HcRed, a Genetically Encoded Fluorescent Binary Cross-Linking Agent for Cross-Linking of Mitochondrial ATP Synthase in Saccharomyces cerevisiae

PubMed Central

Gong, Lan; Ramm, Georg; Devenish, Rodney J.; Prescott, Mark

2012-01-01

Genetically encoded fluorescent cross-linking agents represent powerful tools useful both for visualising and modulating protein interactions in living cells. The far-red fluorescent protein HcRed, which is fluorescent only in a dimer form, can be used to promote the homo-dimerisation of target proteins, and thereby yield useful information about biological processes. We have in yeast cells expressed HcRed fused to a subunit of mitochondrial ATP synthase (mtATPase). This resulted in cross-linking of the large multi-subunit mtATPase complex within the inner-membrane of the mitochondrion. Fluorescence microscopy revealed aberrant mitochondrial morphology, and mtATPase complexes isolated from mitochondria were recovered as fluorescent dimers under conditions where complexes from control mitochondria were recovered as monomers. When viewed by electron microscopy normal cristae were absent from mitochondria in cells in which mATPase complexes were cross-linked. mtATPase dimers are believed to be the building blocks that are assembled into supramolecular mtATPase ribbons that promote the formation of mitochondrial cristae. We propose that HcRed cross-links mATPase complexes in the mitochondrial membrane hindering the normal assembly/disassembly of the supramolecular forms of mtATPase. PMID:22496895

Change in single cystathionine β-synthase domain-containing protein from a bent to flat conformation upon adenosine monophosphate binding.

PubMed

Jeong, Byung-Cheon; Park, Si Hoon; Yoo, Kyoung Shin; Shin, Jeong Sheop; Song, Hyun Kyu

2013-07-01

Cystathionine β-synthase (CBS) domains are small intracellular modules that can act as binding domains for adenosine derivatives, and they may regulate the activity of associated enzymes or other functional domains. Among these, the single CBS domain-containing proteins, CBSXs, from Arabidopsis thaliana, have recently been identified as redox regulators of the thioredoxin system. Here, the crystal structure of CBSX2 in complex with adenosine monophosphate (AMP) is reported at 2.2Å resolution. The structure of dimeric CBSX2 with bound-AMP is shown to be approximately flat, which is in stark contrast to the bent form of apo-CBSXs. This conformational change in quaternary structure is triggered by a local structural change of the unique α5 helix, and by moving each loop P into an open conformation to accommodate incoming ligands. Furthermore, subtle rearrangement of the dimer interface triggers movement of all subunits, and consequently, the bent structure of the CBSX2 dimer becomes a flat structure. This reshaping of the structure upon complex formation with adenosine-containing ligand provides evidence that ligand-induced conformational reorganization of antiparallel CBS domains is an important regulatory mechanism. Copyright © 2013 Elsevier Inc. All rights reserved.

PhaM Is the Physiological Activator of Poly(3-Hydroxybutyrate) (PHB) Synthase (PhaC1) in Ralstonia eutropha

PubMed Central

Pfeiffer, Daniel

2014-01-01

Poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) is the key enzyme of PHB synthesis in Ralstonia eutropha and other PHB-accumulating bacteria and catalyzes the polymerization of 3-hydroxybutyryl-CoA to PHB. Activity assays of R. eutropha PHB synthase are characterized by the presence of lag phases and by low specific activity. It is assumed that the lag phase is caused by the time necessary to convert the inactive PhaC1 monomer into the active dimeric form by an unknown priming process. The lag phase can be reduced by addition of nonionic detergents such as hecameg [6-O-(N-heptyl-carbamoyl)-methyl-α-d-glucopyranoside], which apparently accelerates the formation of PhaC1 dimers. We identified the PHB granule-associated protein (PGAP) PhaM as the natural primer (activator) of PHB synthase activity. PhaM was recently discovered as a novel type of PGAP with multiple functions in PHB metabolism. Addition of PhaM to PHB synthase assays resulted in immediate polymerization of 3HB coenzyme A with high specific activity and without a significant lag phase. The effect of PhaM on (i) PhaC1 activity, (ii) oligomerization of PhaC1, (iii) complex formation with PhaC1, and (iv) PHB granule formation in vitro and in vivo was shown by cross-linking experiments of purified proteins (PhaM, PhaC1) with glutardialdehyde, by size exclusion chromatography, and by fluorescence microscopic detection of de novo-synthesized PHB granules. PMID:24212577

PhaM is the physiological activator of poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) in Ralstonia eutropha.

PubMed

Pfeiffer, Daniel; Jendrossek, Dieter

2014-01-01

Poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) is the key enzyme of PHB synthesis in Ralstonia eutropha and other PHB-accumulating bacteria and catalyzes the polymerization of 3-hydroxybutyryl-CoA to PHB. Activity assays of R. eutropha PHB synthase are characterized by the presence of lag phases and by low specific activity. It is assumed that the lag phase is caused by the time necessary to convert the inactive PhaC1 monomer into the active dimeric form by an unknown priming process. The lag phase can be reduced by addition of nonionic detergents such as hecameg [6-O-(N-heptyl-carbamoyl)-methyl-α-D-glucopyranoside], which apparently accelerates the formation of PhaC1 dimers. We identified the PHB granule-associated protein (PGAP) PhaM as the natural primer (activator) of PHB synthase activity. PhaM was recently discovered as a novel type of PGAP with multiple functions in PHB metabolism. Addition of PhaM to PHB synthase assays resulted in immediate polymerization of 3HB coenzyme A with high specific activity and without a significant lag phase. The effect of PhaM on (i) PhaC1 activity, (ii) oligomerization of PhaC1, (iii) complex formation with PhaC1, and (iv) PHB granule formation in vitro and in vivo was shown by cross-linking experiments of purified proteins (PhaM, PhaC1) with glutardialdehyde, by size exclusion chromatography, and by fluorescence microscopic detection of de novo-synthesized PHB granules.

Peroxynitrite induces destruction of the tetrahydrobiopterin and heme in endothelial nitric oxide synthase: transition from reversible to irreversible enzyme inhibition†

PubMed Central

Chen, Weiguo; Druhan, Lawrence J.; Chen, Chun-An; Hemann, Craig; Chen, Yeong-Renn; Berka, Vladimir; Tsai, Ah-Lim; Zweier, Jay L.

2010-01-01

Endothelial nitric oxide synthase (eNOS) is an important regulator of vascular and cardiac function. Peroxynitrite (ONOO−) inactivates eNOS, but questions remain regarding the mechanisms of this process. It has been reported that inactivation is due to oxidation of the eNOS zinc-thiolate cluster, rather than the cofactor tetrahydrobiopterin (BH4); however, this remains highly controversial. Therefore, we investigated the mechanisms of ONOO−-induced eNOS dysfunction and their dose-dependence. Exposure of human eNOS to ONOO− resulted in a dose-dependent loss of activity with a marked destabilization of the eNOS dimer. HPLC analysis indicated that both free and eNOS-bound BH4 were oxidized during exposure to ONOO−; however, full oxidation of protein bound biopterin required higher ONOO− levels. Additionally, ONOO− triggered changes in UV/Visible spectrum and heme content of the enzyme. Pre-incubation of eNOS with BH4 decreased dimer destabilization and heme alteration. Addition of BH4 to the ONOO−-destabilized eNOS dimer only partially rescued enzyme function. In contrast to ONOO− treatment, incubation with the zinc chelator TPEN with removal of enzyme-bound zinc did not change the eNOS activity or stability of the SDS-resistant eNOS dimer, demonstrating that the dimer stabilization induced by BH4 does not require zinc occupancy of the zinc-thiolate cluster. While ONOO− treatment was observed to induce loss of Zn-binding this can not account for the loss of enzyme activity. Therefore, ONOO−-induced eNOS inactivation is primarily due to oxidation of BH4 and irreversible destruction of the heme/heme-center. PMID:20184376

Structures of human thymidylate synthase R163K with dUMP, FdUMP and glutathione show asymmetric ligand binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gibson, Lydia M.; Celeste, Lesa R.; Lovelace, Leslie L.

Thymidylate synthase (TS) is a well validated target in cancer chemotherapy. Here, a new crystal form of the R163K variant of human TS (hTS) with five subunits per asymmetric part of the unit cell, all with loop 181-197 in the active conformation, is reported. This form allows binding studies by soaking crystals in artificial mother liquors containing ligands that bind in the active site. Using this approach, crystal structures of hTS complexes with FdUMP and dUMP were obtained, indicating that this form should facilitate high-throughput analysis of hTS complexes with drug candidates. Crystal soaking experiments using oxidized glutathione revealed thatmore » hTS binds this ligand. Interestingly, the two types of binding observed are both asymmetric. In one subunit of the physiological dimer covalent modification of the catalytic nucleophile Cys195 takes place, while in another dimer a noncovalent adduct with reduced glutathione is formed in one of the active sites.« less

ATP synthase promotes germ cell differentiation independent of oxidative phosphorylation

PubMed Central

Teixeira, Felipe K.; Sanchez, Carlos G.; Hurd, Thomas R.; Seifert, Jessica R. K.; Czech, Benjamin; Preall, Jonathan B.; Hannon, Gregory J.; Lehmann, Ruth

2015-01-01

The differentiation of stem cells is a tightly regulated process essential for animal development and tissue homeostasis. Through this process, attainment of new identity and function is achieved by marked changes in cellular properties. Intrinsic cellular mechanisms governing stem cell differentiation remain largely unknown, in part because systematic forward genetic approaches to the problem have not been widely used1,2. Analysing genes required for germline stem cell differentiation in the Drosophila ovary, we find that the mitochondrial ATP synthase plays a critical role in this process. Unexpectedly, the ATP synthesizing function of this complex was not necessary for differentiation, as knockdown of other members of the oxidative phosphorylation system did not disrupt the process. Instead, the ATP synthase acted to promote the maturation of mitochondrial cristae during differentiation through dimerization and specific upregulation of the ATP synthase complex. Taken together, our results suggest that ATP synthase-dependent crista maturation is a key developmental process required for differentiation independent of oxidative phosphorylation. PMID:25915123

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi,J.; Sivaraman, J.; Song, J.

Unlike 3C protease, the severe acute respiratory syndrome coronavirus (SARS-CoV) 3C-like protease (3CLpro) is only enzymatically active as a homodimer and its catalysis is under extensive regulation by the unique extra domain. Despite intense studies, two puzzles still remain: (i) how the dimer-monomer switch is controlled and (ii) why dimerization is absolutely required for catalysis. Here we report the monomeric crystal structure of the SARS-CoV 3CLpro mutant R298A at a resolution of 1.75 Angstroms . Detailed analysis reveals that Arg298 serves as a key component for maintaining dimerization, and consequently, its mutation will trigger a cooperative switch from a dimermore » to a monomer. The monomeric enzyme is irreversibly inactivated because its catalytic machinery is frozen in the collapsed state, characteristic of the formation of a short 310-helix from an active-site loop. Remarkably, dimerization appears to be coupled to catalysis in 3CLpro through the use of overlapped residues for two networks, one for dimerization and another for the catalysis.« less

Dimer model for Tau proteins bound in microtubule bundles

NASA Astrophysics Data System (ADS)

Hall, Natalie; Kluber, Alexander; Hayre, N. Robert; Singh, Rajiv; Cox, Daniel

2013-03-01

The microtubule associated protein tau is important in nucleating and maintaining microtubule spacing and structure in neuronal axons. Modification of tau is implicated as a later stage process in Alzheimer's disease, but little is known about the structure of tau in microtubule bundles. We present preliminary work on a proposed model for tau dimers in microtubule bundles (dimers are the minimal units since there is one microtubule binding domain per tau). First, a model of tau monomer was created and its characteristics explored using implicit solvent molecular dynamics simulation. Multiple simulations yield a partially collapsed form with separate positively/negatively charged clumps, but which are a factor of two smaller than required by observed microtubule spacing. We argue that this will elongate in dimer form to lower electrostatic energy at a cost of entropic ``spring'' energy. We will present preliminary results on steered molecular dynamics runs on tau dimers to estimate the actual force constant. Supported by US NSF Grant DMR 1207624.

Loop-driven conformational transition between the alternative and collapsed form of prethrombin-2: targeted molecular dynamics study.

PubMed

Wu, Sangwook

2017-01-01

Two distinct crystal structures of prethrombin-2, the alternative and collapsed forms, are elucidated by X-ray crystallogrphy. We analyzed the conformational transition from the alternative to the collapsed form employing targeted molecular dynamics (TMD) simulation. Despite small RMSD difference in the two X-ray crystal structures, some hydrophobic residues (W60d, W148, W215, and F227) show a significant difference between the two conformations. TMD simulation shows that the four hydrophobic residues undergo concerted movement from dimer to trimer transition via tetramer state in the conformational change from the alternative to the collapsed form. We reveal that the concerted movement of the four hydrophobic residues is controlled by movement of specific loop regions behind. In this paper, we propose a sequential scenario for the conformational transition from the alternative form to the collapsed form, which is partially supported by the mutant W148A simulation.

Structural Basis for a Reciprocating Mechanism of Negative Cooperativity in Dimeric Phosphagen Kinase Activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, X.; Ye, S; Guo, S

Phosphagen kinase (PK) family members catalyze the reversible phosphoryl transfer between phosphagen and ADP to reserve or release energy in cell energy metabolism. The structures of classic quaternary complexes of dimeric creatine kinase (CK) revealed asymmetric ligand binding states of two protomers, but the significance and mechanism remain unclear. To understand this negative cooperativity further, we determined the first structure of dimeric arginine kinase (dAK), another PK family member, at 1.75 {angstrom}, as well as the structure of its ternary complex with AMPPNP and arginine. Further structural analysis shows that the ligand-free protomer in a ligand-bound dimer opens more widelymore » than the protomers in a ligand-free dimer, which leads to three different states of a dAK protomer. The unexpected allostery of the ligand-free protomer in a ligand-bound dimer should be relayed from the ligand-binding-induced allostery of its adjacent protomer. Mutations that weaken the interprotomer connections dramatically reduced the catalytic activities of dAK, indicating the importance of the allosteric propagation mediated by the homodimer interface. These results suggest a reciprocating mechanism of dimeric PK, which is shared by other ATP related oligomeric enzymes, e.g., ATP synthase. - Wu, X., Ye, S., Guo, S., Yan, W., Bartlam, M., Rao, Z. Structural basis for a reciprocating mechanism of negative cooperativity in dimeric phosphagen kinase activity.« less

The N-terminal Part of Arabidopsis thaliana Starch Synthase 4 Determines the Localization and Activity of the Enzyme.

PubMed

Raynaud, Sandy; Ragel, Paula; Rojas, Tomás; Mérida, Ángel

2016-05-13

Starch synthase 4 (SS4) plays a specific role in starch synthesis because it controls the number of starch granules synthesized in the chloroplast and is involved in the initiation of the starch granule. We showed previously that SS4 interacts with fibrillins 1 and is associated with plastoglobules, suborganelle compartments physically attached to the thylakoid membrane in chloroplasts. Both SS4 localization and its interaction with fibrillins 1 were mediated by the N-terminal part of SS4. Here we show that the coiled-coil region within the N-terminal portion of SS4 is involved in both processes. Elimination of this region prevents SS4 from binding to fibrillins 1 and alters SS4 localization in the chloroplast. We also show that SS4 forms dimers, which depends on a region located between the coiled-coil region and the glycosyltransferase domain of SS4. This region is highly conserved between all SS4 enzymes sequenced to date. We show that the dimerization seems to be necessary for the activity of the enzyme. Both dimerization and the functionality of the coiled-coil region are conserved among SS4 proteins from phylogenetically distant species, such as Arabidopsis and Brachypodium This finding suggests that the mechanism of action of SS4 is conserved among different plant species. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

A heterodimer of human 3'-phospho-adenosine-5'-phosphosulphate (PAPS) synthases is a new sulphate activating complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grum, Daniel, E-mail: daniel.grum@uni-due.de; Boom, Johannes van den, E-mail: johannes.van-den-boom@stud.uni-due.de; Neumann, Daniel, E-mail: dneuman@gwdg.de

2010-05-07

3'-Phospho-adenosine-5'-phosphosulphate (PAPS) synthases are fundamental to mammalian sulphate metabolism. These enzymes have recently been linked to a rising number of human diseases. Despite many studies, it is not yet understood how the mammalian PAPS synthases 1 and 2 interact with each other. We provide first evidence for heterodimerisation of these two enzymes by pull-down assays and Foerster resonance energy transfer (FRET) measurements. Kinetics of dimer dissociation/association indicates that these heterodimers form as soon as PAPSS1 and -S2 encounter each other in solution. Affinity of the homo- and heterodimers were found to be in the low nanomolar range using anisotropy measurementsmore » employing proteins labelled with the fluorescent dye IAEDANS that - in spite of its low quantum yield - is well suited for anisotropy due to its large Stokes shift. Within its kinase domain, the PAPS synthase heterodimer displays similar substrate inhibition by adenosine-5'-phosphosulphate (APS) as the homodimers. Due to divergent catalytic efficacies of PAPSS1 and -S2, the heterodimer might be a way of regulating PAPS synthase function within mammalian cells.« less

Crystal Structure of Toxoplasma gondii Porphobilinogen Synthase

PubMed Central

Jaffe, Eileen K.; Shanmugam, Dhanasekaran; Gardberg, Anna; Dieterich, Shellie; Sankaran, Banumathi; Stewart, Lance J.; Myler, Peter J.; Roos, David S.

2011-01-01

Porphobilinogen synthase (PBGS) is essential for heme biosynthesis, but the enzyme of the protozoan parasite Toxoplasma gondii (TgPBGS) differs from that of its human host in several important respects, including subcellular localization, metal ion dependence, and quaternary structural dynamics. We have solved the crystal structure of TgPBGS, which contains an octamer in the crystallographic asymmetric unit. Crystallized in the presence of substrate, each active site contains one molecule of the product porphobilinogen. Unlike prior structures containing a substrate-derived heterocycle directly bound to an active site zinc ion, the product-bound TgPBGS active site contains neither zinc nor magnesium, placing in question the common notion that all PBGS enzymes require an active site metal ion. Unlike human PBGS, the TgPBGS octamer contains magnesium ions at the intersections between pro-octamer dimers, which are presumed to function in allosteric regulation. TgPBGS includes N- and C-terminal regions that differ considerably from previously solved crystal structures. In particular, the C-terminal extension found in all apicomplexan PBGS enzymes forms an intersubunit β-sheet, stabilizing a pro-octamer dimer and preventing formation of hexamers that can form in human PBGS. The TgPBGS structure suggests strategies for the development of parasite-selective PBGS inhibitors. PMID:21383008

Simulations of cellulose translocation in the bacterial cellulose synthase suggest a regulatory mechanism for the dimeric structure of cellulose.

PubMed

Knott, Brandon C; Crowley, Michael F; Himmel, Michael E; Zimmer, Jochen; Beckham, Gregg T

2016-05-01

The processive cycle of the bacterial cellulose synthase (Bcs) includes the addition of a single glucose moiety to the end of a growing cellulose chain followed by the translocation of the nascent chain across the plasma membrane. The mechanism of this translocation and its precise location within the processive cycle are not well understood. In particular, the molecular details of how a polymer (cellulose) whose basic structural unit is a dimer (cellobiose) can be constructed by adding one monomer (glucose) at a time are yet to be elucidated. Here, we have utilized molecular dynamics simulations and free energy calculations to the shed light on these questions. We find that translocation forward by one glucose unit is quite favorable energetically, giving a free energy stabilization of greater than 10 kcal/mol. In addition, there is only a small barrier to translocation, implying that translocation is not rate limiting within the Bcs processive cycle (given experimental rates for cellulose synthesis in vitro ). Perhaps most significantly, our results also indicate that steric constraints at the transmembrane tunnel entrance regulate the dimeric structure of cellulose. Namely, when a glucose molecule is added to the cellulose chain in the same orientation as the acceptor glucose, the terminal glucose freely rotates upon forward motion, thus suggesting a regulatory mechanism for the dimeric structure of cellulose. We characterize both the conserved and non-conserved enzyme-polysaccharide interactions that drive translocation, and find that 20 of the 25 residues that strongly interact with the translocating cellulose chain in the simulations are well conserved, mostly with polar or aromatic side chains. Our results also allow for a dynamical analysis of the role of the so-called `finger helix' in cellulose translocation that has been observed structurally. Taken together, these findings aid in the elucidation of the translocation steps of the Bcs processive cycle and may be widely relevant to polysaccharide synthesizing or degrading enzymes that couple catalysis with chain translocation.

Structures of the N-acetyltransferase domain of Xylella fastidiosa N-acetyl-L-glutamate synthase/kinase with and without a His tag bound to N-acetyl-L-glutamate.

PubMed

Zhao, Gengxiang; Jin, Zhongmin; Allewell, Norma M; Tuchman, Mendel; Shi, Dashuang

2015-01-01

Structures of the catalytic N-acetyltransferase (NAT) domain of the bifunctional N-acetyl-L-glutamate synthase/kinase (NAGS/K) from Xylella fastidiosa bound to N-acetyl-L-glutamate (NAG) with and without an N-terminal His tag have been solved and refined at 1.7 and 1.4 Å resolution, respectively. The NAT domain with an N-terminal His tag crystallized in space group P4(1)2(1)2, with unit-cell parameters a=b=51.72, c=242.31 Å. Two subunits form a molecular dimer in the asymmetric unit, which contains ∼41% solvent. The NAT domain without an N-terminal His tag crystallized in space group P21, with unit-cell parameters a=63.48, b=122.34, c=75.88 Å, β=107.6°. Eight subunits, which form four molecular dimers, were identified in the asymmetric unit, which contains ∼38% solvent. The structures with and without the N-terminal His tag provide an opportunity to evaluate how the His tag affects structure and function. Furthermore, multiple subunits in different packing environments allow an assessment of the plasticity of the NAG binding site, which might be relevant to substrate binding and product release. The dimeric structure of the X. fastidiosa N-acetytransferase (xfNAT) domain is very similar to that of human N-acetyltransferase (hNAT), reinforcing the notion that mammalian NAGS is evolutionally derived from bifunctional bacterial NAGS/K.

Mechanism for controlling the monomer-dimer conversion of SARS coronavirus main protease.

PubMed

Wu, Cheng Guo; Cheng, Shu Chun; Chen, Shiang Chuan; Li, Juo Yan; Fang, Yi Hsuan; Chen, Yau Hung; Chou, Chi Yuan

2013-05-01

The Severe acute respiratory syndrome coronavirus (SARS-CoV) main protease (M(pro)) cleaves two virion polyproteins (pp1a and pp1ab); this essential process represents an attractive target for the development of anti-SARS drugs. The functional unit of M(pro) is a homodimer and each subunit contains a His41/Cys145 catalytic dyad. Large amounts of biochemical and structural information are available on M(pro); nevertheless, the mechanism by which monomeric M(pro) is converted into a dimer during maturation still remains poorly understood. Previous studies have suggested that a C-terminal residue, Arg298, interacts with Ser123 of the other monomer in the dimer, and mutation of Arg298 results in a monomeric structure with a collapsed substrate-binding pocket. Interestingly, the R298A mutant of M(pro) shows a reversible substrate-induced dimerization that is essential for catalysis. Here, the conformational change that occurs during substrate-induced dimerization is delineated by X-ray crystallography. A dimer with a mutual orientation of the monomers that differs from that of the wild-type protease is present in the asymmetric unit. The presence of a complete substrate-binding pocket and oxyanion hole in both protomers suggests that they are both catalytically active, while the two domain IIIs show minor reorganization. This structural information offers valuable insights into the molecular mechanism associated with substrate-induced dimerization and has important implications with respect to the maturation of the enzyme.

Snyder-Robinson Syndrome: Rescuing the Disease-Causing Effect of G56S mutant by Small Molecule Binding

NASA Astrophysics Data System (ADS)

Zhang, Zhe; Martiny, Virginie; Lagorce, David; Alexov, Emil; Miteva, Maria; Clemson University Team; Université Paris Diderot Team

2013-03-01

Snyder-Robinson Syndrome (SRS) is an X-linked mental retardation disorder, which is caused by defects in a particular gene coding for the spermine synthase (SMS) protein. Among the missense mutations known to be disease-causing is the G56S, which is positioned at the interface of the SMS homo-dimer. Previous computational and experimental investigations have shown that G56S mutation destabilizes the homo-dimer and thus greatly reduces the SMS enzymatic activity. In this study, we explore the possibility of mitigating the effect of G56S mutation by binding small molecules to suitable pockets around the mutation site. It is done by combined efforts of molecular dynamics simulations and in silico screening. The binding of selected molecules was calculated to fully compensate the effect of the mutation and rescue the wild type dimer affinity. This work was supported by NIH, NLM grant. No. 1R03LM009748

Domain analysis of 3 Keto Acyl-CoA synthase for structural variations in Vitis vinifera and Oryza brachyantha using comparative modelling.

PubMed

Sagar, Mamta; Pandey, Neetesh; Qamar, Naseha; Singh, Brijendra; Shukla, Akanksha

2015-03-01

The long chain fatty acids incorporated into plant lipids are derived from the iterative addition of C2 units which is provided by malonyl-CoA to an acyl-CoA after interactions with 3-ketoacyl-CoA synthase (KCS), found in several plants. This study provides functional characterization of three 3 ketoacyl CoA synthase like proteins in Vitis vinifera (one) and Oryza brachyantha (two proteins). Sequence analysis reveals that protein of Oryza brachyantha shows 96% similarity to a hypothetical protein in Sorghum bicolor; total 11 homologs were predicted in Sorghum bicolor. Conserved domain prediction confirm the presence of FAE1/Type III polyketide synthase-like protein, Thiolase-like, subgroup; Thiolase-like and 3-Oxoacyl-ACP synthase III, C-terminal and chalcone synthase like domain but very long chain 3-keto acyl CoA domain is absent. All three proteins were found to have Chalcone and stilbene synthases C terminal domain which is similar to domain of thiolase and β keto acyl synthase. Its N terminal domain is absent in J3M9Z7 protein of Oryza brachyantha and F6HH63 protein of Vitis vinifera. Differences in N-terminal domain is responsible for distinguish activity. The J3MF16 protein of Oryza brachyantha contains N terminal domain and C terminal domain and characterized using annotation of these domains. Domains Gcs (streptomyces coelicolor) and Chalcone-stilbene synthases (KAS) in 2-pyrone synthase (Gerbera hybrid) and chalcone synthase 2 (Medicago sativa) were found to be present in three proteins. This similarity points toward anthocyanin biosynthetic process. Similarity to chalcone synthase 2 reveals its possible role in Naringenine and Chalcone synthase like activity. In 3 keto acyl CoA synthase of Oryza brachyantha. Active site residues C-240, H-407, N-447 are present in J3MF16 protein that are common in these three protein at different positions. Structural variations among dimer interface, product binding site, malonyl-CoA binding sites, were predicted in localized combination of conserved residues.

Selecting the swimming mechanisms of colloidal particles: bubble propulsion versus self-diffusiophoresis.

PubMed

Wang, Sijia; Wu, Ning

2014-04-01

Bubble propulsion and self-diffusiophoresis are two common mechanisms that can drive autonomous motion of microparticles in hydrogen peroxide. Although microtubular particles, when coated with platinum in their interior concave surfaces, can propel due to the formation and release of bubbles from one end, the convex Janus particles usually do not generate any visible bubble. They move primarily due to the self-diffusiophoresis. Coincidentally, the platinum films on those particles were typically coated by physical evaporation. In this paper, we use a simple chemical deposition method to make platinum-polystyrene Janus dimers. Surprisingly, those particles are propelled by periodic growth and collapse of bubbles on the platinum-coated lobes. We find that both high catalytic activity and rough surface are necessary to change the propulsion mode from self-diffusiophoresis to bubble propulsion. Our Janus dimers, with combined geometric and interfacial anisotropy, also exhibit distinctive motions at the respective stages of bubble growth and collapse, which differ by 5-6 orders of magnitude in time. Our study not only provides insight into the link between self-diffusiophoresis and bubble propulsion but also reveals the intriguing impacts of the combined geometric and interfacial anisotropy on self-propulsion of particles.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aripirala, Srinivas; Gonzalez-Pacanowska, Dolores; Oldfield, Eric

Structural insights into L. major farnesyl diphosphate synthase, a key enzyme in the mevalonate pathway, are described. Farnesyl diphosphate synthase (FPPS) is an essential enzyme involved in the biosynthesis of sterols (cholesterol in humans and ergosterol in yeasts, fungi and trypanosomatid parasites) as well as in protein prenylation. It is inhibited by bisphosphonates, a class of drugs used in humans to treat diverse bone-related diseases. The development of bisphosphonates as antiparasitic compounds targeting ergosterol biosynthesis has become an important route for therapeutic intervention. Here, the X-ray crystallographic structures of complexes of FPPS from Leishmania major (the causative agent of cutaneousmore » leishmaniasis) with three bisphosphonates determined at resolutions of 1.8, 1.9 and 2.3 Å are reported. Two of the inhibitors, 1-(2-hydroxy-2,2-diphosphonoethyl)-3-phenylpyridinium (300B) and 3-butyl-1-(2,2-diphosphonoethyl)pyridinium (476A), co-crystallize with the homoallylic substrate isopentenyl diphosphate (IPP) and three Ca{sup 2+} ions. A third inhibitor, 3-fluoro-1-(2-hydroxy-2,2-diphosphonoethyl)pyridinium (46I), was found to bind two Mg{sup 2+} ions but not IPP. Calorimetric studies showed that binding of the inhibitors is entropically driven. Comparison of the structures of L. major FPPS (LmFPPS) and human FPPS provides new information for the design of bisphosphonates that will be more specific for inhibition of LmFPPS. The asymmetric structure of the LmFPPS–46I homodimer indicates that binding of the allylic substrate to both monomers of the dimer results in an asymmetric dimer with one open and one closed homoallylic site. It is proposed that IPP first binds to the open site, which then closes, opening the site on the other monomer, which closes after binding the second IPP, leading to the symmetric fully occupied FPPS dimer observed in other structures.« less

Man o' War Mutation in UDP-α-D-Xylose Synthase Favors the Abortive Catalytic Cycle and Uncovers a Latent Potential for Hexamer Formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Walsh, Jr., Richard M.; Polizzi, Samuel J.; Kadirvelraj, Renuka

The man o’ war (mow) phenotype in zebrafish is characterized by severe craniofacial defects due to a missense mutation in UDP-α-D-xylose synthase (UXS), an essential enzyme in proteoglycan biosynthesis. The mow mutation is located in the UXS dimer interface ~16 Å away from the active site, suggesting an indirect effect on the enzyme mechanism. We have examined the structural and catalytic consequences of the mow mutation (R236H) in the soluble fragment of human UXS (hUXS), which shares 93% sequence identity with the zebrafish enzyme. In solution, hUXS dimers undergo a concentration-dependent association to form a tetramer. Sedimentation velocity studies showmore » that the R236H substitution induces the formation of a new hexameric species. Using two new crystal structures of the hexamer, we show that R236H and R236A substitutions cause a local unfolding of the active site that allows for a rotation of the dimer interface necessary to form the hexamer. The disordered active sites in the R236H and R236A mutant constructs displace Y231, the essential acid/base catalyst in the UXS reaction mechanism. The loss of Y231 favors an abortive catalytic cycle in which the reaction intermediate, UDP-α-D-4-keto-xylose, is not reduced to the final product, UDP-α-D-xylose. Surprisingly, the mow-induced hexamer is almost identical to the hexamers formed by the deeply divergent UXS homologues from Staphylococcus aureus and Helicobacter pylori (21% and 16% sequence identity, respectively). The persistence of a latent hexamer-building interface in the human enzyme suggests that the ancestral UXS may have been a hexamer.« less

Carbon monoxide releasing molecule induces endothelial nitric oxide synthase activation through a calcium and phosphatidylinositol 3-kinase/Akt mechanism.

PubMed

Yang, Po-Min; Huang, Yu-Ting; Zhang, Yu-Qi; Hsieh, Chia-Wen; Wung, Being-Sun

2016-12-01

The production of nitric oxide (NO) by endothelial NO synthase (eNOS) plays a major role in maintaining vascular homeostasis. This study elucidated the potential role of carbon monoxide (CO)-releasing molecules (CORMs) in NO production and explored the underlying mechanisms in endothelial cells. We observed that 25μM CORM-2 could increase NO production and stimulate an increase in the intracellular Ca 2+ level. Furthermore, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetra acetic acid caused CORM-2-induced NO production, which was abolished by 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetraacetoxy-methyl ester (BAPTA-AM), indicating that intracellular Ca 2+ release plays a major role in eNOS activation. The inhibition of the IP3 receptor diminished the CORM-2-induced intracellular Ca 2+ increase and NO production. Furthermore, CORM-2 induced eNOS Ser 1179 phosphorylation and eNOS dimerization, but it did not alter eNOS expression. CORM-2 (25μM) also prolonged Akt phosphorylation, lasting for at least 12h. Pretreatment with phosphatidylinositol 3-kinase inhibitors (wortmannin or LY294002) inhibited the increases in NO production and phosphorylation but did not affect eNOS dimerization. CORM-2-induced eNOS Ser 1179 phosphorylation was intracellularly calcium-dependent, because pretreatment with an intracellular Ca 2+ chelator (BAPTA-AM) inhibited this process. Although CORM-2 increases intracellular reactive oxygen species (ROS), pretreatment with antioxidant enzyme catalase and N-acetyl-cysteine did not abolish the CORM-2-induced eNOS activity or phosphorylation, signifying that ROS is not involved in this activity. Hence, CORM-2 enhances eNOS activation through intracellular calcium release, Akt phosphorylation, and eNOS dimerization. Copyright © 2016 Elsevier Inc. All rights reserved.

Doxorubicin-induced nitrosative stress is mitigated by vitamin C via the modulation of nitric oxide synthases.

