mutations being able to assess their impact on oligomerization stability. In conclusion, our results showed the usefulness of coarse grained simulations with Martini force field to study in an unbiased manner higher order transmembrane oligomerization.
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Yu, Yang, Liangnan Cui, Xianbin Liu, Yuwen Wang, Chenchen Song, UnHak Pak, Kevin H. Mayo, Lin Sun, and Yifa Zhou. "Determining Methyl-Esterification Patterns in Plant-Derived Homogalacturonan Pectins." Frontiers in Nutrition 9 (July 1, 2022). http://dx.doi.org/10.3389/fnut.2022.925050.
Full textAbstract:
Homogalacturonan (HG)-type pectins are nutrient components in plants and are widely used in the food industry. The methyl-esterification pattern is a crucial structural parameter used to assess HG pectins in terms of their nutraceutical activity. To better understand the methyl-esterification pattern of natural HG pectins from different plants, we purified twenty HG pectin-rich fractions from twelve plants and classified them by their monosaccharide composition, Fourier transform-infrared spectroscopy (FT-IR) signatures, and NMR analysis. FT-IR shows that these HG pectins are all minimally esterified, with the degree of methyl-esterification (DM) being 5 to 40%. To examine their methyl-esterification pattern by enzymatic fingerprinting, we hydrolyzed the HG pectins using endo-polygalacturonase. Hydrolyzed oligomers were derivatized with 2-aminobenzamide and subjected to liquid chromatography-fluorescence-tandem mass spectrometry (HILIC-FLR-MSn). Twenty-one types of mono-/oligo-galacturonides having DP values of 1–10 were found to contain nonesterified monomers, dimers, and trimers, as well as oligomers with 1 to 6 methyl-ester groups. In these oligo-galacturonides, MSn analysis demonstrated that the number of methyl-ester groups in the continuous sequence was 2 to 5. Mono- and di-esterified oligomers had higher percentages in total methyl-esterified groups, suggesting that these are a random methyl-esterification pattern in these HG pectins. Our study analyzes the characteristics of the methyl-esterification pattern in naturally occurring plant-derived HG pectins and findings that will be useful for further studying HG structure-function relationships.