PubMed

Akolkar, Gauri; Bagchi, Ashim K; Ayyappan, Prathapan; Jassal, Davinder S; Singal, Pawan K

2017-04-01

An increase in oxidative stress is suggested to be the main cause in Doxorubicin (Dox)-induced cardiotoxicity. However, there is now evidence that activation of inducible nitric oxide synthase (iNOS) and nitrosative stress are also involved. The role of vitamin C (Vit C) in the regulation of nitric oxide synthase (NOS) and reduction of nitrosative stress in Dox-induced cardiotoxicity is unknown. The present study investigated the effects of Vit C in the mitigation of Dox-induced changes in the levels of nitric oxide (NO), NOS activity, protein expression of NOS isoforms, and nitrosative stress as well as cytokines TNF-α and IL-10 in isolated cardiomyocytes. Cardiomyocytes isolated from adult Sprague-Dawley rats were segregated into four groups: 1 ) control, 2 ) Vit C (25 µM), 3 ) Dox (10 µM), and 4 ) Vit C + Dox. Dox caused a significant increase in the generation of superoxide radical (O 2 ·- ), peroxynitrite, and NO, and these effects of Dox were blunted by Vit C. Dox increased the expression of iNOS and altered protein expression as well as activation of endothelial NOS (eNOS). These changes were prevented by Vit C. Dox induced an increase in the ratio of monomeric/dimeric eNOS, promoting the production of O 2 ·- , which was prevented by Vit C by increasing the stability of the dimeric form of eNOS. Vit C protected against the Dox-induced increase in TNFα as well as a reduction in IL-10. These results suggest that Vit C provides cardioprotection by reducing oxidative/nitrosative stress and inflammation via a modulation of Dox-induced increase in the NO levels and NOS activity. Copyright © 2017 the American Physiological Society.

Enhanced Si-O Bond Breaking in Silica Glass by Water Dimer: A Hybrid Quantum-Classical Simulation Study

NASA Astrophysics Data System (ADS)

Kouno, Takahisa; Ogata, Shuji; Shimada, Takaaki; Tamura, Tomoyuki; Kobayashi, Ryo

2016-05-01

A hybrid quantum-classical simulation of a 4,608-atom silica glass is performed at a temperature of 400 K with either a water monomer or dimer inserted in a void. The quantum region that includes the water and the surrounding atoms is treated by the density-functional theory (DFT). During a simulation, the silica glass is gradually compressed or expanded. No Si-O bond breaking occurs with a water monomer until the silica glass collapses. With a water dimer, we find that Si-O bond breaking occurs through three steps in 3 out of 24 compression cases: (i) H-transfer as 2H2O → OH- + H3O+ accompanied by the adsorption of OH- at a strained Si to make it five-coordinated, (ii) breaking of a Si-O bond that originates from the five-coordinated Si, and (iii) H-transfer from H3O+ to the O of the broken Si-O bond. A separate DFT calculation confirms that the barrier energy of the bond breaking with a water dimer under compression is smaller than that with a water monomer and that the barrier energy decreases significantly when the silica glass is compressed further.

Competition between spin-orbit coupling, magnetism, and dimerization in the honeycomb iridates: α -Li2IrO3 under pressure

NASA Astrophysics Data System (ADS)

Hermann, V.; Altmeyer, M.; Ebad-Allah, J.; Freund, F.; Jesche, A.; Tsirlin, A. A.; Hanfland, M.; Gegenwart, P.; Mazin, I. I.; Khomskii, D. I.; Valentí, R.; Kuntscher, C. A.

2018-02-01

Single-crystal x-ray diffraction studies with synchrotron radiation on the honeycomb iridate α -Li2IrO3 reveal a pressure-induced structural phase transition with symmetry lowering from monoclinic to triclinic at a critical pressure of Pc=3.8 GPa. According to the evolution of the lattice parameters with pressure, the transition mainly affects the a b plane and thereby the Ir hexagon network, leading to the formation of Ir-Ir dimers. These observations are independently predicted and corroborated by our ab initio density functional theory calculations where we find that the appearance of Ir-Ir dimers at finite pressure is a consequence of a subtle interplay between magnetism, correlation, spin-orbit coupling, and covalent bonding. Our results further suggest that at Pc the system undergoes a magnetic collapse. Finally we provide a general picture of competing interactions for the honeycomb lattices A2M O3 with A =Li , Na and M =Ir , Ru.

Identification of a new binding site in E. coli FabH using Molecular dynamics simulations: validation by computational alanine mutagenesis and docking studies.

PubMed

Ramamoorthy, Divya; Turos, Edward; Guida, Wayne C

2013-05-24

FabH (Fatty acid biosynthesis, enzyme H, also referred to as β-ketoacyl-ACP-synthase III) is a key condensing enzyme in the type II fatty acid synthesis (FAS) system. The FAS pathway in bacteria is essential for growth and survival and vastly differs from the human FAS pathway. Enzymes involved in this pathway have arisen as promising biomolecular targets for discovery of new antibacterial drugs. However, currently there are no clinical drugs that selectively target FabH, and known inhibitors of FabH all act within the active site. FabH exerts its catalytic function as a dimer, which could potentially be exploited in developing new strategies for inhibitor design. The aim of this study was to elucidate structural details of the dimer interface region by means of computational modeling, including molecular dynamics (MD) simulations, in order to derive information for the structure-based design of new FabH inhibitors. The dimer interface region was analyzed by MD simulations, trajectory snapshots were collected for further analyses, and docking studies were performed with potential small molecule disruptors. Alanine mutation and docking studies strongly suggest that the dimer interface could be a potential target for anti-infection drug discovery.

S-Nitrosation and regulation of inducible nitric oxide synthase.

PubMed

Mitchell, Douglas A; Erwin, Phillip A; Michel, Thomas; Marletta, Michael A

2005-03-29

The inducible isoform of nitric oxide synthase (iNOS) and three zinc tetrathiolate mutants (C104A, C109A, and C104A/C109A) were expressed in Escherichia coli and purified. The mutants were found by ICP-AES and the zinc-specific PAR colorimetric assay to be zinc free, whereas the wild-type iNOS zinc content was 0.38 +/- 0.01 mol of Zn/mol of iNOS dimer. The cysteine mutants (C104A and C109A) had an activity within error of wild-type iNOS (2.24 +/- 0.12 micromol of NO min(-1) mg(-1)), but the double cysteine mutant had a modestly decreased activity (1.75 +/- 0.14 micromol of NO min(-1) mg(-1)). To determine if NO could stimulate release of zinc and dimer dissociation, wild-type protein was allowed to react with an NO donor, DEA/NO, followed by buffer exchange. ICP-AES of samples treated with 10 microM DEA/NO showed a decrease in zinc content (0.23 +/- 0.01 to 0.09 +/- 0.01 mol of Zn/mol of iNOS dimer) with no loss of heme iron. Gel filtration of wild-type iNOS treated similarly resulted in approximately 20% more monomeric iNOS compared to a DEA-treated sample. Only wild-type iNOS had decreased activity (42 +/- 2%) after reaction with 50 microM DEA/NO compared to a control sample. Using the biotin switch method under the same conditions, only wild-type iNOS had increased levels of S-biotinylation. S-Biotinylation was mapped to C104 and C109 on wild-type iNOS using LysC digestion and MALDI-TOF/TOF MS. Immunoprecipitation of iNOS from the mouse macrophage cell line, RAW-264.7, and the biotin switch method were used to confirm endogenous S-nitrosation of iNOS. The data show that S-nitrosation of the zinc tetrathiolate cysteine results in zinc release from the dimer interface and formation of inactive monomers, suggesting that this mode of inhibition might occur in vivo.

Simulations of cellulose translocation in the bacterial cellulose synthase suggest a regulatory mechanism for the dimeric structure of cellulose

DOE PAGES

Knott, Brandon C.; Crowley, Michael F.; Himmel, Michael E.; ...

2016-01-29

The processive cycle of the bacterial cellulose synthase (Bcs) includes the addition of a single glucose moiety to the end of a growing cellulose chain followed by the translocation of the nascent chain across the plasma membrane. The mechanism of this translocation and its precise location within the processive cycle are not well understood. In particular, the molecular details of how a polymer (cellulose) whose basic structural unit is a dimer (cellobiose) can be constructed by adding one monomer (glucose) at a time are yet to be elucidated. Here, we have utilized molecular dynamics simulations and free energy calculations tomore » the shed light on these questions. We find that translocation forward by one glucose unit is quite favorable energetically, giving a free energy stabilization of greater than 10 kcal mol-1. In addition, there is only a small barrier to translocation, implying that translocation is not rate limiting within the Bcs processive cycle (given experimental rates for cellulose synthesis in vitro). Perhaps most significantly, our results also indicate that steric constraints at the transmembrane tunnel entrance regulate the dimeric structure of cellulose. Namely, when a glucose molecule is added to the cellulose chain in the same orientation as the acceptor glucose, the terminal glucose freely rotates upon forward motion, thus suggesting a regulatory mechanism for the dimeric structure of cellulose. We characterize both the conserved and non-conserved enzyme-polysaccharide interactions that drive translocation, and find that 20 of the 25 residues that strongly interact with the translocating cellulose chain in the simulations are well conserved, mostly with polar or aromatic side chains. Our results also allow for a dynamical analysis of the role of the so-called 'finger helix' in cellulose translocation that has been observed structurally. Taken together, these findings aid in the elucidation of the translocation steps of the Bcs processive cycle and may be widely relevant to polysaccharide synthesizing or degrading enzymes that couple catalysis with chain translocation.« less

PSD-95 uncoupling from NMDA receptors by Tat- N-dimer ameliorates neuronal depolarization in cortical spreading depression.

PubMed

Kucharz, Krzysztof; Søndergaard Rasmussen, Ida; Bach, Anders; Strømgaard, Kristian; Lauritzen, Martin

2017-05-01

Cortical spreading depression is associated with activation of NMDA receptors, which interact with the postsynaptic density protein 95 (PSD-95) that binds to nitric oxide synthase (nNOS). Here, we tested whether inhibition of the nNOS/PSD-95/NMDA receptor complex formation by anti-ischemic compound, UCCB01-144 (Tat- N-dimer) ameliorates the persistent effects of cortical spreading depression on cortical function. Using in vivo two-photon microscopy in somatosensory cortex in mice, we show that fluorescently labelled Tat- N-dimer readily crosses blood-brain barrier and accumulates in nerve cells during the first hour after i.v. injection. The Tat- N-dimer suppressed stimulation-evoked synaptic activity by 2-20%, while cortical blood flow and cerebral oxygen metabolic (CMRO 2 ) responses were preserved. During cortical spreading depression, the Tat- N-dimer reduced the average amplitude of the negative shift in direct current potential by 33% (4.1 mV). Furthermore, the compound diminished the average depression of spontaneous electrocorticographic activity by 11% during first 40 min of post-cortical spreading depression recovery, but did not mitigate the suppressing effect of cortical spreading depression on cortical blood flow and CMRO 2 . We suggest that uncoupling of PSD-95 from NMDA receptors reduces overall neuronal excitability and the amplitude of the spreading depolarization wave. These findings may be of interest for understanding the neuroprotective effects of the nNOS/PSD-95 uncoupling in stroke.

Nitric-oxide Synthase Forms N-NO-pterin and S-NO-Cys

PubMed Central

Rosenfeld, Robin J.; Bonaventura, Joseph; Szymczyna, Blair R.; MacCoss, Michael J.; Arvai, Andrew S.; Yates, John R.; Tainer, John A.; Getzoff, Elizabeth D.

2010-01-01

Inducible nitric-oxide synthase (iNOS) produces biologically stressful levels of nitric oxide (NO) as a potent mediator of cellular cytotoxicity or signaling. Yet, how this nitrosative stress affects iNOS function in vivo is poorly understood. Here we define two specific non-heme iNOS nitrosation sites discovered by combining UV-visible spectroscopy, chemiluminescence, mass spectrometry, and x-ray crystallography. We detected auto-S-nitrosylation during enzymatic turnover by using chemiluminescence. Selective S-nitrosylation of the ZnS4 site, which bridges the dimer interface, promoted a dimer-destabilizing order-to-disorder transition. The nitrosated iNOS crystal structure revealed an unexpected N-NO modification on the pterin cofactor. Furthermore, the structurally defined N-NO moiety is solvent-exposed and available to transfer NO to a partner. We investigated glutathione (GSH) as a potential transnitrosation partner because the intracellular GSH concentration is high and NOS can form S-nitrosoglutathione. Our computational results predicted a GSH binding site adjacent to the N-NO-pterin. Moreover, we detected GSH binding to iNOS with saturation transfer difference NMR spectroscopy. Collectively, these observations resolve previous paradoxes regarding this uncommon pterin cofactor in NOS and suggest means for regulating iNOS activity via N-NO-pterin and S-NO-Cys modifications. The iNOS self-nitrosation characterized here appears appropriate to help control NO production in response to cellular conditions. PMID:20659888

Integration of energy and electron transfer processes in the photosynthetic membrane of Rhodobacter sphaeroides

DOE PAGES

Cartron, Michaël L.; Olsen, John D.; Sener, Melih; ...

2014-02-13

Photosynthesis converts absorbed solar energy to a protonmotive force, which drives ATP synthesis. The membrane network of chlorophyll–protein complexes responsible for light absorption, photochemistry and quinol (QH 2) production has been mapped in the purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides using atomic force microscopy (AFM), but the membrane location of the cytochrome bc 1 (cytbc 1) complexes that oxidise QH 2 to quinone (Q) to generate a protonmotive force is unknown. We labelled cytbc 1 complexes with gold nanobeads, each attached by a Histidine 10 (His 10)-tag to the C-terminus of cytc1. Electron microscopy (EM) of negatively stained chromatophore vesiclesmore » showed that the majority of the cytbc 1 complexes occur as dimers in the membrane. The cytbc 1 complexes appeared to be adjacent to reaction centre light-harvesting 1-PufX (RC-LH1-PufX) complexes, consistent with AFM topographs of a gold-labelled membrane. His-tagged cytbc1 complexes were retrieved from chromatophores partially solubilised by detergent; RC-LH1-PufX complexes tended to co-purify with cytbc 1, whereas LH2 complexes became detached, consistent with clusters of cytbc1 complexes close to RC-LH1-PufX arrays, but not with a fixed, stoichiometric cytbc 1-RC-LH1- PufX supercomplex. This information was combined with a quantitative mass spectrometry (MS) analysis of the RC, cytbc 1, ATP synthase, cytaa 3 and cytcbb 3 membrane protein complexes, to construct an atomic-level model of a chromatophore vesicle comprising 67 LH2 complexes, 11 LH1-RC-PufX dimers & 2 RC-LH1-PufX monomers, 4 cytbc 1 dimers and 2 ATP synthases. In conclusion, simulation of the interconnected energy, electron and proton transfer processes showed a halfmaximal ATP turnover rate for a light intensity equivalent to only 1% of bright sunlight. Thus, the photosystem architecture of the chromatophore is optimised for growth at low light intensities.« less

Characterization of Recombinant UDP- and ADP-Glucose Pyrophosphorylases and Glycogen Synthase To Elucidate Glucose-1-Phosphate Partitioning into Oligo- and Polysaccharides in Streptomyces coelicolor

PubMed Central

Asención Diez, Matías D.; Peirú, Salvador; Demonte, Ana M.; Gramajo, Hugo

2012-01-01

Streptomyces coelicolor exhibits a major secondary metabolism, deriving important amounts of glucose to synthesize pigmented antibiotics. Understanding the pathways occurring in the bacterium with respect to synthesis of oligo- and polysaccharides is of relevance to determine a plausible scenario for the partitioning of glucose-1-phosphate into different metabolic fates. We report the molecular cloning of the genes coding for UDP- and ADP-glucose pyrophosphorylases as well as for glycogen synthase from genomic DNA of S. coelicolor A3(2). Each gene was heterologously expressed in Escherichia coli cells to produce and purify to electrophoretic homogeneity the respective enzymes. UDP-glucose pyrophosphorylase (UDP-Glc PPase) was characterized as a dimer exhibiting a relatively high Vmax in catalyzing UDP-glucose synthesis (270 units/mg) and with respect to dTDP-glucose (94 units/mg). ADP-glucose pyrophosphorylase (ADP-Glc PPase) was found to be tetrameric in structure and specific in utilizing ATP as a substrate, reaching similar activities in the directions of ADP-glucose synthesis or pyrophosphorolysis (Vmax of 0.15 and 0.27 units/mg, respectively). Glycogen synthase was arranged as a dimer and exhibited specificity in the use of ADP-glucose to elongate α-1,4-glucan chains in the polysaccharide. ADP-Glc PPase was the only of the three enzymes exhibiting sensitivity to allosteric regulation by different metabolites. Mannose-6-phosphate, phosphoenolpyruvate, fructose-6-phosphate, and glucose-6-phosphate behaved as major activators, whereas NADPH was a main inhibitor of ADP-Glc PPase. The results support a metabolic picture where glycogen synthesis occurs via ADP-glucose in S. coelicolor, with the pathway being strictly regulated in connection with other routes involved with oligo- and polysaccharides, as well as with antibiotic synthesis in the bacterium. PMID:22210767

Sex differences in nitrosative stress during renal ischemia.

PubMed

Rodríguez, Francisca; Nieto-Cerón, Susana; Fenoy, Francisco J; López, Bernardo; Hernández, Isabel; Martinez, Raquel Rodado; Soriano, Ma José González; Salom, Miguel G

2010-11-01

Females suffer a less severe ischemic acute renal failure than males, apparently because of higher nitric oxide (NO) bioavailability and/or lower levels of oxidative stress. Because the renal ischemic injury is associated with outer medullary (OM) endothelial dysfunction, the present study evaluated sex differences in OM changes of NO and peroxynitrite levels (by differential pulse voltammetry and amperometry, respectively) during 45 min of ischemia and 60 min of reperfusion in anesthetized Sprague-Dawley rats. Endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) protein expression and their phosphorylated forms [peNOS(Ser1177) and pnNOS(Ser1417)], 3-nitrotyrosine, reduced sulfhydryl groups (-SH), and glomerular filtration rate (GFR) were also determined. No sex differences were observed in monomeric eNOS and nNOS expression, NO, or 3-nitrotyrosine levels in nonischemic kidneys, but renal -SH content was higher in females. Ischemia increased dimeric/monomeric eNOS and nNOS ratio more in females, but the dimeric phosphorylated peNOS(Ser1177) and pnNOS(Ser1417) forms rose similarly in both sexes, indicating no sex differences in nitric oxide synthase activation. However, NO levels increased more in females than in males (6,406.0 ± 742.5 and 4,058.2 ± 272.35 nmol/l respectively, P < 0.05), together with a lower increase in peroxynitrite current (5.5 ± 0.7 vs. 12.7 ± 1.5 nA, P < 0.05) and 3-nitrotyrosine concentration, (28.7 ± 3.7 vs. 48.7 ± 3.7 nmol/mg protein, P < 0.05) in females than in males and a better preserved GFR after ischemia in females than in males (689.7 ± 135.0 and 221.4 ± 52.5 μl·min(-1)·g kidney wt(-1), P < 0.01). Pretreatment with the antioxidants N-acetyl-L-cysteine or ebselen abolished sex differences in peroxynitrite, nitrotyrosine, and GFR, suggesting that a greater oxidative and nitrosative stress worsens renal damage in males.

Structural analyses of human thymidylate synthase reveal a site that may control conformational switching between active and inactive states.

PubMed

Chen, Dan; Jansson, Anna; Sim, Daniel; Larsson, Andreas; Nordlund, Pär

2017-08-11

Thymidylate synthase (TS) is the sole enzyme responsible for de novo biosynthesis of thymidylate (TMP) and is essential for cell proliferation and survival. Inhibition of human TS (hTS) has been extensively investigated for cancer chemotherapy, but several aspects of its activity and regulation are still uncertain. In this study, we performed comprehensive structural and biophysical studies of hTS using crystallography and thermal shift assay and provided the first detailed structural information on the conformational changes induced by ligand binding to the hTS active site. We found that upon binding of the antifolate agents raltitrexed and nolatrexed, the two insert regions in hTS, the functions of which are unclear, undergo positional shifts toward the catalytic center. We investigated the inactive conformation of hTS and found that the two insert regions are also involved in the conformational transition between the active and inactive state of hTS. Moreover, we identified a ligand-binding site in the dimer interface, suggesting that the cavity in the dimer interface could serve as an allosteric site of hTS to regulate the conformational switching between the active and inactive states. On the basis of these findings, we propose a regulatory mechanism of hTS activity that involves allosteric regulation of interactions of hTS with its own mRNA depending on cellular demands for TMP. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

SUCROSE SYNTHASE: ELUCIDATION OF COMPLEX POST-TRANSLATIONAL REGULATORY MECHANISMS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steven C. Huber

2009-05-12

Studies have focused on the enzyme sucrose synthase, which plays an important role in the metabolism of sucrose in seeds and tubers. There are three isoforms of SUS in maize, referred to as SUS1, SUS-SH1, and SUS2. SUS is generally considered to be tetrameric protein but recent evidence suggests that SUS can also occur as a dimeric protein. The formation of tetrameric SUS is regulated by sucrose concentration in vitro and this could also be an important factor in the cellular localization of the protein. We found that high sucrose concentrations, which promote tetramer formation, also inhibit the binding ofmore » SUS1 to actin filaments in vitro. Previously, high sucrose concentrations were shown to promote SUS association with the plasma membrane. The specific regions of the SUS molecule involved in oligomerization are not known, but we identified a region of the SUS1 moelcule by bioinformatic analysis that was predicted to form a coiled coil. We demonstrated that this sequence could, in fact, self-associate as predicted for a coiled coil, but truncation analysis with the full-length recombinant protein suggested that it was not responsible for formation of dimers or tetramers. However, the coiled coil may function in binding of other proteins to SUS1. Overall, sugar availability may differentially influence the binding of SUS to cellular structures, and these effects may be mediated by changes in the oligomeric nature of the enzyme.« less

Coexistence of Phases in a Protein Heterodimer

PubMed Central

Krokhotin, Andrey; Liwo, Adam; Niemi, Antti J.; Scheraga, Harold A.

2012-01-01

A heterodimer consisting of two or more different kinds of proteins can display an enormous number of distinct molecular architectures. The conformational entropy is an essential ingredient in the Helmholtz free energy and, consequently, these heterodimers can have a very complex phase structure. Here, it is proposed that there is a state of proteins, in which the different components of a heterodimer exist in different phases. For this purpose, the structures in the protein data bank (PDB) have been analyzed, with radius of gyration as the order parameter. Two major classes of heterodimers with their protein components coexisting in different phases have been identified. An example is the PDB structure 3DXC. This is a transcriptionally active dimer. One of the components is an isoform of the intra-cellular domain of the Alzheimer-disease related amyloid precursor protein (AICD), and the other is a nuclear multidomain adaptor protein in the Fe65 family. It is concluded from the radius of gyration that neither of the two components in this dimer is in its own collapsed phase, corresponding to a biologically active protein. The UNRES energy function has been utilized to confirm that, if the two components are separated from each other, each of them collapses. The results presented in this work show that heterodimers whose protein components coexist in different phases, can have intriguing physical properties with potentially important biological consequences. PMID:22830730

Acetylation Suppresses the Proapoptotic Activity of GD3 Ganglioside

PubMed Central

Malisan, Florence; Franchi, Luigi; Tomassini, Barbara; Ventura, Natascia; Condò, Ivano; Rippo, Maria Rita; Rufini, Alessandra; Liberati, Laura; Nachtigall, Claudia; Kniep, Bernhard; Testi, Roberto

2002-01-01

GD3 synthase is rapidly activated in different cell types after specific apoptotic stimuli. De novo synthesized GD3 accumulates and contributes to the apoptotic program by relocating to mitochondrial membranes and inducing the release of apoptogenic factors. We found that sialic acid acetylation suppresses the proapoptotic activity of GD3. In fact, unlike GD3, 9-O-acetyl-GD3 is completely ineffective in inducing cytochrome c release and caspase-9 activation on isolated mitochondria and fails to induce the collapse of mitochondrial transmembrane potential and cellular apoptosis. Moreover, cells which are resistant to the overexpression of the GD3 synthase, actively convert de novo synthesized GD3 to 9-O-acetyl-GD3. The coexpression of GD3 synthase with a viral 9-O-acetyl esterase, which prevents 9-O-acetyl-GD3 accumulation, reconstitutes GD3 responsiveness and apoptosis. Finally, the expression of the 9-O-acetyl esterase is sufficient to induce apoptosis of glioblastomas which express high levels of 9-O-acetyl-GD3. Thus, sialic acid acetylation critically controls the proapoptotic activity of GD3. PMID:12486096

The role of the ATPase inhibitor factor 1 (IF1) in cancer cells adaptation to hypoxia and anoxia.

PubMed

Sgarbi, G; Barbato, S; Costanzini, A; Solaini, G; Baracca, A

2018-02-01

The physiological role of the mitochondrial ATP synthase complex is to generate ATP through oxidative phosphorylation. Indeed, the enzyme can reverse its activity and hydrolyze ATP under ischemic conditions, as shown in isolated mitochondria and in mammalian heart and liver. However, what occurs when cancer cells experience hypoxia or anoxia has not been well explored. In the present study, we investigated the bioenergetics of cancer cells under hypoxic/anoxic conditions with particular emphasis on ATP synthase, and the conditions driving it to work in reverse. In this context, we further examined the role exerted by its endogenous inhibitor factor, IF 1 , that it is overexpressed in cancer cells. Metabolic and bioenergetic analysis of cancer cells exposed to severe hypoxia (down to 0.1% O 2 ) unexpectedly showed that Δψ m is preserved independently of the presence of IF 1 and that ATP synthase still phosphorylates ADP though at a much lower rate than in normoxia. However, when we induced an anoxia-mimicking condition by collapsing Δμ Η + with the FCCP uncoupler, the IF 1 -silenced clones only reversed the ATP synthase activity hydrolyzing ATP in order to reconstitute the electrochemical proton gradient. Notably, in cancer cells IF 1 overexpression fully prevents ATP synthase hydrolytic activity activation under uncoupling conditions. Therefore, our results suggest that IF 1 overexpression promotes cancer cells survival under temporary anoxic conditions by preserving cellular ATP despite mitochondria dysfunction. Copyright © 2017 Elsevier B.V. All rights reserved.

Protein disulfide isomerase mediates glutathione depletion-induced cytotoxicity.

PubMed

Okada, Kazushi; Fukui, Masayuki; Zhu, Bao-Ting

2016-08-26

Glutathione depletion is a distinct cause underlying many forms of pathogenesis associated with oxidative stress and cytotoxicity. Earlier studies showed that glutamate-induced glutathione depletion in immortalized murine HT22 hippocampal neuronal cells leads to accumulation of reactive oxygen species (ROS) and ultimately cell death, but the precise mechanism underlying these processes is not clear. Here we show that during the induction of glutathione depletion, nitric oxide (NO) accumulation precedes ROS accumulation. While neuronal NO synthase (nNOS) in untreated HT22 cells exists mostly as a monomer, glutathione depletion results in increased formation of the dimer nNOS, accompanied by increases in the catalytic activity. We identified that nNOS dimerization is catalyzed by protein disulfide isomerase (PDI). Inhibition of PDI's isomerase activity effectively abrogates glutathione depletion-induced conversion of monomer nNOS into dimer nNOS, accumulation of NO and ROS, and cytotoxicity. Furthermore, we found that PDI is present in untreated cells in an inactive S-nitrosylated form, which becomes activated following glutathione depletion via S-denitrosylation. These results reveal a novel role for PDI in mediating glutathione depletion-induced oxidative cytotoxicity, as well as its role as a valuable therapeutic target for protection against oxidative cytotoxicity. Copyright © 2016. Published by Elsevier Inc.

The crystal structure of N-acetyl-L-glutamate synthase from Neisseria gonorrhoeae provides insights into mechanisms of catalysis and regulation.

PubMed

Shi, Dashuang; Sagar, Vatsala; Jin, Zhongmin; Yu, Xiaolin; Caldovic, Ljubica; Morizono, Hiroki; Allewell, Norma M; Tuchman, Mendel

2008-03-14

The crystal structures of N-acetylglutamate synthase (NAGS) in the arginine biosynthetic pathway of Neisseria gonorrhoeae complexed with acetyl-CoA and with CoA plus N-acetylglutamate have been determined at 2.5- and 2.6-A resolution, respectively. The monomer consists of two separately folded domains, an amino acid kinase (AAK) domain and an N-acetyltransferase (NAT) domain connected through a 10-A linker. The monomers assemble into a hexameric ring that consists of a trimer of dimers with 32-point symmetry, inner and outer ring diameters of 20 and 100A, respectively, and a height of 110A(.) Each AAK domain interacts with the cognate domains of two adjacent monomers across two 2-fold symmetry axes and with the NAT domain from a second monomer of the adjacent dimer in the ring. The catalytic sites are located within the NAT domains. Three active site residues, Arg316, Arg425, and Ser427, anchor N-acetylglutamate in a position at the active site to form hydrogen bond interactions to the main chain nitrogen atoms of Cys356 and Leu314, and hydrophobic interactions to the side chains of Leu313 and Leu314. The mode of binding of acetyl-CoA and CoA is similar to other NAT family proteins. The AAK domain, although catalytically inactive, appears to bind arginine. This is the first reported crystal structure of any NAGS, and it provides insights into the catalytic function and arginine regulation of NAGS enzymes.

The Crystal Structure of N-Acetyl-L-glutamate Synthase from Neisseria gonorrhoeae Provides Insights into Mechanisms of Catalysis and Regulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Dashuang; Sagar, Vatsala; Jin, Zhongmin

2010-01-07

The crystal structures of N-acetylglutamate synthase (NAGS) in the arginine biosynthetic pathway of Neisseria gonorrhoeae complexed with acetyl-CoA and with CoA plus N-acetylglutamate have been determined at 2.5- and 2.6-A resolution, respectively. The monomer consists of two separately folded domains, an amino acid kinase (AAK) domain and an N-acetyltransferase (NAT) domain connected through a 10-A linker. The monomers assemble into a hexameric ring that consists of a trimer of dimers with 32-point symmetry, inner and outer ring diameters of 20 and 100A, respectively, and a height of 110A(.) Each AAK domain interacts with the cognate domains of two adjacent monomersmore » across two 2-fold symmetry axes and with the NAT domain from a second monomer of the adjacent dimer in the ring. The catalytic sites are located within the NAT domains. Three active site residues, Arg316, Arg425, and Ser427, anchor N-acetylglutamate in a position at the active site to form hydrogen bond interactions to the main chain nitrogen atoms of Cys356 and Leu314, and hydrophobic interactions to the side chains of Leu313 and Leu314. The mode of binding of acetyl-CoA and CoA is similar to other NAT family proteins. The AAK domain, although catalytically inactive, appears to bind arginine. This is the first reported crystal structure of any NAGS, and it provides insights into the catalytic function and arginine regulation of NAGS enzymes.« less

Energy exchange dynamics of the discrete nonlinear Schrödinger equation lattice and intrinsic formation of strongly localized states

NASA Astrophysics Data System (ADS)

Hennig, D.

1997-09-01

We study the dynamics of excitation energy transfer along a lattice chain modeled by the discrete nonlinear Schrödinger (DNLS) equation. We prove that a segment carrying resonant motion can be decoupled from the remainder of the chain supporting quasiperiodic dynamics. The resonant segment from the extended chain is taken to be a four-site element, viz., a tetramer. First, we focus interest on the energy exchange dynamics along the tetramer viewed as two weakly coupled DNLS dimers. Hamiltonian methods are used to investigate the phase-space dynamics. We pay special attention to the role of the diffusion of the action variables inside resonance layers for the energy migration. When distributing the energy initially equally between the two dimers one observes a directed irreversible flow of energy from one dimer into the other assisted by action diffusion. Eventually on one dimer a stable self-trapped excitation of large amplitude forms at a single site while the other dimer exhibits equal energy partition over its two sites. Finally, we study the formation of localized structure on an extended DNLS lattice chain. In particular we explore the stability of the so-called even-parity and odd-parity localized modes, respectively, and explain their different stability properties by means of phase-space dynamics. The global instability of the even-parity mode is shown. For the excited even-parity mode a symmetry-breaking perturbation of the pattern leads to an intrinsic collapse of the even-parity mode to the odd-parity one. The latter remains stable with respect to symmetry-breaking perturbations. In this way we demonstrate that the favored stable localized states for the DNLS lattice chain correspond to one-site localized excitations.

Dissecting the dynamic conformations of the metamorphic protein lymphotactin.

PubMed

Harvey, Sophie R; Porrini, Massimiliano; Konijnenberg, Albert; Clarke, David J; Tyler, Robert C; Langridge-Smith, Patrick R R; MacPhee, Cait E; Volkman, Brian F; Barran, Perdita E

2014-10-30

A mass spectrometer provides an ideal laboratory to probe the structure and stability of isolated protein ions. Interrogation of each discrete mass/charge-separated species enables the determination of the intrinsic stability of a protein fold, gaining snapshots of unfolding pathways. In solution, the metamorphic protein lymphotactin (Ltn) exists in equilibrium between two distinct conformations, a monomeric (Ltn10) and a dimeric (Ltn40) fold. Here, we use electron capture dissociation (ECD) and drift tube ion mobility-mass spectrometry (DT IM-MS) to analyze both forms and use molecular dynamics (MD) to consider how the solution fold alters in a solvent-free environment. DT IM-MS reveals significant conformational flexibility for the monomer, while the dimer appears more conformationally restricted. These findings are supported by MD calculations, which reveal how salt bridges stabilize the conformers in vacuo. Following ECD experiments, a distinctive fragmentation pattern is obtained for both the monomer and dimer. Monomer fragmentation becomes more pronounced with increasing charge state especially in the disordered regions and C-terminal α-helix in the solution fold. Lower levels of fragmentation are seen in the β-sheet regions and in regions that contain salt bridges, identified by MD simulations. The lowest charge state of the dimer for which we obtain ECD data ([D+9H](9+)) exhibits extensive fragmentation with no relationship to the solution fold and has a smaller collision cross section (CCS) than charge states 10-13+, suggesting a "collapsed" encounter complex. Other charge states of the dimer, as for the monomer, are resistant to fragmentation in regions of β-sheets in the solution fold. This study provides evidence for preservation and loss of global fold and secondary structural elements, providing a tantalizing glimpse into the power of the emerging field of native top-down mass spectrometry.

Structure of the Mitochondrial Aminolevulinic Acid Synthase, a Key Heme Biosynthetic Enzyme.

PubMed

Brown, Breann L; Kardon, Julia R; Sauer, Robert T; Baker, Tania A

2018-04-03

5-Aminolevulinic acid synthase (ALAS) catalyzes the first step in heme biosynthesis. We present the crystal structure of a eukaryotic ALAS from Saccharomyces cerevisiae. In this homodimeric structure, one ALAS subunit contains covalently bound cofactor, pyridoxal 5'-phosphate (PLP), whereas the second is PLP free. Comparison between the subunits reveals PLP-coupled reordering of the active site and of additional regions to achieve the active conformation of the enzyme. The eukaryotic C-terminal extension, a region altered in multiple human disease alleles, wraps around the dimer and contacts active-site-proximal residues. Mutational analysis demonstrates that this C-terminal region that engages the active site is important for ALAS activity. Our discovery of structural elements that change conformation upon PLP binding and of direct contact between the C-terminal extension and the active site thus provides a structural basis for investigation of disruptions in the first step of heme biosynthesis and resulting human disorders. Copyright © 2018 Elsevier Ltd. All rights reserved.

The C2'- and C3'-endo equilibrium for AMP molecules bound in the cystathionine-beta-synthase domain.

PubMed

Feng, Na; Qi, Chao; Hou, Yan-Jie; Zhang, Ying; Wang, Da-Cheng; Li, De-Feng

2018-03-04

The equilibrium between C2'- and C3'-endo conformations of nucleotides in solution, as well as their polymers DNA and RNA, has been well studied in previous work. However, this equilibrium of nucleotides in their binding state remains unclear. We observed two AMP molecules, in C3'- and C2'-endo conformations respectively, simultaneously bound to a cystathionine-beta-synthase (CBS) domain dimer of the magnesium and cobalt efflux protein CorC in the crystallographic study. The C2'-endo AMP molecule assumes the higher sugar pucker energy and one more hydrogen bond with the protein than the C3'-endo molecule does. The balance between the high sugar pucker energy and the low binding energy suggests an equilibrium or switch between C2'- and C3'-endo conformations of the bound nucleotides. Our work challenge the previous hypothesis that the ribose of the bound nucleotides would be locked in a fixed conformation. Copyright © 2018 Elsevier Inc. All rights reserved.

Cobalamin in inflammation III — glutathionylcobalamin and methylcobalamin/adenosylcobalamin coenzymes: the sword in the stone? How cobalamin may directly regulate the nitric oxide synthases

PubMed Central

Wheatley, Carmen

2007-01-01

Several mysteries surround the structure and function of the nitric oxide synthases (NOS). The NOS oxygenase domain structure is unusually open with a large area of solvent that could accommodate an unidentified ligand. The exact mechanism of the two-step five-electron monoxygenation of arginine to NG-hydroxy-L-arginine, thence to citrulline and nitric oxide (NO), is not clear, particularly as arginine/NG-hydroxy-L-arginine is bound at a great distance to the supposed catalytic heme Fe [III], as the anti-stereoisomer. The Return of the Scarlet Pimpernel Paper proposed that cobalamin is a primary indirect regulator of the NOS. An additional direct regulatory effect of the ‘base-off’ dimethylbenzimidazole of glutathionylcobalamin (GSCbl), which may act as a sixth ligand to the heme iron, promote Co-oriented, BH4/BH3 radical catalysed oxidation of L-arginine to NO, and possibly regulate the rate of inducible NOS/NO production by the NOS dimers, is further advanced. The absence of homology between the NOS and methionine synthase/methylmalonyl CoA mutase may enable GSCbl to regulate both sets of enzymes simultaneously by completely separate mechanisms. Thus, cobalamin may exert central control over both pro-and anti-inflammatory systems. PMID:18923642

The 1-aminocyclopropane-1-carboxylate synthase of Cucurbita. Purification, properties, expression in Escherichia coli, and primary structure determination by DNA sequence analysis.

PubMed

Sato, T; Oeller, P W; Theologis, A

1991-02-25

The key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene is 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.4.1.14). We have partially purified ACC synthase 6,000-fold from Cucurbita fruit tissue treated with indoleacetic acid + benzyladenine + aminooxyacetic acid + LiCl. The enzyme has a specific activity of 35,000 nmol/h/mg protein, a pH optimum of 9.5, an isoelectric point of 5.0, a Km of 17 microM with respect to S-adenosylmethionine, and is a dimer of two identical subunits of approximately 46,000 Da each. The subunit exists in vivo as a 55,000-Da species similar in size to the primary in vitro translation product. DNA sequence analysis of the cDNA clone pACC1 revealed that the coding region of the ACC synthase mRNA spans 493 amino acids corresponding to a 55,779-Da polypeptide; and expression of the coding sequence (pACC1) in Escherichia coli as a COOH terminus hybrid of beta-galactosidase or as a nonhybrid polypeptide catalyzed the conversion of S-adenosylmethionine to ACC (Sato, T., and Theologis, A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6621-6625). Immunoblotting experiments herein show that the molecular mass of the beta-galactosidase hybrid polypeptide is 170,000 Da, and the size of the largest nonhybrid polypeptide is 53,000 Da. The data suggest that the enzyme is post-translationally processed during protein purification.

Insight on an Arginine Synthesis Metabolon from the Tetrameric Structure of Yeast Acetylglutamate Kinase

PubMed Central

de Cima, Sergio; Gil-Ortiz, Fernando; Crabeel, Marjolaine; Fita, Ignacio; Rubio, Vicente

2012-01-01

N-acetyl-L-glutamate kinase (NAGK) catalyzes the second, generally controlling, step of arginine biosynthesis. In yeasts, NAGK exists either alone or forming a metabolon with N-acetyl-L-glutamate synthase (NAGS), which catalyzes the first step and exists only within the metabolon. Yeast NAGK (yNAGK) has, in addition to the amino acid kinase (AAK) domain found in other NAGKs, a ∼150-residue C-terminal domain of unclear significance belonging to the DUF619 domain family. We deleted this domain, proving that it stabilizes yNAGK, slows catalysis and modulates feed-back inhibition by arginine. We determined the crystal structures of both the DUF619 domain-lacking yNAGK, ligand-free as well as complexed with acetylglutamate or acetylglutamate and arginine, and of complete mature yNAGK. While all other known arginine-inhibitable NAGKs are doughnut-like hexameric trimers of dimers of AAK domains, yNAGK has as central structure a flat tetramer formed by two dimers of AAK domains. These dimers differ from canonical AAK dimers in the −110° rotation of one subunit with respect to the other. In the hexameric enzymes, an N-terminal extension, found in all arginine-inhibitable NAGKs, forms a protruding helix that interlaces the dimers. In yNAGK, however, it conforms a two-helix platform that mediates interdimeric interactions. Arginine appears to freeze an open inactive AAK domain conformation. In the complete yNAGK structure, two pairs of DUF619 domains flank the AAK domain tetramer, providing a mechanism for the DUF619 domain modulatory functions. The DUF619 domain exhibits the histone acetyltransferase fold, resembling the catalytic domain of bacterial NAGS. However, the putative acetyl CoA site is blocked, explaining the lack of NAGS activity of yNAGK. We conclude that the tetrameric architecture is an adaptation to metabolon formation and propose an organization for this metabolon, suggesting that yNAGK may be a good model also for yeast and human NAGSs. PMID:22529931

Insight on an arginine synthesis metabolon from the tetrameric structure of yeast acetylglutamate kinase.

PubMed

de Cima, Sergio; Gil-Ortiz, Fernando; Crabeel, Marjolaine; Fita, Ignacio; Rubio, Vicente

2012-01-01

N-acetyl-L-glutamate kinase (NAGK) catalyzes the second, generally controlling, step of arginine biosynthesis. In yeasts, NAGK exists either alone or forming a metabolon with N-acetyl-L-glutamate synthase (NAGS), which catalyzes the first step and exists only within the metabolon. Yeast NAGK (yNAGK) has, in addition to the amino acid kinase (AAK) domain found in other NAGKs, a ~150-residue C-terminal domain of unclear significance belonging to the DUF619 domain family. We deleted this domain, proving that it stabilizes yNAGK, slows catalysis and modulates feed-back inhibition by arginine. We determined the crystal structures of both the DUF619 domain-lacking yNAGK, ligand-free as well as complexed with acetylglutamate or acetylglutamate and arginine, and of complete mature yNAGK. While all other known arginine-inhibitable NAGKs are doughnut-like hexameric trimers of dimers of AAK domains, yNAGK has as central structure a flat tetramer formed by two dimers of AAK domains. These dimers differ from canonical AAK dimers in the -110° rotation of one subunit with respect to the other. In the hexameric enzymes, an N-terminal extension, found in all arginine-inhibitable NAGKs, forms a protruding helix that interlaces the dimers. In yNAGK, however, it conforms a two-helix platform that mediates interdimeric interactions. Arginine appears to freeze an open inactive AAK domain conformation. In the complete yNAGK structure, two pairs of DUF619 domains flank the AAK domain tetramer, providing a mechanism for the DUF619 domain modulatory functions. The DUF619 domain exhibits the histone acetyltransferase fold, resembling the catalytic domain of bacterial NAGS. However, the putative acetyl CoA site is blocked, explaining the lack of NAGS activity of yNAGK. We conclude that the tetrameric architecture is an adaptation to metabolon formation and propose an organization for this metabolon, suggesting that yNAGK may be a good model also for yeast and human NAGSs.

Structural insight into arginine methylation by the mouse protein arginine methyltransferase 7: a zinc finger freezes the mimic of the dimeric state into a single active site.

PubMed

Cura, Vincent; Troffer-Charlier, Nathalie; Wurtz, Jean Marie; Bonnefond, Luc; Cavarelli, Jean

2014-09-01

Protein arginine methyltransferase 7 (PRMT7) is a type III arginine methyltransferase which has been implicated in several biological processes such as transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation and metastasis. PRMT7 is a unique but less characterized member of the family of PRMTs. The crystal structure of full-length PRMT7 from Mus musculus refined at 1.7 Å resolution is described. The PRMT7 structure is composed of two catalytic modules in tandem forming a pseudo-dimer and contains only one AdoHcy molecule bound to the N-terminal module. The high-resolution crystal structure presented here revealed several structural features showing that the second active site is frozen in an inactive state by a conserved zinc finger located at the junction between the two PRMT modules and by the collapse of two degenerated AdoMet-binding loops.

Arginase Inhibition Restores Peroxynitrite-Induced Endothelial Dysfunction via L-Arginine-Dependent Endothelial Nitric Oxide Synthase Phosphorylation

PubMed Central

Nguyen, Minh Cong; Park, Jong Taek; Jeon, Yeong Gwan; Jeon, Byeong Hwa; Hoe, Kwang Lae; Kim, Young Myeong

2016-01-01

Purpose Peroxynitrite plays a critical role in vascular pathophysiology by increasing arginase activity and decreasing endothelial nitric oxide synthase (eNOS) activity. Therefore, the aims of this study were to investigate whether arginase inhibition and L-arginine supplement could restore peroxynitrite-induced endothelial dysfunction and determine the involved mechanism. Materials and Methods Human umbilical vein endothelial cells (HUVECs) were treated with SIN-1, a peroxynitrite generator, and arginase activity, nitrite/nitrate production, and expression levels of proteins were measured. eNOS activation was evaluated via Western blot and dimer blot analysis. We also tested nitric oxide (NO) and reactive oxygen species (ROS) production and performed a vascular tension assay. Results SIN-1 treatment increased arginase activity in a time- and dose-dependent manner and reciprocally decreased nitrite/nitrate production that was prevented by peroxynitrite scavenger in HUVECs. Furthermore, SIN-1 induced an increase in the expression level of arginase I and II, though not in eNOS protein. The decreased eNOS phosphorylation at Ser1177 and the increased at Thr495 by SIN-1 were restored with arginase inhibitor and L-arginine. The changed eNOS phosphorylation was consistent in the stability of eNOS dimers. SIN-1 decreased NO production and increased ROS generation in the aortic endothelium, all of which was reversed by arginase inhibitor or L-arginine. NG-Nitro-L-arginine methyl ester (L-NAME) prevented SIN-1-induced ROS generation. In the vascular tension assay, SIN-1 enhanced vasoconstrictor responses to U46619 and attenuated vasorelaxant responses to acetylcholine that were reversed by arginase inhibition. Conclusion These findings may explain the beneficial effect of arginase inhibition and L-arginine supplement on endothelial dysfunction under redox imbalance-dependent pathophysiological conditions. PMID:27593859

Arginase Inhibition Restores Peroxynitrite-Induced Endothelial Dysfunction via L-Arginine-Dependent Endothelial Nitric Oxide Synthase Phosphorylation.

PubMed

Nguyen, Minh Cong; Park, Jong Taek; Jeon, Yeong Gwan; Jeon, Byeong Hwa; Hoe, Kwang Lae; Kim, Young Myeong; Lim, Hyun Kyo; Ryoo, Sungwoo

2016-11-01

Peroxynitrite plays a critical role in vascular pathophysiology by increasing arginase activity and decreasing endothelial nitric oxide synthase (eNOS) activity. Therefore, the aims of this study were to investigate whether arginase inhibition and L-arginine supplement could restore peroxynitrite-induced endothelial dysfunction and determine the involved mechanism. Human umbilical vein endothelial cells (HUVECs) were treated with SIN-1, a peroxynitrite generator, and arginase activity, nitrite/nitrate production, and expression levels of proteins were measured. eNOS activation was evaluated via Western blot and dimer blot analysis. We also tested nitric oxide (NO) and reactive oxygen species (ROS) production and performed a vascular tension assay. SIN-1 treatment increased arginase activity in a time- and dose-dependent manner and reciprocally decreased nitrite/nitrate production that was prevented by peroxynitrite scavenger in HUVECs. Furthermore, SIN-1 induced an increase in the expression level of arginase I and II, though not in eNOS protein. The decreased eNOS phosphorylation at Ser1177 and the increased at Thr495 by SIN-1 were restored with arginase inhibitor and L-arginine. The changed eNOS phosphorylation was consistent in the stability of eNOS dimers. SIN-1 decreased NO production and increased ROS generation in the aortic endothelium, all of which was reversed by arginase inhibitor or L-arginine. N(G)-Nitro-L-arginine methyl ester (L-NAME) prevented SIN-1-induced ROS generation. In the vascular tension assay, SIN-1 enhanced vasoconstrictor responses to U46619 and attenuated vasorelaxant responses to acetylcholine that were reversed by arginase inhibition. These findings may explain the beneficial effect of arginase inhibition and L-arginine supplement on endothelial dysfunction under redox imbalance-dependent pathophysiological conditions.

Feast/famine regulatory proteins (FFRPs): Escherichia coli Lrp, AsnC and related archaeal transcription factors.

PubMed

Yokoyama, Katsushi; Ishijima, Sanae A; Clowney, Lester; Koike, Hideaki; Aramaki, Hironori; Tanaka, Chikako; Makino, Kozo; Suzuki, Masashi

2006-01-01

Feast/famine regulatory proteins comprise a diverse family of transcription factors, which have been referred to in various individual identifications, including Escherichia coli leucine-responsive regulatory protein and asparagine synthase C gene product. A full length feast/famine regulatory protein consists of the N-terminal DNA-binding domain and the C-domain, which is involved in dimerization and further assembly, thereby producing, for example, a disc or a chromatin-like cylinder. Various ligands of the size of amino acids bind at the interface between feast/famine regulatory protein dimers, thereby altering their assembly forms. Also, the combination of feast/famine regulatory protein subunits forming the same assembly is altered. In this way, a small number of feast/famine regulatory proteins are able to regulate a large number of genes in response to various environmental changes. Because feast/famine regulatory proteins are shared by archaea and eubacteria, the genome-wide regulation by feast/famine regulatory proteins is traceable back to their common ancestor, being the prototype of highly differentiated transcription regulatory mechanisms found in organisms nowadays.

Seed-specific overexpression of an endogenous Arabidopsis phytoene synthase gene results in delayed germination and increased levels of carotenoids, chlorophyll, and abscisic acid.

PubMed

Lindgren, L Ove; Stålberg, Kjell G; Höglund, Anna-Stina

2003-06-01

Phytoene synthase catalyzes the dimerization of two molecules of geranylgeranyl pyrophosphate to phytoene and has been shown to be rate limiting for the synthesis of carotenoids. To elucidate if the capacity to produce phytoene is limiting also in the seed of Arabidopsis (Wassilewskija), a gene coding for an endogenous phytoene synthase was cloned and coupled to a seed-specific promoter, and the effects of the overexpression were examined. The resulting transgenic plants produced darker seeds, and extracts from the seed of five overexpressing plants had a 43-fold average increase of beta-carotene and a total average amount of beta-carotene of approximately 260 microg g-1 fresh weight. Lutein, violaxanthin, and chlorophyll were significantly increased, whereas the levels of zeaxanthin only increased by a factor 1.1. In addition, substantial levels of lycopene and alpha-carotene were produced in the seeds, whereas only trace amounts were found in the control plants. Seeds from the transgenic plants exhibited delayed germination, and the degree of delay was positively correlated with the increased levels of carotenoids. The abscisic acid levels followed the increase of the carotenoids, and plants having the highest carotenoid levels also had the highest abscisic acid content. Addition of gibberellic acid to the growth medium only partly restored germination of the transgenic seeds.

Chaperone activity of human small heat shock protein-GST fusion proteins.

PubMed

Arbach, Hannah; Butler, Caley; McMenimen, Kathryn A

2017-07-01

Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST fusion proteins with MDH and CS, is modulated by both sHsp oligomeric conformation and by variations of sHsp sequences.

DOE Office of Scientific and Technical Information (OSTI.GOV)

French, Jarrod B.; Yates, Phillip A.; Soysa, D.Radika

The final two steps of de novo uridine 5'-monophosphate (UMP) biosynthesis are catalyzed by orotate phosphoribosyltransferase (OPRT) and orotidine 5'-monophosphate decarboxylase (OMPDC). In most prokaryotes and simple eukaryotes these two enzymes are encoded by separate genes, whereas in mammals they are expressed as a bifunctional gene product called UMP synthase (UMPS), with OPRT at the N terminus and OMPDC at the C terminus. Leishmania and some closely related organisms also express a bifunctional enzyme for these two steps, but the domain order is reversed relative to mammalian UMPS. In this work we demonstrate that L. donovani UMPS (LdUMPS) is anmore » essential enzyme in promastigotes and that it is sequestered in the parasite glycosome. We also present the crystal structure of the LdUMPS in complex with its product, UMP. This structure reveals an unusual tetramer with two head to head and two tail to tail interactions, resulting in two dimeric OMPDC and two dimeric OPRT functional domains. In addition, we provide structural and biochemical evidence that oligomerization of LdUMPS is controlled by product binding at the OPRT active site. We propose a model for the assembly of the catalytically relevant LdUMPS tetramer and discuss the implications for the structure of mammalian UMPS.« less

Evaporation of Lennard-Jones fluids.

PubMed

Cheng, Shengfeng; Lechman, Jeremy B; Plimpton, Steven J; Grest, Gary S

2011-06-14

Evaporation and condensation at a liquid/vapor interface are ubiquitous interphase mass and energy transfer phenomena that are still not well understood. We have carried out large scale molecular dynamics simulations of Lennard-Jones (LJ) fluids composed of monomers, dimers, or trimers to investigate these processes with molecular detail. For LJ monomers in contact with a vacuum, the evaporation rate is found to be very high with significant evaporative cooling and an accompanying density gradient in the liquid domain near the liquid/vapor interface. Increasing the chain length to just dimers significantly reduces the evaporation rate. We confirm that mechanical equilibrium plays a key role in determining the evaporation rate and the density and temperature profiles across the liquid/vapor interface. The velocity distributions of evaporated molecules and the evaporation and condensation coefficients are measured and compared to the predictions of an existing model based on kinetic theory of gases. Our results indicate that for both monatomic and polyatomic molecules, the evaporation and condensation coefficients are equal when systems are not far from equilibrium and smaller than one, and decrease with increasing temperature. For the same reduced temperature T/T(c), where T(c) is the critical temperature, these two coefficients are higher for LJ dimers and trimers than for monomers, in contrast to the traditional viewpoint that they are close to unity for monatomic molecules and decrease for polyatomic molecules. Furthermore, data for the two coefficients collapse onto a master curve when plotted against a translational length ratio between the liquid and vapor phase.

Role of CBS and Bateman Domains in Phosphorylation-Dependent Regulation of a CLC Anion Channel.

PubMed

Yamada, Toshiki; Krzeminski, Mickael; Bozoky, Zoltan; Forman-Kay, Julie D; Strange, Kevin

2016-11-01

Eukaryotic CLC anion channels and transporters are homodimeric proteins composed of multiple α-helical membrane domains and large cytoplasmic C-termini containing two cystathionine-β-synthase domains (CBS1 and CBS2) that dimerize to form a Bateman domain. The Bateman domains of adjacent CLC subunits interact to form a Bateman domain dimer. The functions of CLC CBS and Bateman domains are poorly understood. We utilized the Caenorhabditis elegans CLC-1/2/Ka/Kb anion channel homolog CLH-3b to characterize the regulatory roles of CLC cytoplasmic domains. CLH-3b activity is reduced by phosphorylation or deletion of a 14-amino-acid activation domain (AD) located on the linker connecting CBS1 and CBS2. We demonstrate here that phosphorylation-dependent reductions in channel activity require an intact Bateman domain dimer and concomitant phosphorylation or deletion of both ADs. Regulation of a CLH-3b AD deletion mutant is reconstituted by intracellular perfusion with recombinant 14-amino-acid AD peptides. The sulfhydryl reactive reagent 2-(trimethylammonium)ethyl methanethiosulfonate bromide (MTSET) alters in a phosphorylation-dependent manner the activity of channels containing single cysteine residues that are engineered into the short intracellular loop connecting membrane α-helices H and I (H-I loop), the AD, CBS1, and CBS2. In contrast, MTSET has no effect on channels in which cysteine residues are engineered into intracellular regions that are dispensable for regulation. These studies together with our previous work suggest that binding and unbinding of the AD to the Bateman domain dimer induces conformational changes that are transduced to channel membrane domains via the H-I loop. Our findings provide new, to our knowledge, insights into the roles of CLC Bateman domains and the structure-function relationships that govern the regulation of CLC protein activity by diverse ligands and signaling pathways. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

An insertion in the methyltransferase domain of P. falciparum trimethylguanosine synthase harbors a classical nuclear localization signal.

PubMed

Babar, Prasad H; Dey, Vishakha; Jaiswar, Praveen; Patankar, Swati

Many Plasmodium falciparum proteins do not share homology with, and are generally longer than their respective orthologs. This, to some extent, can be attributed to insertions. Here, we studied a P. falciparum RNA hypermethylase, trimethylguanosine synthase (PfTGS1) that harbors a 76 amino acid insertion in its methyltransferase domain. Bioinformatics analysis revealed that this insertion was present in TGS1 orthologs from other Plasmodium species as well. Interestingly, a classical nuclear localization signal (NLS) was predicted in the insertions of primate parasite TGS1 proteins. To check whether these predicted NLS are functional, we developed an in vivo heterologous system using S. cerevisiae. The predicted NLS when fused to dimeric GFP were able to localize the fusion protein to the nucleus in yeast indicating that it is indeed recognized by the yeast nuclear import machinery. We further showed that the PfTGS1 NLS binds to P. falciparum importin-α in vitro, confirming that the NLS is also recognized by the P. falciparum classical nuclear import machinery. Thus, in this study we report a novel function of the insertion in PfTGS1. Copyright © 2016 Elsevier B.V. All rights reserved.

Structural and Kinetic Properties of Lumazine Synthase Isoenzymes in the Order Rhizobiales

DOE Office of Scientific and Technical Information (OSTI.GOV)

Klinke,S.; Zylberman, V.; Bonomi, H.

6, 7-Dimethyl-8-ribityllumazine synthase (lumazine synthase; LS) catalyzes the penultimate step in the biosynthesis of riboflavin in plants and microorganisms. This protein is known to exhibit different quaternary assemblies between species, existing as free pentamers, decamers (dimers of pentamers) and icosahedrally arranged dodecamers of pentamers. A phylogenetic analysis on eubacterial, fungal and plant LSs allowed us to classify them into two categories: Type I LSs (pentameric or icosahedral) and Type II LSs (decameric). The Rhizobiales represent an order of ?-proteobacteria that includes, among others, the genera Mesorhizobium, Agrobacterium and Brucella. Here, we present structural and kinetic studies on several LSs frommore » Rhizobiales. Interestingly, Mesorhizobium and Brucella encode both a Type-I LS and a Type-II LS called RibH1 and RibH2, respectively. We show that Type II LSs appear to be almost inactive, whereas Type I LSs present a highly variable catalytic activity according to the genus. Additionally, we have solved four RibH1/RibH2 crystallographic structures from the genera Mesorhizobium and Brucella. The relationship between the active-site architecture and catalytic properties in these isoenzymes is discussed, and a model that describes the enzymatic behavior is proposed. Furthermore, sequence alignment studies allowed us to extend our results to the genus Agrobacterium. Our results suggest that the selective pressure controlling the riboflavin pathway favored the evolution of catalysts with low reaction rates, since the excess of flavins in the intracellular pool in Rhizobiales could act as a negative factor when these bacteria are exposed to oxidative or nitrosative stress.« less

An active site–tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murphy, Jesse R.; Donini, Stefano; Kappock, T. Joseph, E-mail: kappock@purdue.edu

2015-09-23

Citrate synthase from the thermophilic euryarchaeon T. acidophilum fused to a hexahistidine tag was purified and biochemically characterized. The structure of the unliganded enzyme at 2.2 Å resolution contains tail–active site contacts in half of the active sites. Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that ‘close’ the active site play an important role in the assembly of a catalytically competent condensation active site. CS from themore » thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an ‘open’ structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand, the active site–tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS.« less

Placental Vesicles Carry Active Endothelial Nitric Oxide Synthase and Their Activity is Reduced in Preeclampsia.

PubMed

Motta-Mejia, Carolina; Kandzija, Neva; Zhang, Wei; Mhlomi, Vuyane; Cerdeira, Ana Sofia; Burdujan, Alexandra; Tannetta, Dionne; Dragovic, Rebecca; Sargent, Ian L; Redman, Christopher W; Kishore, Uday; Vatish, Manu

2017-08-01

Preeclampsia, a multisystem hypertensive disorder of pregnancy, is associated with increased systemic vascular resistance. Placentae from patients with preeclampsia have reduced levels of endothelial nitric oxide synthase (eNOS) and, thus, less nitric oxide (NO). Syncytiotrophoblast extracellular vesicles (STBEV), comprising microvesicles (STBMV) and exosomes, carry signals from the syncytiotrophoblast to the mother. We hypothesized that STBEV-bound eNOS (STBEV-eNOS), capable of producing NO, are released into the maternal circulation. Dual-lobe ex vivo placental perfusion and differential centrifugation was used to isolate STBEV from preeclampsia (n=8) and normal pregnancies (NP; n=11). Plasma samples of gestational age-matched preeclampsia and NP (n=6) were used to isolate circulating STBMV. STBEV expressed placental alkaline phosphatase, confirming placental origin. STBEV coexpressed eNOS, but not inducible nitric oxide synthase, confirmed using Western blot, flow cytometry, and immunodepletion. STBEV-eNOS produced NO, which was significantly inhibited by N   G -nitro-l-arginine methyl ester (eNOS inhibitor; P <0.05) but not by N -(3-(aminomethyl) bezyl) acetamidine) (inducible nitric oxide synthase inhibitor). STBEV-eNOS catalytic activity was confirmed by visualizing eNOS dimerization. STBEV-eNOS was more abundant in uterine vein compared with peripheral blood, indicating placental origin. STBEV isolated from preeclampsia-perfused placentae had lower levels of STBEV-eNOS (STBMV; P <0.05) and overall lower NO activity (STBMV, not significant; syncytiotrophoblast extracellular exosomes, P <0.05) compared with those from NP. Circulating plasma STBMV from preeclampsia women had lower STBEV-eNOS expression compared with that from NP women ( P <0.01). This is the first observation of functional eNOS expressed on STBEV from NP and preeclampsia placentae, as well as in plasma. The lower STBEV-eNOS NO production seen in preeclampsia may contribute to the decreased NO bioavailability in this disease. © 2017 The Authors.

ReaxFF molecular dynamics simulation of thermal stability of a Cu3(BTC)2 metal-organic framework.

PubMed

Huang, Liangliang; Joshi, Kaushik L; van Duin, Adri C T; Bandosz, Teresa J; Gubbins, Keith E

2012-08-28

The thermal stability of a dehydrated Cu(3)(BTC)(2) (copper(II) benzene 1,3,5-tricarboxylate) metal-organic framework was studied by molecular dynamics simulation with a ReaxFF reactive force field. The results show that Cu(3)(BTC)(2) is thermally stable up to 565 K. When the temperature increases between 600 K and 700 K, the framework starts to partially collapse. The RDF analysis shows that the long range correlations between Cu dimers disappear, indicating the loss of the main channels of Cu(3)(BTC)(2). When the temperature is above 800 K, we find the decomposition of the Cu(3)(BTC)(2) framework. CO is the major product, and we also observe the release of CO(2), O(2), 1,3,5-benzenetricarboxylate (C(6)H(3)(CO(2))(3), BTC) and glassy carbon. The Cu dimer is stable up to 1100 K, but we find the formation of new copper oxide clusters at 1100 K. These results are consistent with experimental findings, and provide valuable information for future theoretical investigations of Cu(3)(BTC)(2) and its application in adsorption, separation and catalytic processes.

Structure of the Plexin Ectodomain Bound by Semaphorin-Mimicking Antibodies

PubMed Central

Omiya, Ryusuke; Matoba, Kyoko; Baba, Takeshi; Suzuki, Sachiyo; Segawa, Hiroaki; Kumanogoh, Atsushi; Iwasaki, Kenji; Hattori, Kunihiro; Takagi, Junichi

2016-01-01

Semaphorin family proteins act on cells to mediate both repulsive and attractive guidance via binding to plexin family receptors, thereby playing fundamental roles in the morphogenesis and homeostasis of various tissues. Although semaphorin-plexin signaling is implicated in various diseases and is thus a target of intensive research, our mechanistic understanding of how semaphorins activate plexins on the cell surface is limited. Here, we describe unique anti-plexin-A1 antibodies that can induce a collapsed morphology in mouse dendritic cells as efficiently as the semaphorin 3A (Sema3A) ligand. Precise epitope analysis indicates that these “semaphorin-mimicking” antibodies dimerize cell-surface plexin-A1 by binding to the N-terminal sema domain of the plexin at sites away from the interface used by the Sema3A ligand. Structural analysis of plexin-A1 fragments using negative stain electron microscopy further revealed that this agonistic capacity is closely linked to the location and orientation of antibody binding. In addition, the full-length plexin-A1 ectodomain exhibited a highly curved “C” shape, reinforcing the very unusual dimeric receptor conformation of this protein at the cell surface when engaged with Sema3A or agonistic antibodies. PMID:27258772

Inherited STING-activating mutation underlies a familial inflammatory syndrome with lupus-like manifestations

PubMed Central

Jeremiah, Nadia; Neven, Bénédicte; Gentili, Matteo; Callebaut, Isabelle; Maschalidi, Sophia; Stolzenberg, Marie-Claude; Goudin, Nicolas; Frémond, Marie-Louis; Nitschke, Patrick; Molina, Thierry J.; Blanche, Stéphane; Picard, Capucine; Rice, Gillian I.; Crow, Yanick J.; Manel, Nicolas; Fischer, Alain; Bader-Meunier, Brigitte; Rieux-Laucat, Frédéric

2014-01-01

Innate immunity to viral infection involves induction of the type I IFN response; however, dysfunctional regulation of this pathway leads to inappropriate inflammation. Here, we evaluated a nonconsanguineous family of mixed European descent, with 4 members affected by systemic inflammatory and autoimmune conditions, including lupus, with variable clinical expression. We identified a germline dominant gain-of-function mutation in TMEM173, which encodes stimulator of type I IFN gene (STING), in the affected individuals. STING is a key signaling molecule in cytosolic DNA-sensing pathways, and STING activation normally requires dimerization, which is induced by 2′3′ cyclic GMP-AMP (cGAMP) produced by the cGAMP synthase in response to cytosolic DNA. Structural modeling supported constitutive activation of the mutant STING protein based on stabilized dimerization. In agreement with the model predictions, we found that the STING mutant spontaneously localizes in the Golgi of patient fibroblasts and is constitutively active in the absence of exogenous 2′3′-cGAMP in vitro. Accordingly, we observed elevated serum IFN activity and a type I IFN signature in peripheral blood from affected family members. These findings highlight the key role of STING in activating both the innate and adaptive immune responses and implicate aberrant STING activation in features of human lupus. PMID:25401470

An active site–tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase

PubMed Central

Murphy, Jesse R.; Donini, Stefano; Kappock, T. Joseph

2015-01-01

Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that ‘close’ the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an ‘open’ structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand, the active site–tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS. PMID:26457521

An active site-tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase

DOE PAGES

Murphy, Jesse R.; Donini, Stefano; Kappock, T. Joseph

2015-10-01

Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that `close' the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an `open'more » structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. In conclusion, as a polar but almost neutral ligand, the active site-tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS.« less

Structural analysis of a 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase with an N-terminal chorismate mutase-like regulatory domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Light, Samuel H.; Halavaty, Andrei S.; Minasov, George

2012-06-27

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) catalyzes the first step in the biosynthesis of a number of aromatic metabolites. Likely because this reaction is situated at a pivotal biosynthetic gateway, several DAHPS classes distinguished by distinct mechanisms of allosteric regulation have independently evolved. One class of DAHPSs contains a regulatory domain with sequence homology to chorismate mutase - an enzyme further downstream of DAHPS that catalyzes the first committed step in tyrosine/phenylalanine biosynthesis - and is inhibited by chorismate mutase substrate (chorismate) and product (prephenate). Described in this work, structures of the Listeria monocytogenes chorismate/prephenate regulated DAHPS in complex with Mn{sup 2+}more » and Mn{sup 2+} + phosphoenolpyruvate reveal an unusual quaternary architecture: DAHPS domains assemble as a tetramer, from either side of which chorismate mutase-like (CML) regulatory domains asymmetrically emerge to form a pair of dimers. This domain organization suggests that chorismate/prephenate binding promotes a stable interaction between the discrete regulatory and catalytic domains and supports a mechanism of allosteric inhibition similar to tyrosine/phenylalanine control of a related DAHPS class. We argue that the structural similarity of chorismate mutase enzyme and CML regulatory domain provides a unique opportunity for the design of a multitarget antibacterial.« less

An active site-tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase.

PubMed

Murphy, Jesse R; Donini, Stefano; Kappock, T Joseph

2015-10-01

Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that `close' the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an `open' structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand, the active site-tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS.

Redox Switch for the Inhibited State of Yeast Glycogen Synthase Mimics Regulation by Phosphorylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mahalingan, Krishna K.; Baskaran, Sulochanadevi; DePaoli-Roach, Anna A.

Glycogen synthase (GS) is the rate limiting enzyme in the synthesis of glycogen. Eukaryotic GS is negatively regulated by covalent phosphorylation and allosterically activated by glucose-6-phosphate (G-6-P). To gain structural insights into the inhibited state of the enzyme, we solved the crystal structure of yGsy2-R589A/R592A to a resolution of 3.3 Å. The double mutant has an activity ratio similar to the phosphorylated enzyme and also retains the ability to be activated by G-6-P. When compared to the 2.88 Å structure of the wild-type G-6-P activated enzyme, the crystal structure of the low-activity mutant showed that the N-terminal domain of themore » inhibited state is tightly held against the dimer-related interface thereby hindering acceptor access to the catalytic cleft. On the basis of these two structural observations, we developed a reversible redox regulatory feature in yeast GS by substituting cysteine residues for two highly conserved arginine residues. When oxidized, the cysteine mutant enzyme exhibits activity levels similar to the phosphorylated enzyme but cannot be activated by G-6-P. Upon reduction, the cysteine mutant enzyme regains normal activity levels and regulatory response to G-6-P activation.« less

A novel lumazine synthase molecule from Brucella significantly promotes the immune-stimulation effects of antigenic protein.

PubMed

Du, Z Q; Wang, J Y

2015-10-27

Brucella, an intracellular parasite that infects some livestock and humans, can damage or destroy the reproductive system of livestock. The syndrome is referred to as brucellosis and often occurs in pastoral areas; it is contagious from livestock to humans. In this study, the intact Brucella suis outer membrane protein 31 (omp31) gene was cloned, recombinantly expressed, and examined as a subunit vaccine candidate. The intact Brucella lumazine synthase (bls) gene was cloned and recombinantly expressed to study polymerization function in vitro. Non-reducing gel electrophoresis showed that rBs-BLS existed in different forms in vitro, including as a dimer and a pentamer. An enzyme-linked immunosorbent assay result showed that rOmp31 protein could induce production of an antibody in rabbits. However, the rOmp31-BLS fusion protein could elicit a much higher antibody titer in rabbits; this construct involved fusion of the Omp31 molecule with the BLS molecule. Our results indicate that Omp31 is involved in immune stimulation, while BLS has a polymerizing function based on rOmp31-BLS fusion protein immunogenicity. These data suggest that Omp31 is an ideal subunit vaccine candidate and that the BLS molecule is a favorable transport vector for antigenic proteins.

Post-translational Regulation of Neuronal Nitric Oxide Synthase: Implications for sympatho-excitatory states

PubMed Central

Sharma, Neeru M; Patel, Kaushik P

2017-01-01

Introduction Nitric oxide (NO) synthesized via neuronal nitric oxide synthase (nNOS) plays a significant role in regulation/modulation of autonomic control of circulation. Various pathological states are associated with diminished nNOS expression and blunted autonomic effects of NO in the central nervous system (CNS) including heart failure, hypertension, diabetes mellitus, chronic renal failure etc. Therefore, elucidation of the molecular mechanism/s involved in dysregulation of nNOS is essential to understand the pathogenesis of increased sympathoexcitation in these diseased states. Areas Covered nNOS is a highly regulated enzyme, being regulated at transcriptional and posttranslational levels via protein-protein interactions and modifications viz. phosphorylation, ubiquitination, and sumoylation. The enzyme activity of nNOS also depends on the optimal concentration of substrate, cofactors and association with regulatory proteins. This review focuses on the posttranslational regulation of nNOS in the context of normal and diseased states within the CNS. Expert Opinion Gaining insight into the mechanism/s involved in the regulation of nNOS would provide novel strategies for manipulating nNOS directed therapeutic modalities in the future, including catalytically active dimer stabilization and protein-protein interactions with intracellular protein effectors. Ultimately, this is expected to provide tools to improve autonomic dysregulation in various diseases such as heart failure, hypertension, and diabetes. PMID:27885874

Allostery and the dynamic oligomerization of porphobilinogen synthase

PubMed Central

Jaffe, Eileen K.; Lawrence, Sarah H.

2011-01-01

The structural basis for allosteric regulation of porphobilinogen synthase (PBGS) is modulation of a quaternary structure equilibrium between octamer and hexamer (via dimers), which is represented schematically as 8mer ⇔ 2mer ⇔ 2mer* ⇔ 6mer*. The “*” represents a reorientation between two domains of each subunit that occurs in the dissociated state because it is sterically forbidden in the larger multimers. Allosteric effectors of PBGS are both intrinsic and extrinsic and are phylogenetically variable. In some species this equilibrium is modulated intrinsically by magnesium which binds at a site specific to the 8mer. In other species this equilibrium is modulated intrinsically by pH; the guanidinium group of an arginine being spatially equivalent to the allosteric magnesium ion. In humans, disease associated variants all shift the equilibrium toward the 6mer* relative to wild type. The 6mer* has a surface cavity that is not present in the 8mer and is proposed as a small molecule allosteric binding site. In silico and in vitro approaches have revealed species-specific allosteric PBGS inhibitors that stabilize the 6mer*. Some of these inhibitors are drugs in clinical use leading to the hypothesis that extrinsic allosteric inhibition of human PBGS could be a mechanism for drug side effects. PMID:22037356

Correlation-driven insulator-metal transition in near-ideal vanadium dioxide films

DOE PAGES

Gray, A. X.; Jeong, J.; Aetukuri, N. P.; ...

2016-03-18

We use polarization- and temperature-dependent x-ray absorption spectroscopy, in combination with photoelectron microscopy, x-ray diffraction, and electronic transport measurements, to study the driving force behind the insulator-metal transition in VO 2. We show that both the collapse of the insulating gap and the concomitant change in crystal symmetry in homogeneously strained single-crystalline VO 2 films are preceded by the purely electronic softening of Coulomb correlations within V-V singlet dimers. Furthermore, this process starts 7 K (±0.3 K) below the transition temperature, as conventionally defined by electronic transport and x-ray diffraction measurements, and sets the energy scale for driving the near-room-temperaturemore » insulator-metal transition in this technologically promising material.« less

Detailed reaction mechanism of macrophomate synthase. Extraordinary enzyme catalyzing five-step transformation from 2-pyrones to benzoates.

PubMed

Watanabe, K; Mie, T; Ichihara, A; Oikawa, H; Honma, M

2000-12-08

Macrophomate synthase from the fungus Macrophoma commelinae IFO 9570 is a Mg(II)-dependent dimeric enzyme that catalyzes an extraordinary, complex five-step chemical transformation from 2-pyrone and oxalacetate to benzoate involving decarboxylation, C-C bond formation, and dehydration. The catalytic mechanism of the whole pathway was investigated in three separate chemical steps. In the first decarboxylation step, the enzyme loses oxalacetate decarboxylation activity upon incubation with EDTA. Activity is fully restored by addition of Mg(II) and is not restored with other divalent metal cations. The dissociation constant of 0.93 x 10(-)(7) for Mg(II) and atomic absorption analysis established a 1:1 stoichiometric complex. Inhibition of pyruvate formation with 2-pyrone revealed that the actual product in the first step is a pyruvate enolate, which undergoes C-C bond formation in the presence of 2-pyrone. Incubation of substrate analogs provided aberrant adducts that were produced via C-C bond formation and rearrangement. This strongly indicates that the second step is two C-C bond formations, affording a bicyclic intermediate. Based on the stereospecificity, involvement of a Diels-Alder reaction at the second step is proposed. Incubation of the stereospecifically deuterium-labeled malate with 2-pyrones in the presence of malate dehydrogenase provided information for the stereochemical course of the reaction catalyzed by macrophomate synthase, indicating that the first decarboxylation provides pyruvate (Z)-[3-(2)H]enolate and that dehydration at the final step occurs with anti-elimination accompanied by concomitant decarboxylation. Examination of kinetic parameters in the individual steps suggests that the third step is the rate-determining step of the overall transformation.

Biochemical and structural characterization of a novel family of cystathionine beta-synthase domain proteins fused to a Zn ribbon-like domain.

PubMed

Proudfoot, Michael; Sanders, Stephen A; Singer, Alex; Zhang, Rongguang; Brown, Greg; Binkowski, Andrew; Xu, Linda; Lukin, Jonathan A; Murzin, Alexey G; Joachimiak, Andrzej; Arrowsmith, Cheryl H; Edwards, Aled M; Savchenko, Alexei V; Yakunin, Alexander F

2008-01-04

We have identified a novel family of proteins, in which the N-terminal cystathionine beta-synthase (CBS) domain is fused to the C-terminal Zn ribbon domain. Four proteins were overexpressed in Escherichia coli and purified: TA0289 from Thermoplasma acidophilum, TV1335 from Thermoplasma volcanium, PF1953 from Pyrococcus furiosus, and PH0267 from Pyrococcus horikoshii. The purified proteins had a red/purple color in solution and an absorption spectrum typical of rubredoxins (Rds). Metal analysis of purified proteins revealed the presence of several metals, with iron and zinc being the most abundant metals (2-67% of iron and 12-74% of zinc). Crystal structures of both mercury- and iron-bound TA0289 (1.5-2.0 A resolution) revealed a dimeric protein whose intersubunit contacts are formed exclusively by the alpha-helices of two cystathionine beta-synthase subdomains, whereas the C-terminal domain has a classical Zn ribbon planar architecture. All proteins were reversibly reduced by chemical reductants (ascorbate or dithionite) or by the general Rd reductase NorW from E. coli in the presence of NADH. Reduced TA0289 was found to be capable of transferring electrons to cytochrome C from horse heart. Likewise, the purified Zn ribbon protein KTI11 from Saccharomyces cerevisiae had a purple color in solution and an Rd-like absorption spectrum, contained both iron and zinc, and was reduced by the Rd reductase NorW from E. coli. Thus, recombinant Zn ribbon domains from archaea and yeast demonstrate an Rd-like electron carrier activity in vitro. We suggest that, in vivo, some Zn ribbon domains might also bind iron and therefore possess an electron carrier activity, adding another physiological role to this large family of important proteins.

Molecular Simulations of the Synthesis of Periodic Mesoporous Silica Phases at High Surfactant Concentrations

DOE PAGES

Chien, Szu-Chia; Pérez-Sánchez, Germán; Gomes, José R. B.; ...

2017-02-17

Molecular dynamics simulations of a coarse-grained model are used to study the formation mechanism of periodic mesoporous silica over a wide range of cationic surfactant concentrations. This follows up on an earlier study of systems with low surfactant concentrations. We started by studying the phase diagram of the surfactant–water system and found that our model shows good qualitative agreement with experiments with respect to the surfactant concentrations where various phases appear. We then considered the impact of silicate species upon the morphologies formed. We have found that even in concentrated surfactant systems—in the concentration range where pure surfactant solutions yieldmore » a liquid crystal phase—the liquid-crystal templating mechanism is not viable because the preformed liquid crystal collapses as silica monomers are added into the solution. Upon the addition of silica dimers, a new phase-separated hexagonal array is formed. The preformed liquid crystals were found to be unstable in the presence of monomeric silicates. In addition, the silica dimer is found to be essential for mesoscale ordering at both low and high surfactant concentrations. Our results support the view that a cooperative interaction of anionic silica oligomers and cationic surfactants determines the mesostructure formation in the M41S family of materials.« less

Proteomic profiling of tandem affinity purified 14-3-3 protein complexes in Arabidopsis thaliana

PubMed Central

Chang, Ing-Feng; Curran, Amy; Woolsey, Rebekah; Quilici, David; Cushman, John; Mittler, Ron; Harmon, Alice; Harper, Jeffrey

2014-01-01

In eukaryotes, 14-3-3 dimers regulate hundreds of functionally diverse proteins (clients), typically in phosphorylation-dependent interactions. To uncover new clients, a 14-3-3 omega (At1g78300) from Arabidopsis was engineered with a “tandem affinity purification” (TAP) tag and expressed in transgenic plants. Purified complexes were analyzed by tandem mass spectrometry. Results indicate that 14-3-3 omega can dimerize with at least 10 of the 12 14-3-3 isoforms expressed in Arabidopsis. The identification here of 121 putative clients provides support for in vivo 14-3-3 interactions with a diverse array of proteins, including those involved in: (1) Ion transport, such as a K+ channel (GORK), a Cl− channel (CLCg), Ca2+ channels belonging to the glutamate receptor family (GLRs 1.2, 2.1, 2.9, 3.4, 3.7); (2) hormone signaling, such as ACC synthase (isoforms ACS-6, 7 and 8 involved in ethylene synthesis) and the brassinolide receptors BRI1 and BAK1; (3) transcription, such as 7 WRKY family transcription factors; (4) metabolism, such as phosphoenol pyruvate (PEP) carboxylase; and (5) lipid signaling, such as phospholipase D (β, and γ). More than 80% (101) of these putative clients represent previously unidentified 14-3-3 interactors. These results raise the number of putative 14-3-3 clients identified in plants to over 300. PMID:19452453

Proteomic profiling of tandem affinity purified 14-3-3 protein complexes in Arabidopsis thaliana.

PubMed

Chang, Ing-Feng; Curran, Amy; Woolsey, Rebekah; Quilici, David; Cushman, John C; Mittler, Ron; Harmon, Alice; Harper, Jeffrey F

2009-06-01

In eukaryotes, 14-3-3 dimers regulate hundreds of functionally diverse proteins (clients), typically in phosphorylation-dependent interactions. To uncover new clients, 14-3-3 omega (At1g78300) from Arabidopsis was engineered with a "tandem affinity purification" tag and expressed in transgenic plants. Purified complexes were analyzed by tandem MS. Results indicate that 14-3-3 omega can dimerize with at least 10 of the 12 14-3-3 isoforms expressed in Arabidopsis. The identification here of 121 putative clients provides support for in vivo 14-3-3 interactions with a diverse array of proteins, including those involved in: (i) Ion transport, such as a K(+) channel (GORK), a Cl(-) channel (CLCg), Ca(2+) channels belonging to the glutamate receptor family (1.2, 2.1, 2.9, 3.4, 3.7); (ii) hormone signaling, such as ACC synthase (isoforms ACS-6, -7 and -8 involved in ethylene synthesis) and the brassinolide receptors BRI1 and BAK1; (iii) transcription, such as 7 WRKY family transcription factors; (iv) metabolism, such as phosphoenol pyruvate carboxylase; and (v) lipid signaling, such as phospholipase D (beta and gamma). More than 80% (101) of these putative clients represent previously unidentified 14-3-3 interactors. These results raise the number of putative 14-3-3 clients identified in plants to over 300.

Cellular determinants involving mitochondrial dysfunction, oxidative stress and apoptosis correlate with the synergic cytotoxicity of epigallocatechin-3-gallate and menadione in human leukemia Jurkat T cells.

PubMed

Tofolean, Ioana Teodora; Ganea, Constanta; Ionescu, Diana; Filippi, Alexandru; Garaiman, Alexandru; Goicea, Alexandru; Gaman, Mihnea-Alexandru; Dimancea, Alexandru; Baran, Irina

2016-01-01

We have investigated the growth-suppressive action of epigallocatechin-3-gallate (EGCG) on human leukemia Jurkat T cells. Results show a strong correlation between the dose-dependent reduction of clonogenic survival following acute EGCG treatments and the EGCG-induced decline of the mitochondrial level of Ca(2+). The cell killing ability of EGCG was synergistically enhanced by menadione. In addition, the cytotoxic effect of EGCG applied alone or in combination with menadione was accompanied by apoptosis induction. We also observed that in acute treatments EGCG displays strong antioxidant properties in the intracellular milieu, but concurrently triggers some oxidative stress generating mechanisms that can fully develop on a longer timescale. In parallel, EGCG dose-dependently induced mitochondrial depolarization during exposure, but this condition was subsequently reversed to a persistent hyperpolarized mitochondrial state that was dependent on the activity of respiratory Complex I. Fluorimetric measurements suggest that EGCG is a mitochondrial Complex III inhibitor and indicate that EGCG evokes a specific cellular fluorescence with emission at 400nm and two main excitation bands (at 330nm and 350nm) that may originate from a mitochondrial supercomplex containing dimeric Complex III and dimeric ATP-synthase, and therefore could provide a valuable means to characterize the functional properties of the respiratory chain. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mechanism of allosteric inhibition of N-acetyl-L-glutamate synthase by L-arginine.

PubMed

Min, Li; Jin, Zhongmin; Caldovic, Ljubica; Morizono, Hiroki; Allewell, Norma M; Tuchman, Mendel; Shi, Dashuang

2009-02-20

N-Acetylglutamate synthase (NAGS) catalyzes the first committed step in l-arginine biosynthesis in plants and micro-organisms and is subject to feedback inhibition by l-arginine. This study compares the crystal structures of NAGS from Neisseria gonorrhoeae (ngNAGS) in the inactive T-state with l-arginine bound and in the active R-state complexed with CoA and l-glutamate. Under all of the conditions examined, the enzyme consists of two stacked trimers. Each monomer has two domains: an amino acid kinase (AAK) domain with an AAK-like fold but lacking kinase activity and an N-acetyltransferase (NAT) domain homologous to other GCN5-related transferases. Binding of l-arginine to the AAK domain induces a global conformational change that increases the diameter of the hexamer by approximately 10 A and decreases its height by approximately 20A(.) AAK dimers move 5A outward along their 2-fold axes, and their tilt relative to the plane of the hexamer decreases by approximately 4 degrees . The NAT domains rotate approximately 109 degrees relative to AAK domains enabling new interdomain interactions. Interactions between AAK and NAT domains on different subunits also change. Local motions of several loops at the l-arginine-binding site enable the protein to close around the bound ligand, whereas several loops at the NAT active site become disordered, markedly reducing enzymatic specific activity.

Mechanism of Allosteric Inhibition of N-Acetyl-L-glutamate Synthase by L-Arginine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Min, Li; Jin, Zhongmin; Caldovic, Ljubica

2010-01-07

N-Acetylglutamate synthase (NAGS) catalyzes the first committed step in L-arginine biosynthesis in plants and micro-organisms and is subject to feedback inhibition by L-arginine. This study compares the crystal structures of NAGS from Neisseria gonorrhoeae (ngNAGS) in the inactive T-state with L-arginine bound and in the active R-state complexed with CoA and L-glutamate. Under all of the conditions examined, the enzyme consists of two stacked trimers. Each monomer has two domains: an amino acid kinase (AAK) domain with an AAK-like fold but lacking kinase activity and an N-acetyltransferase (NAT) domain homologous to other GCN5-related transferases. Binding of L-arginine to the AAKmore » domain induces a global conformational change that increases the diameter of the hexamer by {approx}10 {angstrom} and decreases its height by {approx}20{angstrom}. AAK dimers move 5{angstrom} outward along their 2-fold axes, and their tilt relative to the plane of the hexamer decreases by {approx}4{sup o}. The NAT domains rotate {approx}109{sup o} relative to AAK domains enabling new interdomain interactions. Interactions between AAK and NAT domains on different subunits also change. Local motions of several loops at the L-arginine-binding site enable the protein to close around the bound ligand, whereas several loops at the NAT active site become disordered, markedly reducing enzymatic specific activity.« less

Silencing CHALCONE SYNTHASE in Maize Impedes the Incorporation of Tricin into Lignin and Increases Lignin Content.

PubMed

Eloy, Nubia B; Voorend, Wannes; Lan, Wu; Saleme, Marina de Lyra Soriano; Cesarino, Igor; Vanholme, Ruben; Smith, Rebecca A; Goeminne, Geert; Pallidis, Andreas; Morreel, Kris; Nicomedes, José; Ralph, John; Boerjan, Wout

2017-02-01

Lignin is a phenolic heteropolymer that is deposited in secondary-thickened cell walls, where it provides mechanical strength. A recent structural characterization of cell walls from monocot species showed that the flavone tricin is part of the native lignin polymer, where it is hypothesized to initiate lignin chains. In this study, we investigated the consequences of altered tricin levels on lignin structure and cell wall recalcitrance by phenolic profiling, nuclear magnetic resonance, and saccharification assays of the naturally silenced maize (Zea mays) C2-Idf (inhibitor diffuse) mutant, defective in the CHALCONE SYNTHASE Colorless2 (C2) gene. We show that the C2-Idf mutant produces highly reduced levels of apigenin- and tricin-related flavonoids, resulting in a strongly reduced incorporation of tricin into the lignin polymer. Moreover, the lignin was enriched in β-β and β-5 units, lending support to the contention that tricin acts to initiate lignin chains and that, in the absence of tricin, more monolignol dimerization reactions occur. In addition, the C2-Idf mutation resulted in strikingly higher Klason lignin levels in the leaves. As a consequence, the leaves of C2-Idf mutants had significantly reduced saccharification efficiencies compared with those of control plants. These findings are instructive for lignin engineering strategies to improve biomass processing and biochemical production. © 2017 American Society of Plant Biologists. All Rights Reserved.

Silencing CHALCONE SYNTHASE in Maize Impedes the Incorporation of Tricin into Lignin and Increases Lignin Content

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eloy, Nubia B.; Voorend, Wannes; Lan, Wu

Lignin is a phenolic heteropolymer that is deposited in secondary-thickened cell walls, where it provides mechanical strength. A recent structural characterization of cell walls from monocot species showed that the flavone tricin is part of the native lignin polymer, where it is hypothesized to initiate lignin chains. In this study, we investigated the consequences of altered tricin levels on lignin structure and cell wall recalcitrance by phenolic profiling, nuclear magnetic resonance, and saccharification assays of the naturally silenced maize (Zea mays) C2-Idf (inhibitor diffuse) mutant, defective in the CHALCONE SYNTHASE Colorless2 (C2) gene. We show that the C2-Idf mutant producesmore » highly reduced levels of apigenin- and tricin-related flavonoids, resulting in a strongly reduced incorporation of tricin into the lignin polymer. Moreover, the lignin was enriched in β-β and β-5 units, lending support to the contention that tricin acts to initiate lignin chains and that, in the absence of tricin, more monolignol dimerization reactions occur. In addition, the C2-Idf mutation resulted in strikingly higher Klason lignin levels in the leaves. As a consequence, the leaves of C2-Idf mutants had significantly reduced saccharification efficiencies compared with those of control plants. These findings are instructive for lignin engineering strategies to improve biomass processing and biochemical production.« less

Silencing CHALCONE SYNTHASE in Maize Impedes the Incorporation of Tricin into Lignin and Increases Lignin Content1[OPEN

PubMed Central

2017-01-01

Lignin is a phenolic heteropolymer that is deposited in secondary-thickened cell walls, where it provides mechanical strength. A recent structural characterization of cell walls from monocot species showed that the flavone tricin is part of the native lignin polymer, where it is hypothesized to initiate lignin chains. In this study, we investigated the consequences of altered tricin levels on lignin structure and cell wall recalcitrance by phenolic profiling, nuclear magnetic resonance, and saccharification assays of the naturally silenced maize (Zea mays) C2-Idf (inhibitor diffuse) mutant, defective in the CHALCONE SYNTHASE Colorless2 (C2) gene. We show that the C2-Idf mutant produces highly reduced levels of apigenin- and tricin-related flavonoids, resulting in a strongly reduced incorporation of tricin into the lignin polymer. Moreover, the lignin was enriched in β-β and β-5 units, lending support to the contention that tricin acts to initiate lignin chains and that, in the absence of tricin, more monolignol dimerization reactions occur. In addition, the C2-Idf mutation resulted in strikingly higher Klason lignin levels in the leaves. As a consequence, the leaves of C2-Idf mutants had significantly reduced saccharification efficiencies compared with those of control plants. These findings are instructive for lignin engineering strategies to improve biomass processing and biochemical production. PMID:27940492

Silencing CHALCONE SYNTHASE in Maize Impedes the Incorporation of Tricin into Lignin and Increases Lignin Content

DOE PAGES

Eloy, Nubia B.; Voorend, Wannes; Lan, Wu; ...

2016-12-09

Lignin is a phenolic heteropolymer that is deposited in secondary-thickened cell walls, where it provides mechanical strength. A recent structural characterization of cell walls from monocot species showed that the flavone tricin is part of the native lignin polymer, where it is hypothesized to initiate lignin chains. In this study, we investigated the consequences of altered tricin levels on lignin structure and cell wall recalcitrance by phenolic profiling, nuclear magnetic resonance, and saccharification assays of the naturally silenced maize (Zea mays) C2-Idf (inhibitor diffuse) mutant, defective in the CHALCONE SYNTHASE Colorless2 (C2) gene. We show that the C2-Idf mutant producesmore » highly reduced levels of apigenin- and tricin-related flavonoids, resulting in a strongly reduced incorporation of tricin into the lignin polymer. Moreover, the lignin was enriched in β-β and β-5 units, lending support to the contention that tricin acts to initiate lignin chains and that, in the absence of tricin, more monolignol dimerization reactions occur. In addition, the C2-Idf mutation resulted in strikingly higher Klason lignin levels in the leaves. As a consequence, the leaves of C2-Idf mutants had significantly reduced saccharification efficiencies compared with those of control plants. These findings are instructive for lignin engineering strategies to improve biomass processing and biochemical production.« less

Catalytic site interactions in yeast OMP synthase.

PubMed

Hansen, Michael Riis; Barr, Eric W; Jensen, Kaj Frank; Willemoës, Martin; Grubmeyer, Charles; Winther, Jakob R

2014-01-15

The enigmatic kinetics, half-of-the-sites binding, and structural asymmetry of the homodimeric microbial OMP synthases (orotate phosphoribosyltransferase, EC 2.4.2.10) have been proposed to result from an alternating site mechanism in these domain-swapped enzymes [R.W. McClard et al., Biochemistry 45 (2006) 5330-5342]. This behavior was investigated in the yeast enzyme by mutations in the conserved catalytic loop and 5-phosphoribosyl-1-diphosphate (PRPP) binding motif. Although the reaction is mechanistically sequential, the wild-type (WT) enzyme shows parallel lines in double reciprocal initial velocity plots. Replacement of Lys106, the postulated intersubunit communication device, produced intersecting lines in kinetic plots with a 2-fold reduction of kcat. Loop (R105G K109S H111G) and PRPP-binding motif (D131N D132N) mutant proteins, each without detectable enzymatic activity and ablated ability to bind PRPP, complemented to produce a heterodimer with a single fully functional active site showing intersecting initial velocity plots. Equilibrium binding of PRPP and orotidine 5'-monophosphate showed a single class of two binding sites per dimer in WT and K106S enzymes. Evidence here shows that the enzyme does not follow half-of-the-sites cooperativity; that interplay between catalytic sites is not an essential feature of the catalytic mechanism; and that parallel lines in steady-state kinetics probably arise from tight substrate binding. Copyright © 2013. Published by Elsevier Inc.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Knott, Brandon C.; Crowley, Michael F.; Himmel, Michael E.

The processive cycle of the bacterial cellulose synthase (Bcs) includes the addition of a single glucose moiety to the end of a growing cellulose chain followed by the translocation of the nascent chain across the plasma membrane. The mechanism of this translocation and its precise location within the processive cycle are not well understood. In particular, the molecular details of how a polymer (cellulose) whose basic structural unit is a dimer (cellobiose) can be constructed by adding one monomer (glucose) at a time are yet to be elucidated. Here, we have utilized molecular dynamics simulations and free energy calculations tomore » the shed light on these questions. We find that translocation forward by one glucose unit is quite favorable energetically, giving a free energy stabilization of greater than 10 kcal mol-1. In addition, there is only a small barrier to translocation, implying that translocation is not rate limiting within the Bcs processive cycle (given experimental rates for cellulose synthesis in vitro). Perhaps most significantly, our results also indicate that steric constraints at the transmembrane tunnel entrance regulate the dimeric structure of cellulose. Namely, when a glucose molecule is added to the cellulose chain in the same orientation as the acceptor glucose, the terminal glucose freely rotates upon forward motion, thus suggesting a regulatory mechanism for the dimeric structure of cellulose. We characterize both the conserved and non-conserved enzyme-polysaccharide interactions that drive translocation, and find that 20 of the 25 residues that strongly interact with the translocating cellulose chain in the simulations are well conserved, mostly with polar or aromatic side chains. Our results also allow for a dynamical analysis of the role of the so-called 'finger helix' in cellulose translocation that has been observed structurally. Taken together, these findings aid in the elucidation of the translocation steps of the Bcs processive cycle and may be widely relevant to polysaccharide synthesizing or degrading enzymes that couple catalysis with chain translocation.« less

Tanshinones and diethyl blechnics with anti-inflammatory and anti-cancer activities from Salvia miltiorrhiza Bunge (Danshen)

NASA Astrophysics Data System (ADS)

Gao, Hongwei; Sun, Wen; Zhao, Jianping; Wu, Xiaxia; Lu, Jin-Jian; Chen, Xiuping; Xu, Qiong-Ming; Khan, Ikhlas A.; Yang, Shilin

2016-09-01

Four novel compounds (1-4) as well as fourteen reported compounds (5-18) were isolated and purified from Salvia miltiorrhiza Bunge (Danshen). The structures of novel compounds were determined by 1D and 2D NMR, HRESIMS data, etc. The anti-inflammatory properties of all the compounds on RAW264.7 macrophages and their cytotoxicity on H1299 and Bel-7402 cell lines coupled with a structure-activity relationship (SAR) were investigated. Compound 4 demonstrated the best anti-inflammatory activity and was chosen for further research. Compound 4 greatly suppressed secretion of nitric oxide (NO), tumor necrosis factor (TNF)-α and interleukin-6 (IL-6) in the RAW264.7 macrophages stimulated by LPS. Additionally, the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was decreased and the nuclear translocation of NF-κB was attenuated after treatment with compound 4 in vitro. Compound 4 was able to dramatically inhibit LPS-induced activation of JNK1/2 and ERK1/2 and remarkably disrupted the TLR4 dimerization in LPS-induced RAW264.7 macrophages. Thus, the new compound 4 suppressed LPS-induced inflammation partially is due to the blocking TLR4 dimerization. In addition, the anti-cancer activity investigation indicated that most of isolated compounds exhibited cytotoxicity and the SAR analysis showed that the intact D ring was indispensable and unsaturated D ring played vital role.

The inner workings of the hydrazine synthase multiprotein complex.

PubMed

Dietl, Andreas; Ferousi, Christina; Maalcke, Wouter J; Menzel, Andreas; de Vries, Simon; Keltjens, Jan T; Jetten, Mike S M; Kartal, Boran; Barends, Thomas R M

2015-11-19

Anaerobic ammonium oxidation (anammox) has a major role in the Earth's nitrogen cycle and is used in energy-efficient wastewater treatment. This bacterial process combines nitrite and ammonium to form dinitrogen (N2) gas, and has been estimated to synthesize up to 50% of the dinitrogen gas emitted into our atmosphere from the oceans. Strikingly, the anammox process relies on the highly unusual, extremely reactive intermediate hydrazine, a compound also used as a rocket fuel because of its high reducing power. So far, the enzymatic mechanism by which hydrazine is synthesized is unknown. Here we report the 2.7 Å resolution crystal structure, as well as biophysical and spectroscopic studies, of a hydrazine synthase multiprotein complex isolated from the anammox organism Kuenenia stuttgartiensis. The structure shows an elongated dimer of heterotrimers, each of which has two unique c-type haem-containing active sites, as well as an interaction point for a redox partner. Furthermore, a system of tunnels connects these active sites. The crystal structure implies a two-step mechanism for hydrazine synthesis: a three-electron reduction of nitric oxide to hydroxylamine at the active site of the γ-subunit and its subsequent condensation with ammonia, yielding hydrazine in the active centre of the α-subunit. Our results provide the first, to our knowledge, detailed structural insight into the mechanism of biological hydrazine synthesis, which is of major significance for our understanding of the conversion of nitrogenous compounds in nature.

Architecture of the nitric-oxide synthase holoenzyme reveals large conformational changes and a calmodulin-driven release of the FMN domain.

PubMed

Yokom, Adam L; Morishima, Yoshihiro; Lau, Miranda; Su, Min; Glukhova, Alisa; Osawa, Yoichi; Southworth, Daniel R

2014-06-13

Nitric-oxide synthase (NOS) is required in mammals to generate NO for regulating blood pressure, synaptic response, and immune defense. NOS is a large homodimer with well characterized reductase and oxygenase domains that coordinate a multistep, interdomain electron transfer mechanism to oxidize l-arginine and generate NO. Ca(2+)-calmodulin (CaM) binds between the reductase and oxygenase domains to activate NO synthesis. Although NOS has long been proposed to adopt distinct conformations that alternate between interflavin and FMN-heme electron transfer steps, structures of the holoenzyme have remained elusive and the CaM-bound arrangement is unknown. Here we have applied single particle electron microscopy (EM) methods to characterize the full-length of the neuronal isoform (nNOS) complex and determine the structural mechanism of CaM activation. We have identified that nNOS adopts an ensemble of open and closed conformational states and that CaM binding induces a dramatic rearrangement of the reductase domain. Our three-dimensional reconstruction of the intact nNOS-CaM complex reveals a closed conformation and a cross-monomer arrangement with the FMN domain rotated away from the NADPH-FAD center, toward the oxygenase dimer. This work captures, for the first time, the reductase-oxygenase structural arrangement and the CaM-dependent release of the FMN domain that coordinates to drive electron transfer across the domains during catalysis. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Mechanism of Orlistat Hydrolysis by the Thioesterase of Human Fatty Acid Synthase

PubMed Central

2015-01-01

Fatty acid synthase (FASN), the sole protein capable of de novo synthesis of free fatty acids, is overexpressed in a wide variety of human cancers and is associated with poor prognosis and aggressiveness of these cancers. Orlistat, an FDA-approved drug for obesity treatment that inhibits pancreatic lipases in the GI tract, also inhibits the thioesterase (TE) of human FASN. The cocrystal structure of TE with orlistat shows a pseudo TE dimer containing two different forms of orlistat in the active site, an intermediate that is covalently bound to a serine residue (Ser2308) and a hydrolyzed and inactivated product. In this study, we attempted to understand the mechanism of TE-catalyzed orlistat hydrolysis by examining the role of the hexyl tail of the covalently bound orlistat in water activation for hydrolysis using molecular dynamics simulations. We found that the hexyl tail of the covalently bound orlistat undergoes a conformational transition, which is accompanied by destabilization of a hydrogen bond between a hydroxyl moiety of orlistat and the catalytic His2481 of TE that in turn leads to an increased hydrogen bonding between water molecules and His2481 and increased chance for water activation to hydrolyze the covalent bond between orlistat and Ser2308. Thus, the conformation of the hexyl tail of orlistat plays an important role in orlistat hydrolysis. Strategies that stabilize the hexyl tail may lead to the design of more potent irreversible inhibitors that target FASN and block TE activity with greater endurance. PMID:25309810

Identification of olivetolic acid cyclase from Cannabis sativa reveals a unique catalytic route to plant polyketides.

PubMed

Gagne, Steve J; Stout, Jake M; Liu, Enwu; Boubakir, Zakia; Clark, Shawn M; Page, Jonathan E

2012-07-31

Δ(9)-Tetrahydrocannabinol (THC) and other cannabinoids are responsible for the psychoactive and medicinal properties of Cannabis sativa L. (marijuana). The first intermediate in the cannabinoid biosynthetic pathway is proposed to be olivetolic acid (OA), an alkylresorcinolic acid that forms the polyketide nucleus of the cannabinoids. OA has been postulated to be synthesized by a type III polyketide synthase (PKS) enzyme, but so far type III PKSs from cannabis have been shown to produce catalytic byproducts instead of OA. We analyzed the transcriptome of glandular trichomes from female cannabis flowers, which are the primary site of cannabinoid biosynthesis, and searched for polyketide cyclase-like enzymes that could assist in OA cyclization. Here, we show that a type III PKS (tetraketide synthase) from cannabis trichomes requires the presence of a polyketide cyclase enzyme, olivetolic acid cyclase (OAC), which catalyzes a C2-C7 intramolecular aldol condensation with carboxylate retention to form OA. OAC is a dimeric α+β barrel (DABB) protein that is structurally similar to polyketide cyclases from Streptomyces species. OAC transcript is present at high levels in glandular trichomes, an expression profile that parallels other cannabinoid pathway enzymes. Our identification of OAC both clarifies the cannabinoid pathway and demonstrates unexpected evolutionary parallels between polyketide biosynthesis in plants and bacteria. In addition, the widespread occurrence of DABB proteins in plants suggests that polyketide cyclases may play an overlooked role in generating plant chemical diversity.

Effects of force fields on the conformational and dynamic properties of amyloid β(1-40) dimer explored by replica exchange molecular dynamics simulations.

PubMed

Watts, Charles R; Gregory, Andrew; Frisbie, Cole; Lovas, Sándor

2018-03-01

The conformational space and structural ensembles of amyloid beta (Aβ) peptides and their oligomers in solution are inherently disordered and proven to be challenging to study. Optimum force field selection for molecular dynamics (MD) simulations and the biophysical relevance of results are still unknown. We compared the conformational space of the Aβ(1-40) dimers by 300 ns replica exchange MD simulations at physiological temperature (310 K) using: the AMBER-ff99sb-ILDN, AMBER-ff99sb*-ILDN, AMBER-ff99sb-NMR, and CHARMM22* force fields. Statistical comparisons of simulation results to experimental data and previously published simulations utilizing the CHARMM22* and CHARMM36 force fields were performed. All force fields yield sampled ensembles of conformations with collision cross sectional areas for the dimer that are statistically significantly larger than experimental results. All force fields, with the exception of AMBER-ff99sb-ILDN (8.8 ± 6.4%) and CHARMM36 (2.7 ± 4.2%), tend to overestimate the α-helical content compared to experimental CD (5.3 ± 5.2%). Using the AMBER-ff99sb-NMR force field resulted in the greatest degree of variance (41.3 ± 12.9%). Except for the AMBER-ff99sb-NMR force field, the others tended to under estimate the expected amount of β-sheet and over estimate the amount of turn/bend/random coil conformations. All force fields, with the exception AMBER-ff99sb-NMR, reproduce a theoretically expected β-sheet-turn-β-sheet conformational motif, however, only the CHARMM22* and CHARMM36 force fields yield results compatible with collapse of the central and C-terminal hydrophobic cores from residues 17-21 and 30-36. Although analyses of essential subspace sampling showed only minor variations between force fields, secondary structures of lowest energy conformers are different. © 2017 Wiley Periodicals, Inc.

A Novel High-Density Phase and Amorphization of Nitrogen-Rich 1H-Tetrazole (CH2N4) under High Pressure

PubMed Central

Li, Wenbo; Huang, Xiaoli; Bao, Kuo; Zhao, Zhonglong; Huang, Yanping; Wang, Lu; Wu, Gang; Zhou, Bo; Duan, Defang; Li, Fangfei; Zhou, Qiang; Liu, Bingbing; Cui, Tian

2017-01-01

The high-pressure behaviors of nitrogen-rich 1H-tetrazole (CH2N4) have been investigated by in situ synchrotron X-ray diffraction (XRD) and Raman scattering up to 75 GPa. A first crystalline-to-crystalline phase transition is observed and identified above ~3 GPa with a large volume collapse (∼18% at 4.4 GPa) from phase I to phase II. The new phase II forms a dimer-like structure, belonging to P1 space group. Then, a crystalline-to-amorphous phase transition takes place over a large pressure range of 13.8 to 50 GPa, which is accompanied by an interphase region approaching paracrystalline state. When decompression from 75 GPa to ambient conditions, the final product keeps an irreversible amorphous state. Our ultraviolet (UV) absorption spectrum suggests the final product exhibits an increase in molecular conjugation. PMID:28218236

Modeling of particle interactions in magnetorheological elastomers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Biller, A. M., E-mail: kam@icmm.ru; Stolbov, O. V., E-mail: oleg100@gmail.com; Raikher, Yu. L., E-mail: raikher@icmm.ru

2014-09-21

The interaction between two particles made of an isotropic linearly polarizable magnetic material and embedded in an elastomer matrix is studied. In this case, when an external field is imposed, the magnetic attraction of the particles, contrary to point dipoles, is almost wraparound. The exact solution of the magnetic problem in the linear polarization case, although existing, is not practical; to circumvent its use, an interpolation formula is proposed. One more interpolation expression is developed for the resistance of the elastic matrix to the field-induced particle displacements. Minimization of the total energy of the pair reveals its configurational bistability inmore » a certain field range. One of the possible equilibrium states corresponds to the particles dwelling at a distance, the other—to their collapse in a tight dimer. This mesoscopic bistability causes magnetomechanical hysteresis which has important implications for the macroscopic behavior of magnetorheological elastomers.« less

A Novel High-Density Phase and Amorphization of Nitrogen-Rich 1H-Tetrazole (CH2N4) under High Pressure.

PubMed

Li, Wenbo; Huang, Xiaoli; Bao, Kuo; Zhao, Zhonglong; Huang, Yanping; Wang, Lu; Wu, Gang; Zhou, Bo; Duan, Defang; Li, Fangfei; Zhou, Qiang; Liu, Bingbing; Cui, Tian

2017-02-20

The high-pressure behaviors of nitrogen-rich 1H-tetrazole (CH 2 N 4 ) have been investigated by in situ synchrotron X-ray diffraction (XRD) and Raman scattering up to 75 GPa. A first crystalline-to-crystalline phase transition is observed and identified above ~3 GPa with a large volume collapse (∼18% at 4.4 GPa) from phase I to phase II. The new phase II forms a dimer-like structure, belonging to P1 space group. Then, a crystalline-to-amorphous phase transition takes place over a large pressure range of 13.8 to 50 GPa, which is accompanied by an interphase region approaching paracrystalline state. When decompression from 75 GPa to ambient conditions, the final product keeps an irreversible amorphous state. Our ultraviolet (UV) absorption spectrum suggests the final product exhibits an increase in molecular conjugation.

Structural and dynamical aspects of Streptococcus gordonii FabH through molecular docking and MD simulations.

PubMed

Shamim, Amen; Abbasi, Sumra Wajid; Azam, Syed Sikander

2015-07-01

β-Ketoacyl-ACP-synthase III (FabH or KAS III) has become an attractive target for the development of new antibacterial agents which can overcome the multidrug resistance. Unraveling the fatty acid biosynthesis (FAB) metabolic pathway and understanding structural coordinates of FabH will provide valuable insights to target Streptococcus gordonii for curing oral infection. In this study, we designed inhibitors against therapeutic target FabH, in order to block the FAB pathway. As compared to other targets, FabH has more interactions with other proteins, located on the leading strand with higher codon adaptation index value and associated with lipid metabolism category of COG. Current study aims to gain in silico insights into the structural and dynamical aspect of S. gordonii FabH via molecular docking and molecular dynamics (MD) simulations. The FabH protein is catalytically active in dimerization while it can lock in monomeric state. Current study highlights two residues Pro88 and Leu315 that are close to each other by dimerization. The active site of FabH is composed of the catalytic triad formed by residues Cys112, His249, and Asn279 in which Cys112 is involved in acetyl transfer, while His249 and Asn279 play an active role in decarboxylation. Docking analysis revealed that among the studied compounds, methyl-CoA disulfide has highest GOLD score (82.75), binding affinity (-11 kcal/mol) and exhibited consistently better interactions. During MD simulations, the FabH structure remained stable with the average RMSD value of 1.7 Å and 1.6 Å for undocked protein and docked complex, respectively. Further, crucial hydrogen bonding of the conserved catalytic triad for exhibiting high affinity between the FabH protein and ligand is observed by RDF analysis. The MD simulation results clearly demonstrated that binding of the inhibitor with S. gordonii FabH enhanced the structure and stabilized the dimeric FabH protein. Therefore, the inhibitor has the potential to become a lead compound. Copyright © 2015 Elsevier Inc. All rights reserved.

Exploring symmetry as an avenue to the computational design of large protein domains.

PubMed

Fortenberry, Carie; Bowman, Elizabeth Anne; Proffitt, Will; Dorr, Brent; Combs, Steven; Harp, Joel; Mizoue, Laura; Meiler, Jens

2011-11-16

It has been demonstrated previously that symmetric, homodimeric proteins are energetically favored, which explains their abundance in nature. It has been proposed that such symmetric homodimers underwent gene duplication and fusion to evolve into protein topologies that have a symmetric arrangement of secondary structure elements--"symmetric superfolds". Here, the ROSETTA protein design software was used to computationally engineer a perfectly symmetric variant of imidazole glycerol phosphate synthase and its corresponding symmetric homodimer. The new protein, termed FLR, adopts the symmetric (βα)(8) TIM-barrel superfold. The protein is soluble and monomeric and exhibits two-fold symmetry not only in the arrangement of secondary structure elements but also in sequence and at atomic detail, as verified by crystallography. When cut in half, FLR dimerizes readily to form the symmetric homodimer. The successful computational design of FLR demonstrates progress in our understanding of the underlying principles of protein stability and presents an attractive strategy for the in silico construction of larger protein domains from smaller pieces.

Black perithecial pigmentation in Fusarium species is due to the accumulation of 5-deoxybostrycoidin-based melanin

PubMed Central

Frandsen, Rasmus J. N.; Rasmussen, Silas A.; Knudsen, Peter B.; Uhlig, Silvio; Petersen, Dirk; Lysøe, Erik; Gotfredsen, Charlotte H.; Giese, Henriette; Larsen, Thomas O.

2016-01-01

Biosynthesis of the black perithecial pigment in the filamentous fungus Fusarium graminearum is dependent on the polyketide synthase PGL1 (oPKS3). A seven-membered PGL1 gene cluster was identified by over-expression of the cluster specific transcription factor pglR. Targeted gene replacement showed that PGL1, pglJ, pglM and pglV were essential for the production of the perithecial pigment. Over-expression of PGL1 resulted in the production of 6-O-demethyl-5-deoxybostrycoidin (1), 5-deoxybostrycoidin (2), and three novel compounds 5-deoxybostrycoidin anthrone (3), 6-O-demethyl-5-deoxybostrycoidin anthrone (4) and purpurfusarin (5). The novel dimeric bostrycoidin purpurfusarin (5) was found to inhibit the growth of Candida albicans with an IC50 of 8.0 +/− 1.9 μM. The results show that Fusarium species with black perithecia have a previously undescribed form of 5-deoxybostrycoidin based melanin in their fruiting bodies. PMID:27193384

Spatial and Temporal Localization of Flavonoid Metabolites in Strawberry Fruit (Fragaria × ananassa).

PubMed

Crecelius, Anna C; Hölscher, Dirk; Hoffmann, Thomas; Schneider, Bernd; Fischer, Thilo C; Hanke, Magda-Viola; Flachowsky, Henryk; Schwab, Wilfried; Schubert, Ulrich S

2017-05-03

Flavonoids are important metabolites in strawberries (Fragaria × ananassa) because they accomplish an extensive collection of physiological functions and are valuable for human health. However, their localization within the fruit tissue has not been extensively explored. Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) was employed to shed light on the spatial distribution of flavonoids during fruit development. One wild-type (WT) and two transgenic lines were compared, wherein the transgenic enzymes anthocyanidin reductase (ANRi) and flavonol synthase (FLSi), respectively, were down-regulated using an RNAi-based silencing approach. In most cases, fruit development led to a reduction of the investigated flavonoids in the fruit tissue; as a consequence, they were exclusively present in the skin of mature red fruits. In the case of (epi)catechin dimer, both the ANRi and the WT phenotypes revealed low levels in mature red fruits, whereas the ANRi line bore the lowest relative concentration, as analyzed by liquid chromatography-electrospray ionization multiple-step mass spectrometry (LC-ESI-MS n ).

An evidence based hypothesis on the existence of two pathways of mitochondrial crista formation

PubMed Central

Harner, Max E; Unger, Ann-Katrin; Geerts, Willie JC; Mari, Muriel; Izawa, Toshiaki; Stenger, Maria; Geimer, Stefan; Reggiori, Fulvio; Westermann, Benedikt; Neupert, Walter

2016-01-01

Metabolic function and architecture of mitochondria are intimately linked. More than 60 years ago, cristae were discovered as characteristic elements of mitochondria that harbor the protein complexes of oxidative phosphorylation, but how cristae are formed, remained an open question. Here we present experimental results obtained with yeast that support a novel hypothesis on the existence of two molecular pathways that lead to the generation of lamellar and tubular cristae. Formation of lamellar cristae depends on the mitochondrial fusion machinery through a pathway that is required also for homeostasis of mitochondria and mitochondrial DNA. Tubular cristae are formed via invaginations of the inner boundary membrane by a pathway independent of the fusion machinery. Dimerization of the F1FO-ATP synthase and the presence of the MICOS complex are necessary for both pathways. The proposed hypothesis is suggested to apply also to higher eukaryotes, since the key components are conserved in structure and function throughout evolution. DOI: http://dx.doi.org/10.7554/eLife.18853.001 PMID:27849155

Crystal structures of nematode (parasitic T. spiralis and free living C. elegans), compared to mammalian, thymidylate synthases (TS). Molecular docking and molecular dynamics simulations in search for nematode-specific inhibitors of TS.

PubMed

Jarmuła, Adam; Wilk, Piotr; Maj, Piotr; Ludwiczak, Jan; Dowierciał, Anna; Banaszak, Katarzyna; Rypniewski, Wojciech; Cieśla, Joanna; Dąbrowska, Magdalena; Frączyk, Tomasz; Bronowska, Agnieszka K; Jakowiecki, Jakub; Filipek, Sławomir; Rode, Wojciech

2017-10-01

Three crystal structures are presented of nematode thymidylate synthases (TS), including Caenorhabditis elegans (Ce) enzyme without ligands and its ternary complex with dUMP and Raltitrexed, and binary complex of Trichinella spiralis (Ts) enzyme with dUMP. In search of differences potentially relevant for the development of species-specific inhibitors of the nematode enzyme, a comparison was made of the present Ce and Ts enzyme structures, as well as binary complex of Ce enzyme with dUMP, with the corresponding mammalian (human, mouse and rat) enzyme crystal structures. To complement the comparison, tCONCOORD computations were performed to evaluate dynamic behaviors of mammalian and nematode TS structures. Finally, comparative molecular docking combined with molecular dynamics and free energy of binding calculations were carried out to search for ligands showing selective affinity to T. spiralis TS. Despite an overall strong similarity in structure and dynamics of nematode vs mammalian TSs, a pool of ligands demonstrating predictively a strong and selective binding to TsTS has been delimited. These compounds, the E63 family, locate in the dimerization interface of TsTS where they exert species-specific interactions with certain non-conserved residues, including hydrogen bonds with Thr174 and hydrophobic contacts with Phe192, Cys191 and Tyr152. The E63 family of ligands opens the possibility of future development of selective inhibitors of TsTS and effective agents against trichinellosis. Copyright © 2017 Elsevier Inc. All rights reserved.

Extracellular purines' action on glomerular albumin permeability in isolated rat glomeruli: insights into the pathogenesis of albuminuria.

PubMed

Kasztan, Małgorzata; Piwkowska, Agnieszka; Kreft, Ewelina; Rogacka, Dorota; Audzeyenka, Irena; Szczepanska-Konkel, Mirosława; Jankowski, Maciej

2016-07-01

Purinoceptors (adrengeric receptors and P2 receptors) are expressed on the cellular components of the glomerular filtration barrier, and their activation may affect glomerular permeability to albumin, which may ultimately lead to albuminuria, a well-established risk factor for the progression of chronic kidney disease and development of cardiovascular diseases. We investigated the mechanisms underlying the in vitro and in vivo purinergic actions on glomerular filter permeability to albumin by measuring convectional albumin permeability (Palb) in a single isolated rat glomerulus based on the video microscopy method. Primary cultured rat podocytes were used for the analysis of Palb, cGMP accumulation, PKG-Iα dimerization, and immunofluorescence. In vitro, natural nucleotides (ATP, ADP, UTP, and UDP) and nonmetabolized ATP analogs (2-meSATP and ATP-γ-S) increased Palb in a time- and concentration-dependent manner. The effects were dependent on P2 receptor activation, nitric oxide synthase, and cytoplasmic guanylate cyclase. ATP analogs significantly increased Palb, cGMP accumulation, and subcortical actin reorganization in a PKG-dependent but nondimer-mediated route in cultured podocytes. In vivo, 2-meSATP and ATP-γ-S increased Palb but did not significantly affect urinary albumin excretion. Both agonists enhanced the clathrin-mediated endocytosis of albumin in podocytes. A product of adenine nucleotides hydrolysis, adenosine, increased the permeability of the glomerular barrier via adrenergic receptors in a dependent and independent manner. Our results suggest that the extracellular nucleotides that stimulate an increase of glomerular Palb involve nitric oxide synthase and cytoplasmic guanylate cyclase with actin reorganization in podocytes. Copyright © 2016 the American Physiological Society.

Structures of mesophilic and extremophilic citrate synthases reveal rigidity and flexibility for function.

PubMed

Wells, Stephen A; Crennell, Susan J; Danson, Michael J

2014-10-01

Citrate synthase (CS) catalyses the entry of carbon into the citric acid cycle and is highly-conserved structurally across the tree of life. Crystal structures of dimeric CSs are known in both "open" and "closed" forms, which differ by a substantial domain motion that closes the substrate-binding clefts. We explore both the static rigidity and the dynamic flexibility of CS structures from mesophilic and extremophilic organisms from all three evolutionary domains. The computational expense of this wide-ranging exploration is kept to a minimum by the use of rigidity analysis and rapid all-atom simulations of flexible motion, combining geometric simulation and elastic network modeling. CS structures from thermophiles display increased structural rigidity compared with the mesophilic enzyme. A CS structure from a psychrophile, stabilized by strong ionic interactions, appears to display likewise increased rigidity in conventional rigidity analysis; however, a novel modified analysis, taking into account the weakening of the hydrophobic effect at low temperatures, shows a more appropriate decreased rigidity. These rigidity variations do not, however, affect the character of the flexible dynamics, which are well conserved across all the structures studied. Simulation trajectories not only duplicate the crystallographically observed symmetric open-to-closed transitions, but also identify motions describing a previously unidentified antisymmetric functional motion. This antisymmetric motion would not be directly observed in crystallography but is revealed as an intrinsic property of the CS structure by modeling of flexible motion. This suggests that the functional motion closing the binding clefts in CS may be independent rather than symmetric and cooperative. © 2014 Wiley Periodicals, Inc.

Stringent Control of NFE2L3 (Nuclear Factor, Erythroid 2-Like 3; NRF3) Protein Degradation by FBW7 (F-box/WD Repeat-containing Protein 7) and Glycogen Synthase Kinase 3 (GSK3)*

PubMed Central

Kannan, Meenakshi B.; Dodard-Friedman, Isadore; Blank, Volker

2015-01-01

The NFE2L3 transcription factor has been implicated in various cellular processes, including carcinogenesis, stress response, differentiation, and inflammation. Previously it has been shown that NFE2L3 has a rapid turnover and is stabilized by proteasomal inhibitors. The mechanisms regulating the degradation of this protein have not been investigated. Here we report ubiquitination of NFE2L3 and demonstrate that F-box/WD repeat-containing protein 7 (FBW7 or FBWX7), a component of Skp1, Cullin 1, F-box containing complex (SCF)-type E3 ligase, is the E3 ligase mediating the degradation of NFE2L3. We showed that FBW7 interacts with NFE2L3 and that dimerization of FBW7 is required for the degradation of the transcription factor. We also demonstrate that the kinase glycogen synthase kinase 3 (GSK3) mediates the FBW7-dependent ubiquitination of NFE2L3. We show phosphorylation of NFE2L3 by GSK3 and its significance in the regulation of NFE2L3 by the tumor suppressor FBW7. FBW7 abrogated NFE2L3-mediated repression of the NAD(P)H:quinone oxidoreductase 1 (NQO1) gene antioxidant response element (ARE). Our findings reveal FBW7 and GSK3 as novel regulators of the NFE2L3 transcription factor and a potential mechanism by which FBW7 might regulate detoxification and the cellular response to stress. PMID:26306035

Dissecting structural and electronic effects in inducible nitric oxide synthase.

PubMed

Hannibal, Luciana; Page, Richard C; Haque, Mohammad Mahfuzul; Bolisetty, Karthik; Yu, Zhihao; Misra, Saurav; Stuehr, Dennis J

2015-04-01

Nitric oxide synthases (NOSs) are haem-thiolate enzymes that catalyse the conversion of L-arginine (L-Arg) into NO and citrulline. Inducible NOS (iNOS) is responsible for delivery of NO in response to stressors during inflammation. The catalytic performance of iNOS is proposed to rely mainly on the haem midpoint potential and the ability of the substrate L-Arg to provide a hydrogen bond for oxygen activation (O-O scission). We present a study of native iNOS compared with iNOS-mesohaem, and investigate the formation of a low-spin ferric haem-aquo or -hydroxo species (P) in iNOS mutant W188H substituted with mesohaem. iNOS-mesohaem and W188H-mesohaem were stable and dimeric, and presented substrate-binding affinities comparable to those of their native counterparts. Single turnover reactions catalysed by iNOSoxy with L-Arg (first reaction step) or N-hydroxy-L-arginine (second reaction step) showed that mesohaem substitution triggered higher rates of Fe(II)O₂ conversion and altered other key kinetic parameters. We elucidated the first crystal structure of a NOS substituted with mesohaem and found essentially identical features compared with the structure of iNOS carrying native haem. This facilitated the dissection of structural and electronic effects. Mesohaem substitution substantially reduced the build-up of species P in W188H iNOS during catalysis, thus increasing its proficiency towards NO synthesis. The marked structural similarities of iNOSoxy containing native haem or mesohaem indicate that the kinetic behaviour observed in mesohaem-substituted iNOS is most heavily influenced by electronic effects rather than structural alterations.

DISSECTING STRUCTURAL AND ELECTRONIC EFFECTS IN INDUCIBLE NITRIC OXIDE SYNTHASE

PubMed Central

Hannibal, Luciana; Page, Richard C.; Haque, Mohammad Mahfuzul; Bolisetty, Karthik; Yu, Zhihao; Misra, Saurav; Stuehr, Dennis J.

2015-01-01

Nitric oxide synthases (NOS) are haem-thiolate enzymes that catalyse the conversion of L-Arginine (LArg) into NO and citrulline. Inducible NOS (iNOS) is responsible for delivery of NO in response to stressors during inflammation. The catalytic performance of iNOS is proposed to rely mainly on the haem midpoint potential and the ability of the substrate L-Arg to provide an H-bond for oxygen activation (O-O scission). We present a comparative study of native iNOS versus iNOS-mesohaem, and investigate the formation of a low-spin ferric haem-aquo or -hydroxo species (P) in iNOS mutant W188H substituted with mesohaem. iNOS-mesohaem and W188H-mesohaem were stable and dimeric, and presented substrate-binding affinities comparable to their native counterparts. Single turnover reactions catalysed by iNOSoxy with LArg (first reaction step) or N-hydroxyarginine (second reaction step) showed that mesohaem substitution triggered faster rates of FeIIO2 conversion and altered other key kinetic parameters. We elucidated the first crystal structure of a NOS substituted with mesohaem and found essentially identical features compared to the structure of iNOS carrying native haem. This facilitated the dissection of structural and electronic effects. Mesohaem substitution substantially reduced the build-up of species P in W188H iNOS during catalysis, thus increasing its proficiency toward NO synthesis. The marked structural similarities of iNOSoxy containing native haem or mesohaem indicate that the kinetic behaviour observed in mesohaem-substituted iNOS is most heavily influenced by electronic effects rather than structural alterations. PMID:25608846

Enhancing Human Spermine Synthase Activity by Engineered Mutations

PubMed Central

Zhang, Zhe; Zheng, Yueli; Petukh, Margo; Pegg, Anthony; Ikeguchi, Yoshihiko; Alexov, Emil

2013-01-01

Spermine synthase (SMS) is an enzyme which function is to convert spermidine into spermine. It was shown that gene defects resulting in amino acid changes of the wild type SMS cause Snyder-Robinson syndrome, which is a mild-to-moderate mental disability associated with osteoporosis, facial asymmetry, thin habitus, hypotonia, and a nonspecific movement disorder. These disease-causing missense mutations were demonstrated, both in silico and in vitro, to affect the wild type function of SMS by either destabilizing the SMS dimer/monomer or directly affecting the hydrogen bond network of the active site of SMS. In contrast to these studies, here we report an artificial engineering of a more efficient SMS variant by transferring sequence information from another organism. It is confirmed experimentally that the variant, bearing four amino acid substitutions, is catalytically more active than the wild type. The increased functionality is attributed to enhanced monomer stability, lowering the pKa of proton donor catalytic residue, optimized spatial distribution of the electrostatic potential around the SMS with respect to substrates, and increase of the frequency of mechanical vibration of the clefts presumed to be the gates toward the active sites. The study demonstrates that wild type SMS is not particularly evolutionarily optimized with respect to the reaction spermidine → spermine. Having in mind that currently there are no variations (non-synonymous single nucleotide polymorphism, nsSNP) detected in healthy individuals, it can be speculated that the human SMS function is precisely tuned toward its wild type and any deviation is unwanted and disease-causing. PMID:23468611

In Silico and In Vitro Investigations of the Mutability of Disease-Causing Missense Mutation Sites in Spermine Synthase

PubMed Central

Zhang, Zhe; Norris, Joy; Schwartz, Charles; Alexov, Emil

2011-01-01

Background Spermine synthase (SMS) is a key enzyme controlling the concentration of spermidine and spermine in the cell. The importance of SMS is manifested by the fact that single missense mutations were found to cause Snyder-Robinson Syndrome (SRS). At the same time, currently there are no non-synonymous single nucleoside polymorphisms, nsSNPs (harmless mutations), found in SMS, which may imply that the SMS does not tolerate amino acid substitutions, i.e. is not mutable. Methodology/Principal Findings To investigate the mutability of the SMS, we carried out in silico analysis and in vitro experiments of the effects of amino acid substitutions at the missense mutation sites (G56, V132 and I150) that have been shown to cause SRS. Our investigation showed that the mutation sites have different degree of mutability depending on their structural micro-environment and involvement in the function and structural integrity of the SMS. It was found that the I150 site does not tolerate any mutation, while V132, despite its key position at the interface of SMS dimer, is quite mutable. The G56 site is in the middle of the spectra, but still quite sensitive to charge residue replacement. Conclusions/Significance The performed analysis showed that mutability depends on the detail of the structural and functional factors and cannot be predicted based on conservation of wild type properties alone. Also, harmless nsSNPs can be expected to occur even at sites at which missense mutations were found to cause diseases. PMID:21647366

A Complex Between Biotin Synthase and The Iron-Sulfur Cluster Assembly Chaperone HscA That Enhances In Vivo Cluster Assembly†

PubMed Central

Reyda, Michael R.; Fugate, Corey J.; Jarrett, Joseph T.

2009-01-01

Biotin synthase (BioB) is an iron-sulfur enzyme that catalyzes the last step in biotin biosynthesis, the insertion of sulfur between the C6 and C9 carbons of dethiobiotin to complete the thiophane ring of biotin. Recent in vitro experiments suggest that the sulfur is derived from a [2Fe-2S]2+ cluster within BioB, and that the remnants of this cluster dissociate from the enzyme following each turnover. In order for BioB to catalyze multiple rounds of biotin synthesis, the [2Fe-2S]2+ cluster in BioB must be reassembled, a process that could be carried out in vivo by the ISC or SUF iron-sulfur cluster assembly systems. The bacterial ISC system includes HscA, an Hsp70-class molecular chaperone, whose yeast homolog has been shown to play an important but nonessential role in assembly of mitochondrial FeS clusters in S. cerevesiae. In the present work we show that in E. coli, HscA significantly improves the efficiency of the in vivo assembly of the [2Fe-2S]2+ cluster on BioB under conditions of low to moderate iron. In vitro, we show that HscA binds with increased affinity to BioB missing one or both FeS clusters, with a maximum of two HscA molecules per BioB dimer. BioB binds to HscA in an ATP/ADP-independent manner and a high affinity complex is also formed with a truncated form of HscA that lacks the nucleotide binding domain. Further, the BioB:HscA complex binds the FeS cluster scaffold protein IscU in a noncompetitive manner, generating a complex that contains all three proteins. We propose that HscA plays a role in facilitating the transfer of FeS clusters from IscU into the appropriate target apoproteins such as biotin synthase, perhaps by enhancing or prolonging the requisite protein:protein interaction. PMID:19821612

PpASCL, the Physcomitrella patens Anther-Specific Chalcone Synthase-Like Enzyme Implicated in Sporopollenin Biosynthesis, Is Needed for Integrity of the Moss Spore Wall and Spore Viability

PubMed Central

Daku, Rhys M.; Rabbi, Fazle; Buttigieg, Josef; Coulson, Ian M.; Horne, Derrick; Martens, Garnet; Ashton, Neil W.; Suh, Dae-Yeon

2016-01-01

Sporopollenin is the main constituent of the exine layer of spore and pollen walls. The anther-specific chalcone synthase-like (ASCL) enzyme of Physcomitrella patens, PpASCL, has previously been implicated in the biosynthesis of sporopollenin, the main constituent of exine and perine, the two outermost layers of the moss spore cell wall. We made targeted knockouts of the corresponding gene, PpASCL, and phenotypically characterized ascl sporophytes and spores at different developmental stages. Ascl plants developed normally until late in sporophytic development, when the spores produced were structurally aberrant and inviable. The development of the ascl spore cell wall appeared to be arrested early in microspore development, resulting in small, collapsed spores with altered surface morphology. The typical stratification of the spore cell wall was absent with only an abnormal perine recognisable above an amorphous layer possibly representing remnants of compromised intine and/or exine. Equivalent resistance of the spore walls of ascl mutants and the control strain to acetolysis suggests the presence of chemically inert, defective sporopollenin in the mutants. Anatomical abnormalities of late-stage ascl sporophytes include a persistent large columella and an air space incompletely filled with spores. Our results indicate that the evolutionarily conserved PpASCL gene is needed for proper construction of the spore wall and for normal maturation and viability of moss spores. PMID:26752629

Sigma- versus Pi-Dimerization Modes of Triangulene.

PubMed

Mou, Zhongyu; Kertesz, Miklos

2018-04-20

We show that the diradicaloid triangulene, a graphene nano-flake molecule, can aggregate in a variety of dimerization modes. We found by density functional theory modeling a number of triangulene dimers including six doubly bonded σ-dimers in addition to the previously reported six pancake bonded π-dimer isomers. The σ-dimers display a wide range of stabilities: the interaction energy of the most stable σ-dimer is -25.17 kcal mol -1 . Besides the doubly bonded σ-dimers with closed shell ground states, we also found an open-shell singly σ-bonded diradicaloid dimer. We found an interesting isomerization route between a doubly bonded σ-dimer, a singly bonded σ-dimer with a low-lying triplet state and two π-bonded dimers with low-lying quintet states. Derivatives of triangulene, trioxo-triangulenes (TOTs) have been previously characterized experimentally. Here, we show the reasons why so far only the π-dimer but not the σ-dimer was experimentally observed for all TOTs. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ligand-mediated Galectin-1 endocytosis prevents intraneural H2O2 production promoting F-actin dynamics reactivation and axonal re-growth.

PubMed

Quintá, Héctor R; Wilson, Carlos; Blidner, Ada G; González-Billault, Christian; Pasquini, Laura A; Rabinovich, Gabriel A; Pasquini, Juana M

2016-09-01

Axonal growth cone collapse following spinal cord injury (SCI) is promoted by semaphorin3A (Sema3A) signaling via PlexinA4 surface receptor. This interaction triggers intracellular signaling events leading to increased hydrogen peroxide levels which in turn promote filamentous actin (F-actin) destabilization and subsequent inhibition of axonal re-growth. In the current study, we demonstrated that treatment with galectin-1 (Gal-1), in its dimeric form, promotes a decrease in hydrogen peroxide (H2O2) levels and F-actin repolimerization in the growth cone and in the filopodium of neuron surfaces. This effect was dependent on the carbohydrate recognition activity of Gal-1, as it was prevented using a Gal-1 mutant lacking carbohydrate-binding activity. Furthermore, Gal-1 promoted its own active ligand-mediated endocytosis together with the PlexinA4 receptor, through mechanisms involving complex branched N-glycans. In summary, our results suggest that Gal-1, mainly in its dimeric form, promotes re-activation of actin cytoskeleton dynamics via internalization of the PlexinA4/Gal-1 complex. This mechanism could explain, at least in part, critical events in axonal regeneration including the full axonal re-growth process, de novo formation of synapse clustering, axonal re-myelination and functional recovery of coordinated locomotor activities in an in vivo acute and chronic SCI model. Axonal regeneration is a response of injured nerve cells critical for nerve repair in human spinal cord injury. Understanding the molecular mechanisms controlling nerve repair by Galectin-1, may be critical for therapeutic intervention. Our results show that Galectin-1; in its dimeric form, interferes with hydrogen peroxide production triggered by Semaphorin3A. The high levels of this reactive oxygen species (ROS) seem to be the main factor preventing axonal regeneration due to promotion of actin depolymerization at the axonal growth cone. Thus, Galectin-1 administration emerges as a novel therapeutic modality for promoting nerve repair and preventing axonal loss. Copyright © 2016 Elsevier Inc. All rights reserved.

Cardiolipin plays a role in KCN-induced necrosis.

PubMed

Tsesin, Natalia; Khalfin, Boris; Nathan, Ilana; Parola, Abraham H

2014-10-01

Cardiolipin (CL) is a unique anionic, dimeric phospholipid found almost exclusively in the inner mitochondrial membrane and is essential for the function of numerous enzymes that are involved in mitochondrial energy metabolism. While the role of cardiolipin in apoptosis is well established, its involvement in necrosis is enigmatic. In the present study, KCN-induced necrosis in U937 cells was used as an experimental model to assess the role of CL in necrosis. KCN addition to U937 cells induced reactive oxygen species (ROS) formation, while the antioxidants inhibited necrosis, indicating that ROS play a role in KCN-induced cell death. Further, CL oxidation was confirmed by the monomer green fluorescence of 10-N-nonyl acridine orange (NAO) and by TLC. Utilizing the red fluorescence of the dimeric NAO, redistribution of CL in mitochondrial membrane during necrosis was revealed. We also showed that the catalytic activity of purified adenosine triphosphate (ATP) synthase complex, known to be modulated by cardiolipin, decreased following KCN treatment. All these events occurred at an early phase of the necrotic process prior to rupture of the cell membrane. Furthermore, CL-deficient HeLa cells were found to be resistant to KCN-induced necrosis as compared with the wild type cells. We suggest that KCN, an effective reversible inhibitor of cytochrome oxidase and thereby of the respiratory chain leads to ROS increase, which in turn oxidizes CL (amongst other membrane phospholipids) and leads to mitochondrial membrane lipid reorganization and loss of CL symmetry. Finally, the resistance of CL-deficient cells to necrosis further supports the notion that CL, which undergoes oxidation during necrotic cell death, is an integral part of the milieu of events taking place in mitochondria leading to membrane disorganization and mitochondrial dysfunction. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

A yeast 2-hybrid analysis of human GTP cyclohydrolase I protein interactions

PubMed Central

Swick, Lance; Kapatos, Gregory

2008-01-01

The yeast 2-hybrid system was used to identify protein domains involved in the oligomerization of human guanosine 5′-triphosphate (GTP) Cyclohydrolase I (GCH1) and the interaction of GCH1 with its regulatory partner, GCH1 feedback regulatory protein (GFRP). When interpreted within the structural framework derived from crystallography, our results indicate that the GCH1 N-terminal α-helices are not the only domains involved in the formation of dimers from monomers and also suggest an important role for the C-terminal α-helix in the assembly of dimers to form decamers. Moreover, a previously unknown role of the extended N-terminal α–helix in the interaction of GCH1 and GFRP was revealed. To discover novel GCH1 protein binding partners, we used the yeast 2-hybrid system to screen a human brain library with GCH1 N-terminal amino acids 1–96 as prey. This protruding extension of GCH1 contains two canonical Type-I Src homology-3 (SH3) ligand domains located within amino acids 1–42. Our screen yielded seven unique clones that were subsequently shown to require amino acids 1–42 for binding to GCH1. The interaction of one of these clones, Activator of Heat Shock 90 kDa Protein (Aha1), with GCH1 was validated by glutathione-s-transferase (GST) pull-down assay. Although the physiological relevance of the Aha1–GCH1 interaction requires further study, Aha1 may recruit GCH1 into the endothelial nitric oxide synthase/heat shock protein (eNOS/Hsp90) complex to support changes in endothelial nitric oxide production through the local synthesis of BH4. PMID:16696853

Spectroscopic and computational studies of a trans-mu-1,2-disulfido-bridged dinickel species, [{(tmc)Ni}(2)(S(2))](OTf)(2): comparison of end-on disulfido and peroxo bonding in (Ni(II))(2) and (Cu(II))(2) species.

PubMed

Van Heuvelen, Katherine M; Kieber-Emmons, Matthew T; Riordan, Charles G; Brunold, Thomas C

2010-04-05

A powerful means of enhancing our understanding of the structures and functions of enzymes that contain nickel-sulfur bonds, such as Ni superoxide dismutase, acetyl-coenzyme A synthase/carbon monoxide dehydrogenase, [NiFe] hydrogenase, and methyl-CoM reductase, involves the investigation of model compounds with similar structural and/or electronic properties. In this study, we have characterized a trans-mu-1,2-disulfido-bridged dinickel(II) species, [{(tmc)Ni}(2)(S(2))](2+) (1, tmc = 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane) by using electronic absorption, magnetic circular dichroism (MCD), and resonance Raman (rR) spectroscopic techniques, as well as density functional theory (DFT) and time-dependent DFT computational methods. Our computational results, validated on the basis of the experimental MCD data and previously reported (1)H NMR spectra, reveal that 1 is best described as containing two antiferromagnetically coupled high-spin Ni(II) centers. A normal coordinate analysis of the rR vibrational data was performed to quantify the core bond strengths, yielding force constants of k(Ni-S) = 2.69 mdyn/A and k(S-S) = 2.40 mdyn/A. These values provide a useful basis for a comparison of metal-S/O bonding in 1 and related Ni(2)(O(2)), Cu(2)(O(2)), and Cu(2)(S(2)) dimers. In both the disulfido and the peroxo species, the lower effective nuclear charge of Ni(II) as compared to Cu(II) results in a decreased covalency, and thus relatively weaker metal-S/O bonding interactions in the Ni(2) dimers than in the Cu(2) complexes.

Effects of Diabetes on Salivary Gland Protein Expression of Tetrahydrobiopterin and Nitric Oxide Synthesis and Function.

PubMed

Stewart, Cassandra R; Obi, Nneka; Epane, Elodie C; Akbari, Alexander A; Halpern, Leslie; Southerland, Janet H; Gangula, Pandu R

2016-06-01

Xerostomia is defined as dry mouth resulting from a change in the amount or composition of saliva and is often a major oral health complication associated with diabetes mellitus (DM). Studies have shown that xerostomia is more common in females at the onset of DM. Evidence suggests that nitric oxide (NO) plays a critical role in healthy salivary gland function. However, the specific mechanisms by which NO regulates salivary gland function at the onset of DM have yet to be determined. This study has two aims: 1) to determine whether protein expression or dimerization of NO synthase enzymes (neuronal [nNOS] and endothelial [eNOS]) are altered in the onset of diabetic xerostomia; and 2) to determine whether the changes in nNOS/eNOS protein expression or dimerization are correlated with changes in NO cofactor tetrahydrobiopterin (BH4) biosynthetic enzymes (guanosine triphosphate cyclohydrolase-1 and dihydrofolate reductase). Functional and Western blot studies were performed in streptozotocin-induced and control Sprague-Dawley female rats with DM (type 1 [t1DM]) using standardized protocols. Confirmation of xerostomia was determined by increased water intake and decreased salivary flow rate. The results showed that in female rats with DM, salivary hypofunction is correlated with decreased submandibular and parotid gland sizes. The results also show a decrease in NOS and BH4 biosynthetic enzyme in submandibular glands. These results indicate that a decrease in submandibular NO-BH4 protein expression may provide insight pertaining to mechanisms for the development of hyposalivation in DM-induced xerostomia. Furthermore, understanding the role of the NO-BH4 pathway may give insight into possible treatment options for the patient with DM experiencing xerostomia.

Epitaxial growth mechanisms of graphene and effects of substrates

NASA Astrophysics Data System (ADS)

Özçelik, V. Ongun; Cahangirov, S.; Ciraci, S.

2012-06-01

The growth process of single layer graphene with and without substrate is investigated using ab initio, finite temperature molecular dynamic calculations within density functional theory. An understanding of the epitaxial graphene growth mechanisms in the atomic level is provided by exploring the transient stages which occur at the growing edges of graphene. These stages are formation and collapse of large carbon rings together with the formation and healing of Stone-Wales like pentagon-heptagon defects. The activation barriers for the healing of these growth induced defects on various substrates are calculated using the climbing image nudge elastic band method and compared with that of the Stone-Wales defect. It is found that the healing of pentagon-heptagon defects occurring near the edge in the course of growth is much easier than that of Stone-Wales defect. The role of the substrate in the epitaxial growth and in the healing of defects are also investigated in detail, along with the effects of using carbon dimers as the building blocks of graphene growth.

Atom loss resonances in a Bose-Einstein condensate.

PubMed

Langmack, Christian; Smith, D Hudson; Braaten, Eric

2013-07-12

Atom loss resonances in ultracold trapped atoms have been observed at scattering lengths near atom-dimer resonances, at which Efimov trimers cross the atom-dimer threshold, and near two-dimer resonances, at which universal tetramers cross the dimer-dimer threshold. We propose a new mechanism for these loss resonances in a Bose-Einstein condensate of atoms. As the scattering length is ramped to the large final value at which the atom loss rate is measured, the time-dependent scattering length generates a small condensate of shallow dimers coherently from the atom condensate. The coexisting atom and dimer condensates can be described by a low-energy effective field theory with universal coefficients that are determined by matching exact results from few-body physics. The classical field equations for the atom and dimer condensates predict narrow enhancements in the atom loss rate near atom-dimer resonances and near two-dimer resonances due to inelastic dimer collisions.

Testing computational prediction of missense mutation phenotypes: Functional characterization of 204 mutations of human cystathionine beta synthase

PubMed Central

Wei, Qiong; Wang, Liqun; Wang, Qiang; Kruger, Warren D.; Dunbrack, Roland L.

2010-01-01

Predicting the phenotypes of missense mutations uncovered by large-scale sequencing projects is an important goal in computational biology. High-confidence predictions can be an aid in focusing experimental and association studies on those mutations most likely to be associated with causative relationships between mutation and disease. As an aid in developing these methods further, we have derived a set of random mutations of the enzymatic domains of human cystathionine beta synthase. This enzyme is a dimeric protein that catalyzes the condensation of serine and homocysteine to produce cystathionine. Yeast missing this enzyme cannot grow on medium lacking a source of cysteine, while transfection of functional human CBS into yeast strains missing endogenous enzyme can successfully complement for the missing gene. We used PCR mutagenesis with error-prone Taq polymerase to produce 948 colonies, and compared cell growth in the presence or absence of a cysteine source as a measure of CBS function. We were able to infer the phenotypes of 204 single-site mutants, 79 of them deleterious and 125 neutral. This set was used to test the accuracy of six publicly available prediction methods for phenotype prediction of missense mutations: SIFT, PolyPhen, PMut, SNPs3D, PhD-SNP, and nsSNPAnalyzer. The top methods are PolyPhen, SIFT, and nsSNPAnalyzer, which have similar performance. Using kernel discriminant functions, we found that the difference in position-specific scoring matrix values is more predictive than the wild-type PSSM score alone, and that the relative surface area in the biologically relevant complex is more predictive than that of the monomeric proteins. PMID:20455263

Cystathionine β-Synthase (CBS) Domain-containing Pyrophosphatase as a Target for Diadenosine Polyphosphates in Bacteria*

PubMed Central

Anashkin, Viktor A.; Salminen, Anu; Tuominen, Heidi K.; Orlov, Victor N.; Lahti, Reijo; Baykov, Alexander A.

2015-01-01

Among numerous proteins containing pairs of regulatory cystathionine β-synthase (CBS) domains, family II pyrophosphatases (CBS-PPases) are unique in that they generally contain an additional DRTGG domain between the CBS domains. Adenine nucleotides bind to the CBS domains in CBS-PPases in a positively cooperative manner, resulting in enzyme inhibition (AMP or ADP) or activation (ATP). Here we show that linear P1,Pn-diadenosine 5′-polyphosphates (ApnAs, where n is the number of phosphate residues) bind with nanomolar affinity to DRTGG domain-containing CBS-PPases of Desulfitobacterium hafniense, Clostridium novyi, and Clostridium perfringens and increase their activity up to 30-, 5-, and 7-fold, respectively. Ap4A, Ap5A, and Ap6A bound noncooperatively and with similarly high affinities to CBS-PPases, whereas Ap3A bound in a positively cooperative manner and with lower affinity, like mononucleotides. All ApnAs abolished kinetic cooperativity (non-Michaelian behavior) of CBS-PPases. The enthalpy change and binding stoichiometry, as determined by isothermal calorimetry, were ∼10 kcal/mol nucleotide and 1 mol/mol enzyme dimer for Ap4A and Ap5A but 5.5 kcal/mol and 2 mol/mol for Ap3A, AMP, ADP, and ATP, suggesting different binding modes for the two nucleotide groups. In contrast, Eggerthella lenta and Moorella thermoacetica CBS-PPases, which contain no DRTGG domain, were not affected by ApnAs and showed no enthalpy change, indicating the importance of the DTRGG domain for ApnA binding. These findings suggest that ApnAs can control CBS-PPase activity and hence affect pyrophosphate level and biosynthetic activity in bacteria. PMID:26400082

Allosteric Inhibition of Human Porphobilinogen Synthase*

PubMed Central

Lawrence, Sarah H.; Ramirez, Ursula D.; Selwood, Trevor; Stith, Linda; Jaffe, Eileen K.

2009-01-01

Porphobilinogen synthase (PBGS) catalyzes the first common step in tetrapyrrole (e.g. heme, chlorophyll) biosynthesis. Human PBGS exists as an equilibrium of high activity octamers, low activity hexamers, and alternate dimer configurations that dictate the stoichiometry and architecture of further assembly. It is posited that small molecules can be found that inhibit human PBGS activity by stabilizing the hexamer. Such molecules, if present in the environment, could potentiate disease states associated with reduced PBGS activity, such as lead poisoning and ALAD porphyria, the latter of which is associated with human PBGS variants whose quaternary structure equilibrium is shifted toward the hexamer (Jaffe, E. K., and Stith, L. (2007) Am. J. Hum. Genet. 80, 329–337). Hexamer-stabilizing inhibitors of human PBGS were identified using in silico prescreening (docking) of ∼111,000 structures to a hexamer-specific surface cavity of a human PBGS crystal structure. Seventy-seven compounds were evaluated in vitro; three provided 90–100% conversion of octamer to hexamer in a native PAGE mobility shift assay. Based on chemical purity, two (ML-3A9 and ML-3H2) were subjected to further evaluation of their effect on the quaternary structure equilibrium and enzymatic activity. Naturally occurring ALAD porphyria-associated human PBGS variants are shown to have an increased susceptibility to inhibition by both ML-3A9 and ML-3H2. ML-3H2 is a structural analog of amebicidal drugs, which have porphyria-like side effects. Data support the hypothesis that human PBGS hexamer stabilization may explain these side effects. The current work identifies allosteric ligands of human PBGS and, thus, identifies human PBGS as a medically relevant allosteric enzyme. PMID:19812033

Neutron Scattering Studies on Correlated Transition-Metal Oxides

NASA Astrophysics Data System (ADS)

Zhu, Mengze

We have explored the collective phenomena of correlated electrons in two different transition-metal oxides, Ruddlesden-Popper type ruthenates (Sr,Ca) n+1RunO3n+1 and inverse-trirutile chromates Cr2MO6 (M = Te, Mo and W), using neutron scattering in combination with various material characterization methods. (Sr,Ca)n+1RunO 3n+1 are 4d transition-metal oxides exhibiting competing magnetic and electronic tendencies. The delicate balance among the competing states can be readily tuned by perturbations, such as chemical doping and magnetic field, which gives rise to emergent phenomena. We have investigated the effects of 3d transition-metal doping on the magnetic and electronic properties of layered ruthenates. For instance, the single-layer (n = 1) Sr2RuO4 is an unconventional superconductor possessing an incommensurate spin density wave instability with a wave vector qic= (0.3 0.3 L) driven by Fermi surface nesting. Upon Fe substitution, we have unveiled an unexpected commensurate spin density wave order with a propagation vector qc= (0.25 0.25 0) in Sr2Ru1-xFexO 4 (x = 0.03 and 0.05), despite the magnetic fluctuations persisting at qic. The latter feature is corroborated by the first principles calculations, which show that Fe doping barely changes the nesting vector of the Fermi surface. These results suggest that in addition to the known incommensurate magnetic instability, Sr2RuO4 is also in proximity to a commensurate magnetic tendency that can be stabilized via Fe doping. We have also studied the effects of a magnetic field. For example, the bilayer (n = 2) Ca3(Ru1-xTi x)2O7 (x = 0.03) is a G-type antiferromagnetic Mott insulator. We have revealed that a modest magnetic field can lead to colossal magnetoresistance arising from an anomalous collapse of the Mott insulating state. Such an insulator-to-metal transition is accompanied by magnetic and structural transitions. These findings call for deeper theoretical studies to reexamine the magnetic field tuning of Mott systems with magnetic and electronic instabilities, as a magnetic field usually stabilizes the insulating ground state in Mott-Hubbard systems. Cr2MO6 (M = Te, W and Mo) are spin dimer systems with the magnetic ions Cr3+ structurally dimerized favoring a singlet ground state. However, all three compounds investigated exhibit long-range antiferromagnetic orders at low temperature owing to the inter-dimer interactions. We have shown that the inter-dimer exchange coupling can be tuned from antiferromagnetic in Cr2TeO6 to ferromagnetic in Cr2WO6 and Cr2MoO6, by altering the degree of d-p orbital hybridization between W(Mo) and O atoms. The tunability of the inter-dimer interactions without introducing additional complexities such as structural distortions and carrier doping offers a rare opportunity to drive the system toward the quantum critical point (QCP) separating the dimer-based quantum disordered state and the classical long-range antiferromagnetic order. Moreover, we have unraveled Higgs amplitude modes in the magnetic excitation spectra of Cr2TeO6 and Cr2WO6, which are generally believed to survive only in systems close to the QCP where the ordered moment is suppressed significantly from its fully saturated value by quantum fluctuations. However, these two compounds are away from the QCP with the ordered moment reduced only by 24%. This study suggests that Higgs amplitude modes are not the privilege of ordered systems in the vicinity of the QCP, but may be common excitation modes in ordered spin dimer systems.

D-dimer test

MedlinePlus

... vein thrombosis - D-dimer; Pulmonary embolism - D-dimer; Blood clot to the lungs - D-dimer ... dimer test if you are showing symptoms of blood clots, such as: Swelling, pain, warmth, and changes in ...

Dimer monomer transition and dimer re-formation play important role for ATM cellular function during DNA repair

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du, Fengxia; Zhang, Minjie; University of Chinese Academy of Sciences, Beijing 100049

2014-10-03

Highlights: • ATM phosphorylates the opposite strand of the dimer in response to DNA damage. • The PETPVFRLT box of ATM plays a key role in its dimer dissociation in DNA repair. • The dephosphorylation of ATM is critical for dimer re-formation after DNA repair. - Abstract: The ATM protein kinase, is a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks, mediates responses to ionizing radiation in mammalian cells. Here we show that ATM is held inactive in unirradiated cells as a dimer and phosphorylates the opposite strand of the dimer in response to DNA damage.more » Cellular irradiation induces rapid intermolecular autophosphorylation of serine 1981 that causes dimer dissociation and initiates cellular ATM kinase activity. ATM cannot phosphorylate the substrates when it could not undergo dimer monomer transition. After DNA repair, the active monomer will undergo dephosphorylation to form dimer again and dephosphorylation is critical for dimer re-formation. Our work reveals novel function of ATM dimer monomer transition and explains why ATM dimer monomer transition plays such important role for ATM cellular activity during DNA repair.« less

Effects of Dimers on Cooperation in the Spatial Prisoner's Dilemma Game

NASA Astrophysics Data System (ADS)

Li, Hai-Hong; Cheng, Hong-Yan; Dai, Qiong-Lin; Ju, Ping; Zhang, Mei; Yang, Jun-Zhong

2011-11-01

We investigate the evolutionary prisoner's dilemma game in structured populations by introducing dimers, which are defined as that two players in each dimer always hold a same strategy. We find that influences of dimers on cooperation depend on the type of dimers and the population structure. For those dimers in which players interact with each other, the cooperation level increases with the number of dimers though the cooperation improvement level depends on the type of network structures. On the other hand, the dimers, in which there are not mutual interactions, will not do any good to the cooperation level in a single community, but interestingly, will improve the cooperation level in a population with two communities. We explore the relationship between dimers and self-interactions and find that the effects of dimers are similar to that of self-interactions. Also, we find that the dimers, which are established over two communities in a multi-community network, act as one type of interaction through which information between communities is communicated by the requirement that two players in a dimer hold a same strategy.

Biophysical Characterization of the Dimer and Tetramer Interface Interactions of the Human Cytosolic Malic Enzyme

PubMed Central

Murugan, Sujithkumar; Hung, Hui-Chih

2012-01-01

The cytosolic NADP+-dependent malic enzyme (c-NADP-ME) has a dimer-dimer quaternary structure in which the dimer interface associates more tightly than the tetramer interface. In this study, the urea-induced unfolding process of the c-NADP-ME interface mutants was monitored using fluorescence and circular dichroism spectroscopy, analytical ultracentrifugation and enzyme activities. Here, we demonstrate the differential protein stability between dimer and tetramer interface interactions of human c-NADP-ME. Our data clearly demonstrate that the protein stability of c-NADP-ME is affected predominantly by disruptions at the dimer interface rather than at the tetramer interface. First, during thermal stability experiments, the melting temperatures of the wild-type and tetramer interface mutants are 8–10°C higher than those of the dimer interface mutants. Second, during urea denaturation experiments, the thermodynamic parameters of the wild-type and tetramer interface mutants are almost identical. However, for the dimer interface mutants, the first transition of the urea unfolding curves shift towards a lower urea concentration, and the unfolding intermediate exist at a lower urea concentration. Third, for tetrameric WT c-NADP-ME, the enzyme is first dissociated from a tetramer to dimers before the 2 M urea treatment, and the dimers then dissociated into monomers before the 2.5 M urea treatment. With a dimeric tetramer interface mutant (H142A/D568A), the dimer completely dissociated into monomers after a 2.5 M urea treatment, while for a dimeric dimer interface mutant (H51A/D90A), the dimer completely dissociated into monomers after a 1.5 M urea treatment, indicating that the interactions of c-NADP-ME at the dimer interface are truly stronger than at the tetramer interface. Thus, this study provides a reasonable explanation for why malic enzymes need to assemble as a dimer of dimers. PMID:23284632

Synthesis and photophysical properties of a single bond linked tetracene dimer

NASA Astrophysics Data System (ADS)

Sun, Tingting; Shen, Li; Liu, Heyuan; Sun, Xuan; Li, Xiyou

2016-07-01

A tetracene dimer linked directly by a single bond has been successfully prepared by using electron withdrawing groups to improve the stability. The molecular structure of this dimer is characterized by 1H NMR, MALDI-TOF mass spectroscopy, and elemental analysis. The minimized molecular structure and X-ray crystallography reveal that the tetracene subunits of this dimer adopt an orthogonal configuration. Its absorption spectrum differs significantly from that of its monomeric counterpart, suggesting the presence of strong interactions between the two tetracene subunits. The excited state of this dimer is delocalized on both two tetracene subunits, which is significantly different from that of orthogonal anthracene dimers, but similar with that observed for orthogonal pentacene dimer. Most of the excited states of this dimer decay by radioactive channels, which is different from the localized twisted charge transfer state (LTCT) channel of anthracene dimers and the singlet fission (SF) channel of pentacene dimers. The results of this research suggest that similar orthogonal configurations caused different propertied for acene dimers with different conjugation length.

Monoterpene synthases from common sage (Salvia officinalis)

DOEpatents

Croteau, Rodney Bruce; Wise, Mitchell Lynn; Katahira, Eva Joy; Savage, Thomas Jonathan

1999-01-01

cDNAs encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase from common sage (Salvia officinalis) have been isolated and sequenced, and the corresponding amino acid sequences has been determined. Accordingly, isolated DNA sequences (SEQ ID No:1; SEQ ID No:3 and SEQ ID No:5) are provided which code for the expression of (+)-bornyl diphosphate synthase (SEQ ID No:2), 1,8-cineole synthase (SEQ ID No:4) and (+)-sabinene synthase SEQ ID No:6), respectively, from sage (Salvia officinalis). In other aspects, replicable recombinant cloning vehicles are provided which code for (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase, or for a base sequence sufficiently complementary to at least a portion of (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (+)-bornyl diphosphate synthase, 1,8-cineole synthase or (+)-sabinene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant monoterpene synthases that may be used to facilitate their production, isolation and purification in significant amounts. Recombinant (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase may be used to obtain expression or enhanced expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase in plants in order to enhance the production of monoterpenoids, or may be otherwise employed for the regulation or expression of (+)-bornyl diphosphate synthase, 1,8-cineole synthase and (+)-sabinene synthase, or the production of their products.

Tubulin Dimer Reversible Dissociation

PubMed Central

Schuck, Peter; Sackett, Dan L.

2016-01-01

Tubulins are evolutionarily conserved proteins that reversibly polymerize and direct intracellular traffic. Of the tubulin family only αβ-tubulin forms stable dimers. We investigated the monomer-dimer equilibrium of rat brain αβ-tubulin using analytical ultracentrifugation and fluorescence anisotropy, observing tubulin in virtually fully monomeric and dimeric states. Monomeric tubulin was stable for a few hours and exchanged into preformed dimers, demonstrating reversibility of dimer dissociation. Global analysis combining sedimentation velocity and fluorescence anisotropy yielded Kd = 84 (54–123) nm. Dimer dissociation kinetics were measured by analyzing the shape of the sedimentation boundary and by the relaxation of fluorescence anisotropy following rapid dilution of labeled tubulin, yielding koff in the range 10−3–10−2 s−1. Thus, tubulin dimers reversibly dissociate with moderately fast kinetics. Monomer-monomer association is much less sensitive than dimer-dimer association to solution changes (GTP/GDP, urea, and trimethylamine oxide). PMID:26934918

Structure of the hexameric HerA ATPase reveals a mechanism of translocation-coupled DNA-end processing in archaea.

PubMed

Rzechorzek, Neil J; Blackwood, John K; Bray, Sian M; Maman, Joseph D; Pellegrini, Luca; Robinson, Nicholas P

2014-11-25

The HerA ATPase cooperates with the NurA nuclease and the Mre11-Rad50 complex for the repair of double-strand DNA breaks in thermophilic archaea. Here we extend our structural knowledge of this minimal end-resection apparatus by presenting the first crystal structure of hexameric HerA. The full-length structure visualizes at atomic resolution the N-terminal HerA-ATP synthase domain and a conserved C-terminal extension, which acts as a physical brace between adjacent protomers. The brace also interacts in trans with nucleotide-binding residues of the neighbouring subunit. Our observations support a model in which the coaxial interaction of the HerA ring with the toroidal NurA dimer generates a continuous channel traversing the complex. HerA-driven translocation would propel the DNA towards the narrow annulus of NurA, leading to duplex melting and nucleolytic digestion. This system differs substantially from the bacterial end-resection paradigms. Our findings suggest a novel mode of DNA-end processing by this integrated archaeal helicase-nuclease machine.

A mitochondrial-focused genetic interaction map reveals a scaffold-like complex required for inner membrane organization in mitochondria

PubMed Central

Hoppins, Suzanne; Collins, Sean R.; Cassidy-Stone, Ann; Hummel, Eric; DeVay, Rachel M.; Lackner, Laura L.; Westermann, Benedikt; Schuldiner, Maya

2011-01-01

To broadly explore mitochondrial structure and function as well as the communication of mitochondria with other cellular pathways, we constructed a quantitative, high-density genetic interaction map (the MITO-MAP) in Saccharomyces cerevisiae. The MITO-MAP provides a comprehensive view of mitochondrial function including insights into the activity of uncharacterized mitochondrial proteins and the functional connection between mitochondria and the ER. The MITO-MAP also reveals a large inner membrane–associated complex, which we term MitOS for mitochondrial organizing structure, comprised of Fcj1/Mitofilin, a conserved inner membrane protein, and five additional components. MitOS physically and functionally interacts with both outer and inner membrane components and localizes to extended structures that wrap around the inner membrane. We show that MitOS acts in concert with ATP synthase dimers to organize the inner membrane and promote normal mitochondrial morphology. We propose that MitOS acts as a conserved mitochondrial skeletal structure that differentiates regions of the inner membrane to establish the normal internal architecture of mitochondria. PMID:21987634

cis-Prenyltransferase: New Insights into Protein Glycosylation, Rubber Synthesis, and Human Diseases.

PubMed

Grabińska, Kariona A; Park, Eon Joo; Sessa, William C

2016-08-26

cis-Prenyltransferases (cis-PTs) constitute a large family of enzymes conserved during evolution and present in all domains of life. cis-PTs catalyze consecutive condensation reactions of allylic diphosphate acceptor with isopentenyl diphosphate (IPP) in the cis (Z) configuration to generate linear polyprenyl diphosphate. The chain lengths of isoprenoid carbon skeletons vary widely from neryl pyrophosphate (C10) to natural rubber (C>10,000). The homo-dimeric bacterial enzyme, undecaprenyl diphosphate synthase (UPPS), has been structurally and mechanistically characterized in great detail and serves as a model for understanding the mode of action of eukaryotic cis-PTs. However, recent experiments have revealed that mammals, fungal, and long-chain plant cis-PTs are heteromeric enzymes composed of two distantly related subunits. In this review, the classification, function, and evolution of cis-PTs will be discussed with a special emphasis on the role of the newly described NgBR/Nus1 subunit and its plants' orthologs as essential, structural components of the cis-PTs activity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Time course of salinity adaptation in a strongly euryhaline estuarine teleost, fundulus heteroclitus: A multivariable approach

USGS Publications Warehouse

Marshall, W.S.; Emberley, T.R.; Singer, T.D.; Bryson, S.E.; McCormick, S.D.

1999-01-01

Freshwater-adapted killifish (Fundulus heteroclitus) were transferred directly from soft fresh water to full-strength sea water for periods of 1h, 3h, 8h and 1, 2, 7, 14 and 30 days. Controls were transferred to fresh water for 24 h. Measured variables included: blood [Na+], osmolality, glucose and cortisol levels, basal and stimulated rates of ion transport and permeability of in vitro opercular epithelium, gill Na+/K+-ATPase and citrate synthase activity and chloride cell ultrastructure. These data were compared with previously published killifish cystic fibrosis transmembrane conductance regulator (kfCFTR) expression in the gills measured over a similar time course. Plasma cortisol levels peaked at 1 h, coincident with a rise in plasma [Na+]. At 8 h after transfer to sea water, a time at which previous work has shown kfCFTR expression to be elevated, blood osmolality and [Na+] were high, and cortisol levels and opercular membrane short-circuit current (I(SC); a measure of Cl- secretion rate) were low. The 24h group, which showed the highest level of kfCFTR expression, had the highest plasma [Na+] and osmolality, elevated plasma cortisol levels, significantly lower opercular membrane resistance, an increased opercular membrane ion secretion rate and collapsed tubule inclusions in mitochondria-rich cells, but no change in gill Na+/K+-ATPase and citrate synthase activity or plasma glucose levels. Apparently, killifish have a rapid (<1h) cortisol response to salinity coupled to subsequent (8-48 h) expression of kfCFTR anion channel proteins in existing mitochondria-rich cells that convert transport from ion uptake to ion secretion.

Regulation of CDP-diacylglycerol synthesis and utilization by inositol and choline in Schizosaccharomyces pombe.

PubMed Central

Gaynor, P M; Greenberg, M L

1992-01-01

CDP-diacylglycerol (CDP-DG) is an important branchpoint intermediate in eucaryotic phospholipid biosynthesis and could be a key regulatory site in phospholipid metabolism. Therefore, we examined the effects of growth phase, phospholipid precursors, and the disruption of phosphatidylcholine (PC) synthesis on the membrane-associated phospholipid biosynthetic enzymes CDP-DG synthase, phosphatidylglycerolphosphate (PGP) synthase, phosphatidylinositol (PI) synthase, and phosphatidylserine (PS) synthase in cell extracts of the fission yeast Schizosaccharomyces pombe. In complete synthetic medium containing inositol, maximal expression of CDP-DG synthase, PGP synthase, PI synthase, and PS synthase in wild-type cells occurred in the exponential phase of growth and decreased two- to fourfold in the stationary phase of growth. In cells starved for inositol, this decrease in PGP synthase, PI synthase, and PS synthase expression was not observed. Starvation for inositol resulted in a twofold derepression of PGP synthase and PS synthase expression, while PI synthase expression decreased initially and then remained constant. Upon the addition of inositol to inositol-starved cells, there was a rapid and continued increase in PI synthase expression. We examined expression of these enzymes in cho2 and cho1 mutants, which are blocked in the methylation pathway for synthesis of PC. Choline starvation resulted in a decrease in PS synthase and CDP-DG synthase expression in cho1 but not cho2 cells. Expression of PGP synthase and PI synthase was not affected by choline starvation. Inositol starvation resulted in a 1.7-fold derepression of PGP synthase expression in cho2 but not cho1 cells when PC was synthesized. PS synthase expression was not depressed, while CDP-DG synthase and PI synthase expression decreased in cho2 and cho1 cells in the absence of inositol. These results demonstrate that (i) CDP-DG synthase, PGP synthase, PI synthase, and PS synthase are similarly regulated by growth phase; (ii) inositol affects the expression of PGP synthase, PI synthase, and PS synthase; (iii) disruption of the methylation pathway results in aberrant patterns of regulation of growth phase and phospholipid precursors. Important differences between S. pombe and Saccharomyces cerevisiae with regard to regulation of these enzymes are discussed. PMID:1324908

Ligand regulation of a constitutively dimeric EGF receptor

NASA Astrophysics Data System (ADS)

Freed, Daniel M.; Alvarado, Diego; Lemmon, Mark A.

2015-06-01

Ligand-induced receptor dimerization has traditionally been viewed as the key event in transmembrane signalling by epidermal growth factor receptors (EGFRs). Here we show that the Caenorhabditis elegans EGFR orthologue LET-23 is constitutively dimeric, yet responds to its ligand LIN-3 without changing oligomerization state. SAXS and mutational analyses further reveal that the preformed dimer of the LET-23 extracellular region is mediated by its domain II dimerization arm and resembles other EGFR extracellular dimers seen in structural studies. Binding of LIN-3 induces only minor structural rearrangements in the LET-23 dimer to promote signalling. Our results therefore argue that EGFR can be regulated by allosteric changes within an existing receptor dimer--resembling signalling by insulin receptor family members, which share similar extracellular domain compositions but form covalent dimers.

Analytical study of avian reticuloendotheliosis virus dimeric RNA generated in vivo and in vitro.

PubMed

Darlix, J L; Gabus, C; Allain, B

1992-12-01

The retroviral genome consists of two identical RNA molecules associated at their 5' ends by a stable structure called the dimer linkage structure. The dimer linkage structure, while maintaining the dimer state of the retroviral genome, might also be involved in packaging and reverse transcription, as well as recombination during proviral DNA synthesis. To study the dimer structure of the retroviral genome and the mechanism of dimerization, we analyzed features of the dimeric genome of reticuloendotheliosis virus (REV) type A and identified elements required for its dimerization. Here we report that the REV dimeric genome extracted from virions and infected cells, as well as that synthesized in vitro, is more resistant to heat denaturation than avian sarcoma and leukemia virus, murine leukemia virus, or human immunodeficiency virus type 1 dimeric RNA. The minimal domain required to form a stable REV RNA dimer in vitro was found to map between positions 268 and 452 (KpnI and SalI sites), thus corresponding to the E encapsidation sequence (J. E. Embretson and H. M. Temin, J. Virol. 61:2675-2683, 1987). In addition, both the 5' and 3' halves of E are necessary in cis for RNA dimerization and the extent of RNA dimerization is influenced by viral sequences flanking E. Rapid and efficient dimerization of REV RNA containing gag sequences in addition to the E sequences and annealing of replication primer tRNA(Pro) to the primer-binding site necessitate the nucleocapsid protein.

Analytical study of avian reticuloendotheliosis virus dimeric RNA generated in vivo and in vitro.

PubMed Central

Darlix, J L; Gabus, C; Allain, B

1992-01-01

The retroviral genome consists of two identical RNA molecules associated at their 5' ends by a stable structure called the dimer linkage structure. The dimer linkage structure, while maintaining the dimer state of the retroviral genome, might also be involved in packaging and reverse transcription, as well as recombination during proviral DNA synthesis. To study the dimer structure of the retroviral genome and the mechanism of dimerization, we analyzed features of the dimeric genome of reticuloendotheliosis virus (REV) type A and identified elements required for its dimerization. Here we report that the REV dimeric genome extracted from virions and infected cells, as well as that synthesized in vitro, is more resistant to heat denaturation than avian sarcoma and leukemia virus, murine leukemia virus, or human immunodeficiency virus type 1 dimeric RNA. The minimal domain required to form a stable REV RNA dimer in vitro was found to map between positions 268 and 452 (KpnI and SalI sites), thus corresponding to the E encapsidation sequence (J. E. Embretson and H. M. Temin, J. Virol. 61:2675-2683, 1987). In addition, both the 5' and 3' halves of E are necessary in cis for RNA dimerization and the extent of RNA dimerization is influenced by viral sequences flanking E. Rapid and efficient dimerization of REV RNA containing gag sequences in addition to the E sequences and annealing of replication primer tRNA(Pro) to the primer-binding site necessitate the nucleocapsid protein. Images PMID:1331519

Dimerization of human immunodeficiency virus (type 1) RNA: stimulation by cations and possible mechanism.

PubMed

Marquet, R; Baudin, F; Gabus, C; Darlix, J L; Mougel, M; Ehresmann, C; Ehresmann, B

1991-05-11

The retroviral genome consists of two identical RNA molecules joined close to their 5' ends by the dimer linkage structure. Recent findings indicated that retroviral RNA dimerization and encapsidation are probably related events during virion assembly. We studied the cation-induced dimerization of HIV-1 RNA and results indicate that all in vitro generated HIV-1 RNAs containing a 100 nucleotide domain downstream from the 5' splice site are able to dimerize. RNA dimerization depends on the concentration of RNA, mono- and multivalent cations, the size of the monovalent cation, temperature, and pH. Up to 75% of HIV-1 RNA is dimeric in the presence of spermidine. HIV-1 RNA dimer is fairly resistant to denaturing agents and unaffected by intercalating drugs. Antisense HIV-1 RNA does not dimerize but heterodimers can be formed between HIV-1 RNA and either MoMuLV or RSV RNA. Therefore retroviral RNA dimerization probably does not simply proceed through mechanisms involving Watson-Crick base-pairing. Neither adenine and cytosine protonation, nor quartets containing only guanines appear to determine the stability of the HIV-1 RNA dimer, while quartets involving both adenine(s) and guanine(s) could account for our results. A consensus sequence PuGGAPuA found in the putative dimerization-encapsidation region of all retroviral genomes examined may participate in the dimerization process.

Dimerization of human immunodeficiency virus (type 1) RNA: stimulation by cations and possible mechanism.

PubMed Central

Marquet, R; Baudin, F; Gabus, C; Darlix, J L; Mougel, M; Ehresmann, C; Ehresmann, B

1991-01-01

The retroviral genome consists of two identical RNA molecules joined close to their 5' ends by the dimer linkage structure. Recent findings indicated that retroviral RNA dimerization and encapsidation are probably related events during virion assembly. We studied the cation-induced dimerization of HIV-1 RNA and results indicate that all in vitro generated HIV-1 RNAs containing a 100 nucleotide domain downstream from the 5' splice site are able to dimerize. RNA dimerization depends on the concentration of RNA, mono- and multivalent cations, the size of the monovalent cation, temperature, and pH. Up to 75% of HIV-1 RNA is dimeric in the presence of spermidine. HIV-1 RNA dimer is fairly resistant to denaturing agents and unaffected by intercalating drugs. Antisense HIV-1 RNA does not dimerize but heterodimers can be formed between HIV-1 RNA and either MoMuLV or RSV RNA. Therefore retroviral RNA dimerization probably does not simply proceed through mechanisms involving Watson-Crick base-pairing. Neither adenine and cytosine protonation, nor quartets containing only guanines appear to determine the stability of the HIV-1 RNA dimer, while quartets involving both adenine(s) and guanine(s) could account for our results. A consensus sequence PuGGAPuA found in the putative dimerization-encapsidation region of all retroviral genomes examined may participate in the dimerization process. Images PMID:1645868

One-pot synthesis of high molecular weight synthetic heteroprotein dimers driven by charge complementarity electrostatic interactions.

PubMed

Hvasanov, David; Nam, Ekaterina V; Peterson, Joshua R; Pornsaksit, Dithepon; Wiedenmann, Jörg; Marquis, Christopher P; Thordarson, Pall

2014-10-17

Despite the importance of protein dimers and dimerization in biology, the formation of protein dimers through synthetic covalent chemistry has not found widespread use. In the case of maleimide-cysteine-based dimerization of proteins, we show here that when the proteins have the same charge, dimerization appears to be inherently difficult with yields around 1% or less, regardless of the nature of the spacer used or whether homo- or heteroprotein dimers are targeted. In contrast, if the proteins have opposing (complementary) charges, the formation of heteroprotein dimers proceeds much more readily, and in the case of one high molecular weight (>80 kDa) synthetic dimer between cytochrome c and bovine serum albumin, a 30% yield of the purified, isolated dimer was achieved. This represents at least a 30-fold increase in yield for protein dimers formed from proteins with complementary charges, compared to when the proteins have the same charge, under otherwise similar conditions. These results illustrate the role of ionic supramolecular interactions in controlling the reactivity of proteins toward bis-functionalized spacers. The strategy here for effective synthetic dimerization of proteins could be very useful for developing novel approaches to study the important role of protein-protein interactions in chemical biology.

Stabilization of a tetrameric malate dehydrogenase by introduction of a disulfide bridge at the dimer-dimer interface.

PubMed

Bjørk, Alexandra; Dalhus, Bjørn; Mantzilas, Dimitrios; Eijsink, Vincent G H; Sirevåg, Reidun

2003-12-05

Malate dehydrogenase (MDH) from the moderately thermophilic bacterium Chloroflexus aurantiacus (CaMDH) is a tetrameric enzyme, while MDHs from mesophilic organisms usually are dimers. To investigate the potential contribution of the extra dimer-dimer interface in CaMDH with respect to thermal stability, we have engineered an intersubunit disulfide bridge designed to strengthen dimer-dimer interactions. The resulting mutant (T187C, containing two 187-187 disulfide bridges in the tetramer) showed a 200-fold increase in half-life at 75 degrees C and an increase of 15 deg. C in apparent melting temperature compared to the wild-type. The crystal structure of the mutant (solved at 1.75 A resolution) was essentially identical with that of the wild-type, with the exception of the added inter-dimer disulfide bridge and the loss of an aromatic intra-dimer contact. Remarkably, the mutant and the wild-type had similar temperature optima and activities at their temperature optima, thus providing a clear case of uncoupling of thermal stability and thermoactivity. The results show that tetramerization may contribute to MDH stability to an extent that depends strongly on the number of stabilizing interactions in the dimer-dimer interface.

Fluxional σ-Bonds of the 2,5,8-Trimethylphenalenyl Dimer: Direct Observation of the Sixfold σ-Bond Shift via a π-Dimer.

PubMed

Uchida, Kazuyuki; Mou, Zhongyu; Kertesz, Miklos; Kubo, Takashi

2016-04-06

Direct evidence for σ-bond fluxionality in a phenalenyl σ-dimer was successfully obtained by a detailed investigation of the solution-state dynamics of 2,5,8-trimethylphenalenyl (TMPLY) using both experimental and theoretical approaches. TMPLY formed three diamagnetic dimers, namely, the σ-dimer (RR/SS), σ-dimer (RS), and π-dimer, which were fully characterized by (1)H NMR spectroscopy and electronic absorption measurements. The experimental findings gave the first quantitative insights into the essential preference of these competitive and unusual dimerization modes. The spectroscopic analyses suggested that the σ-dimer (RR/SS) is the most stable in terms of energy, whereas the others are metastable; the energy differences between these three isomers are less than 1 kcal mol(-1). Furthermore, the intriguing dynamics of the TMPLY dimers in the solution state were fully revealed by means of (1)H-(1)H exchange spectroscopy (EXSY) measurements and variable-temperature (1)H NMR studies. Surprisingly, the σ-dimer (RR/SS) demonstrated a sixfold σ-bond shift between the six sets of α-carbon pairs. This unusual σ-bond fluxionality is ascribed to the presence of a direct interconversion pathway between the σ-dimer (RR/SS) and the π-dimer, which was unambiguously corroborated by the EXSY measurements. The proposed mechanism of the sixfold σ-bond shift based on the experimental findings was well-supported by theoretical calculations.

A study of the dimer formation of Rous sarcoma virus RNA and of its effect on viral protein synthesis in vitro.

PubMed

Bieth, E; Gabus, C; Darlix, J L

1990-01-11

The genetic material of all retroviruses examined so far is an RNA dimer where two identical RNA subunits are joined at their 5' ends by a structure named dimer linkage structure (DLS). Since the precise location and structure of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analysed the dimerization process of Rous sarcoma virus (RSV) RNA. For this purpose we set up an in vitro model for RSV RNA dimerization. Using this model RSV RNA was shown to form dimeric molecules and this dimerization process was greatly activated by nucleocapsid protein (NCp12) of RSV. Furthermore, RSV RNA dimerization was performed in the presence of complementary 5'32P-DNA oligomers in order to probe the monomer and dimer forms of RSV RNA. Data indicated that the DLS of RSV RNA probably maps between positions 544-564 from the 5' end. In an attempt to define sequences needed for the dimerization of RSV RNA, deletion mutageneses were generated in the 5' 600 nt. The results showed that the dimer promoting sequences probably are located within positions 208-270 and 400-600 from the 5' end and hence possibly encompassing the cis-acting elements needed for the specific encapsidation of RSV genomic RNA. Also it is reported that synthesis of the polyprotein precursor Pr76gag is inhibited upon dimerization of RSV RNA. These results suggest that dimerization and encapsidation of genome length RSV RNA might be linked in the course of virion formation since they appear to be under the control of the same cis elements, E and DLS, and the trans-acting factor nucleocapsid protein NCp12.

A study of the dimer formation of Rous sarcoma virus RNA and of its effect on viral protein synthesis in vitro.

PubMed Central

Bieth, E; Gabus, C; Darlix, J L

1990-01-01

The genetic material of all retroviruses examined so far is an RNA dimer where two identical RNA subunits are joined at their 5' ends by a structure named dimer linkage structure (DLS). Since the precise location and structure of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analysed the dimerization process of Rous sarcoma virus (RSV) RNA. For this purpose we set up an in vitro model for RSV RNA dimerization. Using this model RSV RNA was shown to form dimeric molecules and this dimerization process was greatly activated by nucleocapsid protein (NCp12) of RSV. Furthermore, RSV RNA dimerization was performed in the presence of complementary 5'32P-DNA oligomers in order to probe the monomer and dimer forms of RSV RNA. Data indicated that the DLS of RSV RNA probably maps between positions 544-564 from the 5' end. In an attempt to define sequences needed for the dimerization of RSV RNA, deletion mutageneses were generated in the 5' 600 nt. The results showed that the dimer promoting sequences probably are located within positions 208-270 and 400-600 from the 5' end and hence possibly encompassing the cis-acting elements needed for the specific encapsidation of RSV genomic RNA. Also it is reported that synthesis of the polyprotein precursor Pr76gag is inhibited upon dimerization of RSV RNA. These results suggest that dimerization and encapsidation of genome length RSV RNA might be linked in the course of virion formation since they appear to be under the control of the same cis elements, E and DLS, and the trans-acting factor nucleocapsid protein NCp12. Images PMID:2155394

Plasma D-dimer as a predictor of the progression of abdominal aortic aneurysm.

PubMed

Vele, E; Kurtcehajic, A; Zerem, E; Maskovic, J; Alibegovic, E; Hujdurovic, A

2016-11-01

Essentials D-dimer could provide important information about abdominal aortic aneurysm (AAA) progression. The greatest diameter of the infrarenal aorta and the value of plasma D-dimer were determined. AAA progression is correlated with increasing plasma D-dimer levels. The increasing value of plasma D-dimer could be a predictor of aneurysm progression. Background The natural course of abdominal aortic aneurysm (AAA) is mostly asymptomatic and unpredictable. D-dimer could provide potentially important information about subsequent AAA progression. Objectives The aims of this study were to establish the relationship between the progression of an abdominal aortic aneurysm (AAA) and plasma D-dimer concentration over a 12-month period and determine the value of plasma D-dimer in patients with sub-aneurysmal aortic dilatation. Patients/Methods This was a prospective observational study that involved 33 patients with an AAA, 30 patients with sub-aneurysmal aortic dilatation and 30 control subjects. The greatest diameter of the infrarenal aorta, which was assessed by ultrasound, and the value of plasma D-dimer were determined for all subjects at baseline assessment, as well as after 12 months for those with an AAA. Results A positive correlation was found between the diameter of an AAA and plasma D-dimer concentration at the baseline and the control measurement stages. There was a strong positive correlation between AAA progression and increasing plasma D-dimer concentration over a 12-month period. Among patients with sub-aneurysmal aortic dilatation (n = 30), the value of plasma D-dimer was higher compared with matched controls (n = 30). Conclusions There is a strongly positive correlation between AAA progression and increasing plasma D-dimer concentration. The value of plasma D-dimer is higher in patients with sub-aneurysmal aortic dilatation than in control subjects. © 2016 International Society on Thrombosis and Haemostasis.

Unexpected dimerization of isoprene in a gas chromatography inlet. A study by gas chromatography/mass spectrometry coupling.

PubMed

Estevez, Yannick; Gardrat, Christian; Berthelot, Karine; Grau, Etienne; De Jeso, Bernard; Ouardad, Samira; Peruch, Frédéric

2014-02-28

During analysis of pure isoprene by gas chromatography/mass spectrometry (GC-MS) using a programmed temperature vaporization (PTV) inlet, the presence of several isoprene dimers was detected in the total ion chromatograms (TICs). This study intends to determine the part of the instrument where dimerization occurs and the relative importance of the dimer amounts under different experimental conditions. The reference thermal dimerization of isoprene gives four six-membered cyclic dimers and two eight-membered ones. In all samples containing different amounts of freshly distilled isoprene, only peaks corresponding to the former appeared in TICs. For the same temperature, their amounts increase as the concentration of injected isoprene increases. The main products are diprene (from 80 to 100%) of the total dimers and dipentene (from 1 to 14%). The sum of the two other dimers is never higher than 6%. In conclusion, isomeric dimers are produced through a dimerization in the inlet. No dimerization of isoprene occurs in the mass spectrometer source. Then care is needed when analyzing terpenic compounds in the presence of isoprene by GC-MS because structures, retention times and mass spectra of diprene and dipentene are close. Copyright © 2014 Elsevier B.V. All rights reserved.

cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA.

PubMed

Prats, A C; Roy, C; Wang, P A; Erard, M; Housset, V; Gabus, C; Paoletti, C; Darlix, J L

1990-02-01

The genetic material of all retroviruses examined so far consists of two identical RNA molecules joined at their 5' ends by the dimer linkage structure (DLS). Since the precise location of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analyzed the dimerization process of Moloney murine leukemia virus (MoMuLV) genomic RNA. For this purpose we derived an in vitro model for RNA dimerization. By using this model, murine leukemia virus RNA was shown to form dimeric molecules. Deletion mutagenesis in the 620-nucleotide leader of MoMuLV RNA showed that the dimer promoting sequences are located within the encapsidation element Psi between positions 215 and 420. Furthermore, hybridization assays in which DNA oligomers were used to probe monomer and dimer forms of MoMuLV RNA indicated that the DLS probably maps between positions 280 and 330 from the RNA 5' end. Also, retroviral nucleocapsid protein was shown to catalyze dimerization of MoMuLV RNA and to be tightly bound to genomic dimer RNA in virions. These results suggest that MoMuLV RNA dimerization and encapsidation are probably controlled by the same cis element, Psi, and trans-acting factor, nucleocapsid protein, and thus might be linked during virion formation.

cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA.

PubMed Central

Prats, A C; Roy, C; Wang, P A; Erard, M; Housset, V; Gabus, C; Paoletti, C; Darlix, J L

1990-01-01

The genetic material of all retroviruses examined so far consists of two identical RNA molecules joined at their 5' ends by the dimer linkage structure (DLS). Since the precise location of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analyzed the dimerization process of Moloney murine leukemia virus (MoMuLV) genomic RNA. For this purpose we derived an in vitro model for RNA dimerization. By using this model, murine leukemia virus RNA was shown to form dimeric molecules. Deletion mutagenesis in the 620-nucleotide leader of MoMuLV RNA showed that the dimer promoting sequences are located within the encapsidation element Psi between positions 215 and 420. Furthermore, hybridization assays in which DNA oligomers were used to probe monomer and dimer forms of MoMuLV RNA indicated that the DLS probably maps between positions 280 and 330 from the RNA 5' end. Also, retroviral nucleocapsid protein was shown to catalyze dimerization of MoMuLV RNA and to be tightly bound to genomic dimer RNA in virions. These results suggest that MoMuLV RNA dimerization and encapsidation are probably controlled by the same cis element, Psi, and trans-acting factor, nucleocapsid protein, and thus might be linked during virion formation. Images PMID:2153242

D-dimer concentration outliers are not rare in at-term pregnant women.

PubMed

Wang, Yu; Gao, Jie; Du, Juan

2016-06-01

To determine the D-dimer levels in pregnant women at term and the differences between pregnant women with different D-dimer levels. The plasma D-dimer concentrations in pregnant women at term were identified in a cross-sectional study. The clinical indicators that are potentially relevant to D-dimer levels were compared between the pregnant women with different D-dimer levels (i.e., normal, mildly increased, and severely increased). There were always some D-dimer concentration outliers in the pregnant women at term regardless of the presence or absence of complications, and there were no significant differences in maternal age, gestational age, gravidity, parity, blood count, blood coagulation, or liver function between the pregnant women with different D-dimer levels. D-dimer levels may vary significantly during pregnancy for unknown reasons. This variation, particularly in pregnant women at term, might lead to questionable diagnostic information regarding coagulation. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Absence of Local Fluctuating Dimers in Superconducting Ir 1-x(Pt,Rh) xTe 2

DOE PAGES

Yu, Runze; Banerjee, S.; Lei, H. C.; ...

2018-06-01

The compound IrTe2 is known to exhibit a transition to a modulated state featuring Ir-Ir dimers, with large associated atomic displacements. Partial substitution of Pt or Rh for Ir destabilizes the modulated structure and induces superconductivity. It has been proposed that quantum critical dimer fluctuations might be associated with the superconductivity. Here we test for such local dimer correlations and demonstrate their absence. X-ray pair distribution function approach reveals that the local structure of Ir 0.95Pt 0.05Te 2 and Ir 0.8Rh 0.2Te 2 dichalcogenide superconductors with compositions just past the dimer/superconductor boundary is explained well by a dimer-free model downmore » to 10 K, ruling out the possibility of there being nanoscale dimer fluctuations in this regime. This is inconsistent with the proposed quantum-critical-point-like interplay of the dimer state and superconductivity, and precludes scenarios for dimer fluctuations mediated superconducting pairing.« less

Absence of Local Fluctuating Dimers in Superconducting Ir 1-x(Pt,Rh) xTe 2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Runze; Banerjee, S.; Lei, H. C.

The compound IrTe2 is known to exhibit a transition to a modulated state featuring Ir-Ir dimers, with large associated atomic displacements. Partial substitution of Pt or Rh for Ir destabilizes the modulated structure and induces superconductivity. It has been proposed that quantum critical dimer fluctuations might be associated with the superconductivity. Here we test for such local dimer correlations and demonstrate their absence. X-ray pair distribution function approach reveals that the local structure of Ir 0.95Pt 0.05Te 2 and Ir 0.8Rh 0.2Te 2 dichalcogenide superconductors with compositions just past the dimer/superconductor boundary is explained well by a dimer-free model downmore » to 10 K, ruling out the possibility of there being nanoscale dimer fluctuations in this regime. This is inconsistent with the proposed quantum-critical-point-like interplay of the dimer state and superconductivity, and precludes scenarios for dimer fluctuations mediated superconducting pairing.« less

Absence of local fluctuating dimers in superconducting Ir1 -x(Pt,Rh ) xTe2

NASA Astrophysics Data System (ADS)

Yu, Runze; Banerjee, S.; Lei, H. C.; Sinclair, Ryan; Abeykoon, M.; Zhou, H. D.; Petrovic, C.; Guguchia, Z.; Bozin, E. S.

2018-05-01

The compound IrTe2 is known to exhibit a transition to a modulated state featuring Ir-Ir dimers, with large associated atomic displacements. Partial substitution of Pt or Rh for Ir destabilizes the modulated structure and induces superconductivity. It has been proposed that quantum critical dimer fluctuations might be associated with the superconductivity. Here we test for such local dimer correlations and demonstrate their absence. X-ray pair distribution function approach reveals that the local structure of Ir0 :95Pt0 :05Te2 and Ir0 :8Rh0 :2Te2 dichalcogenide superconductors with compositions just past the dimer/superconductor boundary is explained well by a dimer-free model down to 10 K, ruling out the possibility of there being nanoscale dimer fluctuations in this regime. This is inconsistent with the proposed quantum-critical-point-like interplay of the dimer state and superconductivity, and precludes scenarios for dimer fluctuations mediated superconducting pairing.

Structural insights into the intertwined dimer of fyn SH2.

PubMed

Huculeci, Radu; Garcia-Pino, Abel; Buts, Lieven; Lenaerts, Tom; van Nuland, Nico

2015-12-01

Src homology 2 domains are interaction modules dedicated to the recognition of phosphotyrosine sites incorporated in numerous proteins found in intracellular signaling pathways. Here we provide for the first time structural insight into the dimerization of Fyn SH2 both in solution and in crystalline conditions, providing novel crystal structures of both the dimer and peptide-bound structures of Fyn SH2. Using nuclear magnetic resonance chemical shift analysis, we show how the peptide is able to eradicate the dimerization, leading to monomeric SH2 in its bound state. Furthermore, we show that Fyn SH2's dimer form differs from other SH2 dimers reported earlier. Interestingly, the Fyn dimer can be used to construct a completed dimer model of Fyn without any steric clashes. Together these results extend our understanding of SH2 dimerization, giving structural details, on one hand, and suggesting a possible physiological relevance of such behavior, on the other hand. © 2015 The Protein Society.

D-dimer as marker for microcirculatory failure: correlation with LOD and APACHE II scores.

PubMed

Angstwurm, Matthias W A; Reininger, Armin J; Spannagl, Michael

2004-01-01

The relevance of plasma d-dimer levels as marker for morbidity and organ dysfunction in severely ill patients is largely unknown. In a prospective study we determined d-dimer plasma levels of 800 unselected patients at admission to our intensive care unit. In 91% of the patients' samples d-dimer levels were elevated, in some patients up to several hundredfold as compared to normal values. The highest mean d-dimer values were present in the patient group with thromboembolic diseases, and particularly in non-survivors of pulmonary embolism. In patients with circulatory impairment (r=0.794) and in patients with infections (r=0.487) a statistically significant correlation was present between d-dimer levels and the APACHE II score (P<0.001). The logistic organ dysfunction score (LOD, P<0.001) correlated with d-dimer levels only in patients with circulatory impairment (r=0.474). On the contrary, patients without circulatory impairment demonstrated no correlation of d-dimer levels to the APACHE II or LOD score. Taking all patients together, no correlations of d-dimer levels with single organ failure or with indicators of infection could be detected. In conclusion, d-dimer plasma levels strongly correlated with the severity of the disease and organ dysfunction in patients with circulatory impairment or infections suggesting that elevated d-dimer levels may reflect the extent of microcirculatory failure. Thus, a therapeutic strategy to improve the microcirculation in such patients may be monitored using d-dimer plasma levels.

First-principles prediction of the effects of temperature and solvent selection on the dimerization of benzoic acid.

PubMed

Pham, Hieu H; Taylor, Christopher D; Henson, Neil J

2013-01-24

We introduce a procedure of quantum chemical calculations (B3P86/6-31G**) to study carboxylic acid dimerization and its correlation with temperature and properties of the solvent. Benzoic acid is chosen as a model system for studying dimerization via hydrogen bonding. Organic solvents are simulated using the self-consistent reaction field (SCRF) method with the polarized continuum model (PCM). The cyclic dimer is the most stable structure both in gas phase and solution. Dimer mono- and dihydrates could be found in the gas phase if acid molecules are in contact with water vapor. However, the formation of these hydrated conformers is very limited and cyclic dimer is the principal conformer to coexist with monomer acid in solution. Solvation of the cyclic dimer is more favorable compared to other complexes, partially due to the diminishing of hydrogen bonding capability and annihilation of dipole moments. Solvents have a strong effect on inducing dimer dissociation and this dependence is more pronounced at low dielectric constants. By accounting for selected terms in the total free energy of solvation, the solvation entropy could be incorporated to predict the dimer behavior at elevated temperatures. The temperature dependence of benzoic acid dimerization obtained by this technique is in good agreement with available experimental measurements, in which a tendency of dimer to dissociate is observed with increased temperatures. In addition, dimer breakup is more sensitive to temperature in low dielectric environments rather than in solvents with a higher dielectric constant.

A short autocomplementary sequence plays an essential role in avian sarcoma-leukosis virus RNA dimerization.

PubMed

Fossé, P; Motté, N; Roumier, A; Gabus, C; Muriaux, D; Darlix, J L; Paoletti, J

1996-12-24

Retroviral genomes consist of two identical RNA molecules joined noncovalently near their 5'-ends. Recently, two models have been proposed for RNA dimer formation on the basis of results obtained in vitro with human immunodeficiency virus type 1 RNA and Moloney murine leukemia virus RNA. It was first proposed that viral RNA dimerizes by forming an interstrand quadruple helix with purine tetrads. The second model postulates that RNA dimerization is initiated by a loop-loop interaction between the two RNA molecules. In order to better characterize the dimerization process of retroviral genomic RNA, we analyzed the in vitro dimerization of avian sarcoma-leukosis virus (ASLV) RNA using different transcripts. We determined the requirements for heterodimer formation, the thermal dissociation of RNA dimers, and the influence of antisense DNA oligonucleotides on dimer formation. Our results strongly suggest that purine tetrads are not involved in dimer formation. Data show that an autocomplementary sequence located upstream from the splice donor site and within a major packaging signal plays a crucial role in ASLV RNA dimer formation in vitro. This sequence is able to form a stem-loop structure, and phylogenetic analysis reveals that it is conserved in 28 different avian sarcoma and leukosis viruses. These results suggest that dimerization of ASLV RNA is initiated by a loop-loop interaction between two RNA molecules and provide an additional argument for the ubiquity of the dimerization process via loop-loop interaction.

Influence of Poly(ethylenimine) on the Monolayer of Oleic Acid at the Air/Water Interface.

PubMed

Hwan Ha, Tai; Kyu Kim, Dai; Choi, Myung-Un; Kim, Kwan

2000-06-01

The effect of poly(ethylenimine) (PEI) dissolved in water on the surface pressure-area isotherm of oleic acid (OA) at the air/water interface was investigated. On a concentrated PEI solution, the isotherm of the OA monolayers exhibited a noticeable difference as a function of subphase pH. PEI caused the collapse pressure of the OA monolayer to increase up to 45 mN/m, due to a stronger acid-base-type interaction occurring between the amine group of the PEI and the carboxyl group of OA; on a pure water subphase, the collapse pressure was;28 mN/m. On the other hand, owing to a stronger OA-PEI interaction, the OA monolayers favored a liquid-expanded state more on the PEI-containing water subphase than on the pure water. From the QCM measurement, each OA molecule appeared to interact, on average, with 4.3-5.8 ethylenimine repeating units at basic pHs. We also found that OA multilayers could be assembled on a hydrophilic substrate by a Z-type Langmuir-Blodgett (LB) deposition in a PEI-containing subphase at basic pHs. The ATR-IR spectral data revealed that, in a Z-type LB film, the headgroup of OA was mostly present as carboxylate, interacting in an ionic state with the protonated amine groups of PEI. In acidic conditions, neither a Y-type nor a Z-type deposition was really accomplished. Nonetheless, the ATR-IR spectral data suggested that OA molecules should exist in a monomeric state in a LB film assembled at acidic pHs without PEI while they would form intermolecular hydrogen bridges and/or dimers in the presence of PEI. Copyright 2000 Academic Press.

Graded-index optical dimer formed by optical force

DOE PAGES

Akbarzadeh, Alireza; Koschny, Thomas; Kafesaki, Maria; ...

2016-05-30

We propose an optical dimer formed from two spherical lenses bound by the pressure that light exerts on matter. With the help of the method of force tracing, we find the required graded-index profiles of the lenses for the existence of the dimer. We study the dynamics of the opto-mechanical interaction of lenses under the illumination of collimated light beams and quantitatively validate the performance of the proposed dimer. We also examine the stability of the dimer due to the lateral misalignments and we show how restoring forces bring the dimer into lateral equilibrium. The dimer can be employed inmore » various practical applications such as optical manipulation, sensing and imaging.« less

Graded-index optical dimer formed by optical force

DOE Office of Scientific and Technical Information (OSTI.GOV)

Akbarzadeh, Alireza; Koschny, Thomas; Kafesaki, Maria

We propose an optical dimer formed from two spherical lenses bound by the pressure that light exerts on matter. With the help of the method of force tracing, we find the required graded-index profiles of the lenses for the existence of the dimer. We study the dynamics of the opto-mechanical interaction of lenses under the illumination of collimated light beams and quantitatively validate the performance of the proposed dimer. We also examine the stability of the dimer due to the lateral misalignments and we show how restoring forces bring the dimer into lateral equilibrium. The dimer can be employed inmore » various practical applications such as optical manipulation, sensing and imaging.« less

Theoretical investigation on the 2e/12c bond and second hyperpolarizability of azaphenalenyl radical dimers: strength and effect of dimerization.

PubMed

Zhong, Rong-Lin; Xu, Hong-Liang; Sun, Shi-Ling; Qiu, Yong-Qing; Zhao, Liang; Su, Zhong-Min

2013-09-28

An increasing number of chemists have focused on the investigations of two-electron/multicenter bond (2e/mc) that was first introduced to describe the structure of radical dimers. In this work, the dimerization of two isoelectronic radicals, triazaphenalenyl (TAP) and hexaazaphenalenyl (HAP) has been investigated in theory. Results show TAP2 is a stable dimer with stronger 2e/12c bond and larger interaction energy, while HAP2 is a less stable dimer with larger diradical character. Interestingly, the ultraviolet-visible absorption spectra suggest that the dimerization induces a longer wavelength absorption in visible area, which is dependent on the strength of dimerization. Significantly, the amplitude of second hyperpolarizability (γ(yyyy)) of HAP2 is 1.36 × 10(6) a.u. that is larger than 7.79 × 10(4) a.u. of TAP2 because of the larger diradical character of HAP2. Therefore, the results indicate that the strength of radical dimerization can be effectively detected by comparing the magnitude of third order non-linear optical response, which is beneficial for further theoretical and experimental studies on the properties of complexes formed by radical dimerization.

Formic acid dimers in a nitrogen matrix

NASA Astrophysics Data System (ADS)

Lopes, Susy; Fausto, Rui; Khriachtchev, Leonid

2018-01-01

Formic acid (HCOOH) dimers are studied by infrared spectroscopy in a nitrogen matrix and by ab initio calculations. We benefit from the use of a nitrogen matrix where the lifetime of the higher-energy (cis) conformer is very long (˜11 h vs. 7 min in an argon matrix). As a result, in a nitrogen matrix, a large proportion of the cis conformer can be produced by vibrational excitation of the lower-energy (trans) conformer. Three trans-trans, four trans-cis, and three cis-cis dimers are found in the experiments. The spectroscopic information on most of these dimers is enriched compared to the previous studies in an argon matrix. The cis-cis dimers of ordinary formic acid (without deuteration) are reported here for the first time. Several conformational processes are obtained using selective excitation by infrared light, some of them also for the first time. In particular, we report on the formation of cis-cis dimers upon vibrational excitation of trans-cis dimers. Tunneling decays of several dimers have been detected in the dark. The tunneling decay of cis-cis dimers of formic acid as well as the stabilization of cis units in cis-cis dimers is also observed for the first time.

Formic acid dimers in a nitrogen matrix.

PubMed

Lopes, Susy; Fausto, Rui; Khriachtchev, Leonid

2018-01-21

Formic acid (HCOOH) dimers are studied by infrared spectroscopy in a nitrogen matrix and by ab initio calculations. We benefit from the use of a nitrogen matrix where the lifetime of the higher-energy (cis) conformer is very long (∼11 h vs. 7 min in an argon matrix). As a result, in a nitrogen matrix, a large proportion of the cis conformer can be produced by vibrational excitation of the lower-energy (trans) conformer. Three trans-trans, four trans-cis, and three cis-cis dimers are found in the experiments. The spectroscopic information on most of these dimers is enriched compared to the previous studies in an argon matrix. The cis-cis dimers of ordinary formic acid (without deuteration) are reported here for the first time. Several conformational processes are obtained using selective excitation by infrared light, some of them also for the first time. In particular, we report on the formation of cis-cis dimers upon vibrational excitation of trans-cis dimers. Tunneling decays of several dimers have been detected in the dark. The tunneling decay of cis-cis dimers of formic acid as well as the stabilization of cis units in cis-cis dimers is also observed for the first time.

Factors Associated with D-Dimer Levels in HIV-Infected Individuals

PubMed Central

Borges, Álvaro H.; O’Connor, Jemma L.; Phillips, Andrew N.; Baker, Jason V.; Vjecha, Michael J.; Losso, Marcelo H.; Klinker, Hartwig; Lopardo, Gustavo; Williams, Ian; Lundgren, Jens D.

2014-01-01

Background Higher plasma D-dimer levels are strong predictors of mortality in HIV+ individuals. The factors associated with D-dimer levels during HIV infection, however, remain poorly understood. Methods In this cross-sectional study, participants in three randomized controlled trials with measured D-dimer levels were included (N = 9,848). Factors associated with D-dimer were identified by linear regression. Covariates investigated were: age, gender, race, body mass index, nadir and baseline CD4+ count, plasma HIV RNA levels, markers of inflammation (C-reactive protein [CRP], interleukin-6 [IL-6]), antiretroviral therapy (ART) use, ART regimens, co-morbidities (hepatitis B/C, diabetes mellitus, prior cardiovascular disease), smoking, renal function (estimated glomerular filtration rate [eGFR] and cystatin C) and cholesterol. Results Women from all age groups had higher D-dimer levels than men, though a steeper increase of D-dimer with age occurred in men. Hepatitis B/C co-infection was the only co-morbidity associated with higher D-dimer levels. In this subgroup, the degree of hepatic fibrosis, as demonstrated by higher hyaluronic acid levels, but not viral load of hepatitis viruses, was positively correlated with D-dimer. Other factors independently associated with higher D-dimer levels were black race, higher plasma HIV RNA levels, being off ART at baseline, and increased levels of CRP, IL-6 and cystatin C. In contrast, higher baseline CD4+ counts and higher high-density lipoprotein cholesterol were negatively correlated with D-dimer levels. Conclusions D-dimer levels increase with age in HIV+ men, but are already elevated in women at an early age due to reasons other than a higher burden of concomitant diseases. In hepatitis B/C co-infected individuals, hepatic fibrosis, but not hepatitis viral load, was associated with higher D-dimer levels. PMID:24626096

Photochemical dimerization of organic compounds

DOEpatents

Crabtree, Robert H.; Brown, Stephen H.; Muedas, Cesar A.; Ferguson, Richard R.

1992-01-01

At least one of selectivity and reaction rate of photosensitized vapor phase dimerizations, including dehydrodimerizations, hydrodimerizations and cross-dimerizations of saturated and unsaturated organic compounds is improved by conducting the dimerization in the presence of hydrogen or nitrous oxide.

Controlled Patterning of Plasmonic Dimers by Using an Ultrathin Nanoporous Alumina Membrane as a Shadow Mask.

PubMed

Hao, Qi; Huang, Hao; Fan, Xingce; Yin, Yin; Wang, Jiawei; Li, Wan; Qiu, Teng; Ma, Libo; Chu, Paul K; Schmidt, Oliver G

2017-10-18

We report on design and fabrication of patterned plasmonic dimer arrays by using an ultrathin anodic aluminum oxide (AAO) membrane as a shadow mask. This strategy allows for controllable fabrication of plasmonic dimers where the location, size, and orientation of each particle in the dimer pairs can be independently tuned. Particularly, plasmonic dimers with ultrasmall nanogaps down to the sub-10 nm scale as well as a large dimer density up to 1.0 × 10 10 cm -2 are fabricated over a centimeter-sized area. The plasmonic dimers exhibit significant surface-enhanced Raman scattering (SERS) enhancement with a polarization-dependent behavior, which is well interpreted by finite-difference time-domain (FDTD) simulations. Our results reveal a facile approach for controllable fabrication of large-area dimer arrays, which is of fundamental interest for plasmon-based applications in surface-enhanced spectroscopy, biochemical sensing, and optoelectronics.

Dimerization controls the lipid raft partitioning of uPAR/CD87 and regulates its biological functions

PubMed Central

Cunningham, Orla; Andolfo, Annapaola; Santovito, Maria Lisa; Iuzzolino, Lucia; Blasi, Francesco; Sidenius, Nicolai

2003-01-01

The urokinase-type plasminogen activator receptor (uPAR/CD87) is a glycosylphosphatidylinositol-anchored membrane protein with multiple functions in extracellular proteolysis, cell adhesion, cell migration and proliferation. We now report that cell surface uPAR dimerizes and that dimeric uPAR partitions preferentially to detergent-resistant lipid rafts. Dimerization of uPAR did not require raft partitioning as the lowering of membrane cholesterol failed to reduce dimerization and as a transmembrane uPAR chimera, which does not partition to lipid rafts, also dimerized efficiently. While uPA bound to uPAR independently of its membrane localization and dimerization status, uPA-induced uPAR cleavage was strongly accelerated in lipid rafts. In contrast to uPA, the binding of Vn occurred preferentially to raft- associated dimeric uPAR and was completely blocked by cholesterol depletion. PMID:14609946

D-dimer: An Overview of Hemostasis and Fibrinolysis, Assays, and Clinical Applications.

PubMed

Olson, John D

2015-01-01

D-dimer is the smallest fibrinolysis-specific degradation product found in the circulation. The origins, assays, and clinical use of D-dimer will be addressed. Hemostasis (platelet and vascular function, coagulation, fibrinolysis, hemostasis) is briefly reviewed. D-dimer assays are reviewed. The D-dimer is very sensitive to intravascular thrombus and may be markedly elevated in disseminated intravascular coagulation, acute aortic dissection, and pulmonary embolus. Because of its exquisite sensitivity, negative tests are useful in the exclusion venous thromboembolism. Elevations occur in normal pregnancy, rising two- to fourfold by delivery. D-dimer also rises with age, limiting its use in those >80 years old. There is a variable rise in D-dimer in active malignancy and indicates increased thrombosis risk in active disease. Elevated D-dimer following anticoagulation for a thrombotic event indicates increased risk of recurrent thrombosis. These and other issues are addressed. © 2015 Elsevier Inc. All rights reserved.

Conformational Heterogeneity of Bax Helix 9 Dimer for Apoptotic Pore Formation

NASA Astrophysics Data System (ADS)

Liao, Chenyi; Zhang, Zhi; Kale, Justin; Andrews, David W.; Lin, Jialing; Li, Jianing

2016-07-01

Helix α9 of Bax protein can dimerize in the mitochondrial outer membrane (MOM) and lead to apoptotic pores. However, it remains unclear how different conformations of the dimer contribute to the pore formation on the molecular level. Thus we have investigated various conformational states of the α9 dimer in a MOM model — using computer simulations supplemented with site-specific mutagenesis and crosslinking of the α9 helices. Our data not only confirmed the critical membrane environment for the α9 stability and dimerization, but also revealed the distinct lipid-binding preference of the dimer in different conformational states. In our proposed pathway, a crucial iso-parallel dimer that mediates the conformational transition was discovered computationally and validated experimentally. The corroborating evidence from simulations and experiments suggests that, helix α9 assists Bax activation via the dimer heterogeneity and interactions with specific MOM lipids, which eventually facilitate proteolipidic pore formation in apoptosis regulation.

mRNA Molecules Containing Murine Leukemia Virus Packaging Signals Are Encapsidated as Dimers

PubMed Central

Hibbert, Catherine S.; Mirro, Jane; Rein, Alan

2004-01-01

Prior work by others has shown that insertion of ψ (i.e., leader) sequences from the Moloney murine leukemia virus (MLV) genome into the 3′ untranslated region of a nonviral mRNA leads to the specific encapsidation of this RNA in MLV particles. We now report that these RNAs are, like genomic RNAs, encapsidated as dimers. These dimers have the same thermostability as MLV genomic RNA dimers; like them, these dimers are more stable if isolated from mature virions than from immature virions. We characterized encapsidated mRNAs containing deletions or truncations of MLV ψ or with ψ sequences from MLV-related acute transforming viruses. The results indicate that the dimeric linkage in genomic RNA can be completely attributed to the ψ region of the genome. While this conclusion agrees with earlier electron microscopic studies on mature MLV dimers, it is the first evidence as to the site of the linkage in immature dimers for any retrovirus. Since the Ψ+ mRNA is not encapsidated as well as genomic RNA, it is only present in a minority of virions. The fact that it is nevertheless dimeric argues strongly that two of these molecules are packaged into particles together. We also found that the kissing loop is unnecessary for this coencapsidation or for the stability of mature dimers but makes a major contribution to the stability of immature dimers. Our results are consistent with the hypothesis that the packaging signal involves a dimeric structure in which the RNAs are joined by intermolecular interactions between GACG loops. PMID:15452213

Evidence for the Synthesis of ATP by an F0F1 ATP Synthase in Membrane Vesicles from Halorubrum Saccharovorum

NASA Technical Reports Server (NTRS)

Faguy, David; Lawson, Darion; Hochstein, Lawrence I.; Chang, Sherwood (Technical Monitor)

1996-01-01

Vesicles prepared in a buffer containing ADP, Mg(2+) and Pi synthesized ATP at an initial rate of 2 nmols/min/mg protein after acidification of the bulk medium (pH 8 (right arrow) 4). The intravesicular ATP concentration reached a steady state after about 30 seconds and slowly declined thereafter. ATP synthesis was inhibited by low concentrations of dicyclohexylcarbodiimide and m-chlorophenylhydrazone indicating that synthesis took place in response to the proton gradient. NEM and PCMS, which inhibit vacuolar ATPases and the vacuolar-like ATPases of extreme halophiles, did not affect ATP synthesis, and, in fact, produced higher steady state levels of ATP. This suggested that two ATPase activities were present, one which catalyzed ATP synthesis and one that caused its hydrolysis. Azide, a specific inhibitor of F0F1 ATP Synthases, inhibited halobacterial ATP synthesis. The distribution of acridine orange as imposed by a delta pH demonstrated that azide inhibition was not due to the collapse of the proton gradient due to azide acting as a protonophore. Such an effect was observed, but only at azide concentrations higher than those that inhibited ATP synthesis. These results confirm the earler observations with cells of H. saccharovorum and other extreme halophiles that ATP synthesis is inconsistent with the operation of a vacuolar-like ATPase. Therefore, the observation that a vacuolar-like enzyme is responsible for ATP synthesis (and which serves as the basis for imputing ATP synthesis to the vacuolar-like ATPases of the extreme halophiles, and the Archaea in general) should be taken with some degree of caution.

Functional characterization of nine Norway Spruce TPS genes and evolution of gymnosperm terpene synthases of the TPS-d subfamily.

PubMed

Martin, Diane M; Fäldt, Jenny; Bohlmann, Jörg

2004-08-01

Constitutive and induced terpenoids are important defense compounds for many plants against potential herbivores and pathogens. In Norway spruce (Picea abies L. Karst), treatment with methyl jasmonate induces complex chemical and biochemical terpenoid defense responses associated with traumatic resin duct development in stems and volatile terpenoid emissions in needles. The cloning of (+)-3-carene synthase was the first step in characterizing this system at the molecular genetic level. Here we report the isolation and functional characterization of nine additional terpene synthase (TPS) cDNAs from Norway spruce. These cDNAs encode four monoterpene synthases, myrcene synthase, (-)-limonene synthase, (-)-alpha/beta-pinene synthase, and (-)-linalool synthase; three sesquiterpene synthases, longifolene synthase, E,E-alpha-farnesene synthase, and E-alpha-bisabolene synthase; and two diterpene synthases, isopimara-7,15-diene synthase and levopimaradiene/abietadiene synthase, each with a unique product profile. To our knowledge, genes encoding isopimara-7,15-diene synthase and longifolene synthase have not been previously described, and this linalool synthase is the first described from a gymnosperm. These functionally diverse TPS account for much of the structural diversity of constitutive and methyl jasmonate-induced terpenoids in foliage, xylem, bark, and volatile emissions from needles of Norway spruce. Phylogenetic analyses based on the inclusion of these TPS into the TPS-d subfamily revealed that functional specialization of conifer TPS occurred before speciation of Pinaceae. Furthermore, based on TPS enclaves created by distinct branching patterns, the TPS-d subfamily is divided into three groups according to sequence similarities and functional assessment. Similarities of TPS evolution in angiosperms and modeling of TPS protein structures are discussed.

Distribution of Callose Synthase, Cellulose Synthase, and Sucrose Synthase in Tobacco Pollen Tube Is Controlled in Dissimilar Ways by Actin Filaments and Microtubules1[W

PubMed Central

Cai, Giampiero; Faleri, Claudia; Del Casino, Cecilia; Emons, Anne Mie C.; Cresti, Mauro

2011-01-01

Callose and cellulose are fundamental components of the cell wall of pollen tubes and are probably synthesized by distinct enzymes, callose synthase and cellulose synthase, respectively. We examined the distribution of callose synthase and cellulose synthase in tobacco (Nicotiana tabacum) pollen tubes in relation to the dynamics of actin filaments, microtubules, and the endomembrane system using specific antibodies to highly conserved peptide sequences. The role of the cytoskeleton and membrane flow was investigated using specific inhibitors (latrunculin B, 2,3-butanedione monoxime, taxol, oryzalin, and brefeldin A). Both enzymes are associated with the plasma membrane, but cellulose synthase is present along the entire length of pollen tubes (with a higher concentration at the apex) while callose synthase is located in the apex and in distal regions. In longer pollen tubes, callose synthase accumulates consistently around callose plugs, indicating its involvement in plug synthesis. Actin filaments and endomembrane dynamics are critical for the distribution of callose synthase and cellulose synthase, showing that enzymes are transported through Golgi bodies and/or vesicles moving along actin filaments. Conversely, microtubules appear to be critical in the positioning of callose synthase in distal regions and around callose plugs. In contrast, cellulose synthases are only partially coaligned with cortical microtubules and unrelated to callose plugs. Callose synthase also comigrates with tubulin by Blue Native-polyacrylamide gel electrophoresis. Membrane sucrose synthase, which expectedly provides UDP-glucose to callose synthase and cellulose synthase, binds to actin filaments depending on sucrose concentration; its distribution is dependent on the actin cytoskeleton and the endomembrane system but not on microtubules. PMID:21205616

Diagnostic implication of fibrin degradation products and D-dimer in aortic dissection

NASA Astrophysics Data System (ADS)

Dong, Jian; Duan, Xianli; Feng, Rui; Zhao, Zhiqing; Feng, Xiang; Lu, Qingsheng; Jing, Qing; Zhou, Jian; Bao, Junmin; Jing, Zaiping

2017-03-01

Fibrin degradation products (FDP) and D-dimer have been considered to be involved in many vascular diseases. In this study we aimed to explore the diagnostic implication of FDP and D-dimer in aortic dissection patients. 202 aortic dissection patients were collected as the case group, 150 patients with other cardiovascular diseases, including myocardial infarction (MI, n = 45), pulmonary infarction (n = 51) and abdominal aortic aneurysm (n = 54) were collected as non-dissection group, and 27 healthy people were in the blank control group. The FDP and D-dimer levels were detected with immune nephelometry. Logist regression analysis was performed to evaluate the influence of FDP and D-dimer for the aortic dissection patients. ROC curve was used to determine the diagnostic value of FDP and D-dimer. The FDP and D-dimer levels were significantly higher in aortic dissection patients than in non-dissection patients and the healthy controls. FDP and D-dimer were both the risk factors for patients with aortic dissection. From the ROC analysis, diagnostic value of FDP and D-dimer were not high to distinguish aortic dissection patients from the non-dissection patients. However FDP and D-dimer could be valuable diagnostic marker to differentiate aortic dissection patients and healthy controls with both AUC 0.863.

Phosphorylation-related modification at the dimer interface of 14-3-3ω dramatically alters monomer interaction dynamics.

PubMed

Denison, Fiona C; Gökirmak, Tufan; Ferl, Robert J

2014-01-01

14-3-3 proteins are generally believed to function as dimers in a broad range of eukaryotic signaling pathways. The consequences of altering dimer stability are not fully understood. Phosphorylation at Ser58 in the dimer interface of mammalian 14-3-3 isoforms has been reported to destabilise dimers. An equivalent residue, Ser62, is present across most Arabidopsis isoforms but the effects of phosphorylation have not been studied in plants. Here, we assessed the effects of phosphorylation at the dimer interface of Arabidopsis 14-3-3ω. Protein kinase A phosphorylated 14-3-3ω at Ser62 and also at a previously unreported residue, Ser67, resulting in a monomer-sized band on native-PAGE. Phosphorylation at Ser62 alone, or with additional Ser67 phosphorylation, was investigated using phosphomimetic versions of 14-3-3ω. In electrophoretic and chromatographic analyses, these mutants showed mobilities intermediate between dimers and monomers. Mobility was increased by detergents, by reducing protein concentration, or by increasing pH or temperature. Urea gradient gels showed complex structural transitions associated with alterations of dimer stability, including a previously unreported 14-3-3 aggregation phenomenon. Overall, our analyses showed that dimer interface modifications such as phosphorylation reduce dimer stability, dramatically affecting the monomer-dimer equilibrium and denaturation trajectory. These findings may have dramatic implications for 14-3-3 structure and function in vivo. Copyright © 2013 Elsevier Inc. All rights reserved.

The two-state dimer receptor model: a general model for receptor dimers.

PubMed

Franco, Rafael; Casadó, Vicent; Mallol, Josefa; Ferrada, Carla; Ferré, Sergi; Fuxe, Kjell; Cortés, Antoni; Ciruela, Francisco; Lluis, Carmen; Canela, Enric I

2006-06-01

Nonlinear Scatchard plots are often found for agonist binding to G-protein-coupled receptors. Because there is clear evidence of receptor dimerization, these nonlinear Scatchard plots can reflect cooperativity on agonist binding to the two binding sites in the dimer. According to this, the "two-state dimer receptor model" has been recently derived. In this article, the performance of the model has been analyzed in fitting data of agonist binding to A(1) adenosine receptors, which are an example of receptor displaying concave downward Scatchard plots. Analysis of agonist/antagonist competition data for dopamine D(1) receptors using the two-state dimer receptor model has also been performed. Although fitting to the two-state dimer receptor model was similar to the fitting to the "two-independent-site receptor model", the former is simpler, and a discrimination test selects the two-state dimer receptor model as the best. This model was also very robust in fitting data of estrogen binding to the estrogen receptor, for which Scatchard plots are concave upward. On the one hand, the model would predict the already demonstrated existence of estrogen receptor dimers. On the other hand, the model would predict that concave upward Scatchard plots reflect positive cooperativity, which can be neither predicted nor explained by assuming the existence of two different affinity states. In summary, the two-state dimer receptor model is good for fitting data of binding to dimeric receptors displaying either linear, concave upward, or concave downward Scatchard plots.

Association of different biomarkers of renal function with D-dimer levels in patients with type 1 diabetes mellitus (renal biomarkers and D-dimer in diabetes).

PubMed

Domingueti, Caroline Pereira; Fóscolo, Rodrigo Bastos; Dusse, Luci Maria S; Reis, Janice Sepúlveda; Carvalho, Maria das Graças; Gomes, Karina Braga; Fernandes, Ana Paula

2018-02-01

Objective This study aimed to evaluate the association between different renal biomarkers with D-Dimer levels in diabetes mellitus (DM1) patients group classified as: low D-Dimer levels (< 318 ng/mL), which included first and second D-Dimer tertiles, and high D-Dimer levels (≥ 318 ng/mL), which included third D-Dimer tertile. Materials and methods D-Dimer and cystatin C were measured by ELISA. Creatinine and urea were determined by enzymatic method. Estimated glomerular filtration rate (eGFR) was calculated using CKD-EPI equation. Albuminuria was assessed by immunoturbidimetry. Presence of renal disease was evaluated using each renal biomarker: creatinine, urea, cystatin C, eGFR and albuminuria. Bivariate logistic regression analysis was performed to assess which renal biomarkers are associated with high D-Dimer levels and odds ratio was calculated. After, multivariate logistic regression analysis was performed to assess which renal biomarkers are associated with high D-Dimer levels (after adjusting for sex and age) and odds ratio was calculated. Results Cystatin C presented a better association [OR of 9.8 (3.8-25.5)] with high D-Dimer levels than albuminuria, creatinine, eGFR and urea [OR of 5.3 (2.2-12.9), 8.4 (2.5-25.4), 9.1 (2.6-31.4) and 3.5 (1.4-8.4), respectively] after adjusting for sex and age. All biomarkers showed a good association with D-Dimer levels, and consequently, with hypercoagulability status, and cystatin C showed the best association among them. Conclusion Therefore, cystatin C might be useful to detect patients with incipient diabetic kidney disease that present an increased risk of cardiovascular disease, contributing to an early adoption of reno and cardioprotective therapies.

Comparison of clinical probability-adjusted D-dimer and age-adjusted D-dimer interpretation to exclude venous thromboembolism.

PubMed

Takach Lapner, Sarah; Julian, Jim A; Linkins, Lori-Ann; Bates, Shannon; Kearon, Clive

2017-10-05

Two new strategies for interpreting D-dimer results have been proposed: i) using a progressively higher D-dimer threshold with increasing age (age-adjusted strategy) and ii) using a D-dimer threshold in patients with low clinical probability that is twice the threshold used in patients with moderate clinical probability (clinical probability-adjusted strategy). Our objective was to compare the diagnostic accuracy of age-adjusted and clinical probability-adjusted D-dimer interpretation in patients with a low or moderate clinical probability of venous thromboembolism (VTE). We performed a retrospective analysis of clinical data and blood samples from two prospective studies. We compared the negative predictive value (NPV) for VTE, and the proportion of patients with a negative D-dimer result, using two D-dimer interpretation strategies: the age-adjusted strategy, which uses a progressively higher D-dimer threshold with increasing age over 50 years (age in years × 10 µg/L FEU); and the clinical probability-adjusted strategy which uses a D-dimer threshold of 1000 µg/L FEU in patients with low clinical probability and 500 µg/L FEU in patients with moderate clinical probability. A total of 1649 outpatients with low or moderate clinical probability for a first suspected deep vein thrombosis or pulmonary embolism were included. The NPV of both the clinical probability-adjusted strategy (99.7 %) and the age-adjusted strategy (99.6 %) were similar. However, the proportion of patients with a negative result was greater with the clinical probability-adjusted strategy (56.1 % vs, 50.9 %; difference 5.2 %; 95 % CI 3.5 % to 6.8 %). These findings suggest that clinical probability-adjusted D-dimer interpretation is a better way of interpreting D-dimer results compared to age-adjusted interpretation.

EDEM1 targets misfolded HLA-B27 dimers for endoplasmic reticulum associated degradation

PubMed Central

Guiliano, David B.; Fussell, Helen; Lenart, Izabela; Tsao, Edward; Nesbeth, Darren; Fletcher, Adam J.; Campbell, Elaine C.; Yousaf, Nasim; Williams, Sarah; Santos, Susana; Cameron, Amy; Towers, Greg J.; Kellam, Paul; Hebert, Daniel N.; Gould, Keith; Powis, Simon J.; Antoniou, Antony N.

2015-01-01

Objective HLA-B27 forms misfolded heavy chain dimers, which may predispose individuals to inflammatory arthritis by inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). We wanted to define the role of the UPR induced ER associated degradation (ERAD) pathway in the disposal of HLA-B27 dimeric conformers. Methods HeLa cell lines expressing only two copies of a carboxy terminally Sv5 tagged HLA-B27 were generated. The ER stress induced EDEM1 protein was over expressed by transfection and dimer levels monitored by immunoblotting. EDEM1, the UPR associated transcription factor XBP-1, the E3 ubiquitin ligase HRD1, the degradation associated derlin 1 and 2 proteins were inhibited by either short hairpin RNA or dominant negative mutants. The UPR associated ERAD of HLA-B27 was confirmed using ER stress inducing pharamacological agents in kinetic and pulse chase assays. Results We demonstrate that UPR induced machinery can target HLA-B27 dimers, and that dimer formation can be controlled by alterations to expression levels of components of the UPR induced ERAD pathway. HLA-B27 dimers and misfolded MHC class I monomeric molecules were detected bound to EDEM1, with overexpression of EDEM1 inhibiting HLA-B27 dimer formation. EDEM1 inhibition resulted in upregulation of HLA-B27 dimers, whilst UPR induced ERAD of dimers was prevented in the absence of EDEM1. HLA-B27 dimer formation was also enhanced in the absence of XBP-1, HRD1 and derlin1/2. Conclusion The UPR ERAD pathway as described here can dispose of HLA-B27 dimers and presents a potential novel therapeutic target for the modulation of HLA-B27 associated inflammatory disease. PMID:25132672

Model-based Analysis of HER Activation in Cells Co-Expressing EGFR, HER2 and HER3.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shankaran, Harish; Zhang, Yi; Tan, Yunbing

2013-08-22

The HER/ErbB family of receptor tyrosine kinases drive critical responses in normal physiology and cancer, and the expression levels of the various HER receptors are critical determinants of clinical outcomes. HER activation is driven by the formation of various dimer complexes between members of this receptor family. The HER dimer types can have differential effects on downstream signaling and phenotypic outcomes. We constructed an integrated mathematical model of HER activation and trafficking to quantitatively link receptor expression levels to dimerization and activation. We parameterized the model with a comprehensive set of HER phosphorylation and abundance data collected in a panelmore » of human mammary epithelial cells expressing varying levels of EGFR, HER2 and HER3. Although parameter estimation yielded multiple solutions, predictions for dimer phosphorylation were in agreement with each other. We validated the model using experiments where pertuzumab was used to block HER2 dimerization. We used the model to predict HER dimerization and activation patterns in a panel of epithelial cells lines with known HER expression levels. Simulations over the range of expression levels seen in various cell lines indicate that: i) EGFR phosphorylation is driven by HER1/1 and HER1/2 dimers, and not HER1/3 dimers, ii) HER1/2 and HER2/3 dimers both contribute significantly to HER2 activation with the EGFR expression level determining the relative importance of these species, and iii) the HER2/3 dimer is largely responsible for HER3 activation. The model can be used to predict phosphorylated dimer levels for any given HER expression profile. This information in turn can be used to quantify the potencies of the various HER dimers, and can potentially inform personalized therapeutic approaches.« less

Inhibitory effects of p-cresol and p-hydroxy anisole dimers on expression of the cyclooxygenase-2 gene and lipopolysaccharide-stimulated activation of nuclear factor-κB in RAW264.7 cells.

PubMed

Murakami, Yukio; Kawata, Akifumi; Ito, Shigeru; Katayama, Tadashi; Fujisawa, Seiichiro

2014-01-01

Phenolic compounds, particularly dihydroxybiphenyl-related compounds, possess efficient anti-oxidative and anti-inflammatory activity. We investigated the anti-inflammatory activity of 2,2'-dihydroxy-5,5'-dimethylbiphenol (p-cresol dimer), 2,2'-dihydroxy-5,5'-dimethoxybiphenol (pHA dimer), p-cresol, p-hydroxyanisole (pHA) and 2-t-butyl-4-hydroxyanisole (BHA). The cytotoxicity of the investigated compounds against RAW264.7 cells was determined using a cell counting kit (CCK-8). Their inhibitory effects on cyclooxygenase-2 (Cox2) mRNA expression stimulated by lipopolysaccharide (LPS) were determined using northern blot analysis, and their inhibition of LPS-stimulated nuclear factor-kappa B (Nf-κb) activation was evaluated using enzyme-linked immunosorbent assay-like microwell colorimetric transcription factor activity assay. The molecular orbital energy was calculated on the basis of density function theory BLYP/6-31G*. The cytotoxicity of the compounds declined in the order pHA dimer > p-cresol dimer > BHA > p-cresol > pHA. The inhibitory effect on Cox2 expression and Nf-κb activation was enhanced by p-cresol dimer and pHA dimer, particularly the former, suggesting potent anti-inflammatory activity, whereas p-cresol and pHA showed weak activity, and BHA no activity. Both p-cresol dimer and pHA dimer were highly electronegative, as determined by quantum chemical calculations. Dimerization of p-cresol and pHA enhances their anti-inflammatory activity. p-Cresol dimer and pHA dimer, particularly the former, are potent anti-inflammatory agents. Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Multiple regions of Harvey sarcoma virus RNA can dimerize in vitro.

PubMed Central

Feng, Y X; Fu, W; Winter, A J; Levin, J G; Rein, A

1995-01-01

Retroviruses contain a dimeric RNA consisting of two identical molecules of plus-strand genomic RNA. The structure of the linkage between the two monomers is not known, but they are believed to be joined near their 5' ends. Darlix and coworkers have reported that transcripts of retroviral RNA sequences can dimerize spontaneously in vitro (see, for example, E. Bieth, C. Gabus, and J. L. Darlix, Nucleic Acids Res. 18:119-127, 1990). As one approach to identification of sequences which might participate in the linkage, we have mapped sequences derived from the 5' 378 bases of Harvey sarcoma virus (HaSV) RNA which can dimerize in vitro. We found that at least three distinct regions, consisting of nucleotides 37 to 229, 205 to 272, and 271 to 378, can form these dimers. Two of these regions contain nucleotides 205 to 226; computer analysis suggests that this region can form a stem-loop with an inverted repeat in the loop. We propose that this hypothetical structure is involved in dimer formation by these two transcripts. We also compared the thermal stabilities of each of these dimers with that of HaSV viral RNA. Dimers of nucleotides 37 to 229 and 205 to 272 both exhibited melting temperatures near that of viral RNA, while dimers of nucleotides 271 to 378 are quite unstable. We also found that dimers of nucleotides 37 to 378 formed at 37 degrees C are less thermostable than dimers of the same RNA formed at 55 degrees C. It seems possible that bases from all of these regions participate in the dimer linkage present in viral RNA. PMID:7884897

Multiple regions of Harvey sarcoma virus RNA can dimerize in vitro.

PubMed

Feng, Y X; Fu, W; Winter, A J; Levin, J G; Rein, A

1995-04-01

Retroviruses contain a dimeric RNA consisting of two identical molecules of plus-strand genomic RNA. The structure of the linkage between the two monomers is not known, but they are believed to be joined near their 5' ends. Darlix and coworkers have reported that transcripts of retroviral RNA sequences can dimerize spontaneously in vitro (see, for example, E. Bieth, C. Gabus, and J. L. Darlix, Nucleic Acids Res. 18:119-127, 1990). As one approach to identification of sequences which might participate in the linkage, we have mapped sequences derived from the 5' 378 bases of Harvey sarcoma virus (HaSV) RNA which can dimerize in vitro. We found that at least three distinct regions, consisting of nucleotides 37 to 229, 205 to 272, and 271 to 378, can form these dimers. Two of these regions contain nucleotides 205 to 226; computer analysis suggests that this region can form a stem-loop with an inverted repeat in the loop. We propose that this hypothetical structure is involved in dimer formation by these two transcripts. We also compared the thermal stabilities of each of these dimers with that of HaSV viral RNA. Dimers of nucleotides 37 to 229 and 205 to 272 both exhibited melting temperatures near that of viral RNA, while dimers of nucleotides 271 to 378 are quite unstable. We also found that dimers of nucleotides 37 to 378 formed at 37 degrees C are less thermostable than dimers of the same RNA formed at 55 degrees C. It seems possible that bases from all of these regions participate in the dimer linkage present in viral RNA.

Quantitative Experimental Determination of Primer-Dimer Formation Risk by Free-Solution Conjugate Electrophoresis

PubMed Central

Desmarais, Samantha M.; Leitner, Thomas; Barron, Annelise E.

2012-01-01

DNA barcodes are short, unique ssDNA primers that “mark” individual biomolecules. To gain better understanding of biophysical parameters constraining primer-dimer formation between primers that incorporate barcode sequences, we have developed a capillary electrophoresis method that utilizes drag-tag-DNA conjugates to quantify dimerization risk between primer-barcode pairs. Results obtained with this unique free-solution conjugate electrophoresis (FSCE) approach are useful as quantitatively precise input data to parameterize computation models of dimerization risk. A set of fluorescently labeled, model primer-barcode conjugates were designed with complementary regions of differing lengths to quantify heterodimerization as a function of temperature. Primer-dimer cases comprised two 30-mer primers, one of which was covalently conjugated to a lab-made, chemically synthesized poly-N-methoxyethylglycine drag-tag, which reduced electrophoretic mobility of ssDNA to distinguish it from ds primer-dimers. The drag-tags also provided a shift in mobility for the dsDNA species, which allowed us to quantitate primer-dimer formation. In the experimental studies, pairs of oligonucleotide primer-barcodes with fully or partially complementary sequences were annealed, and then separated by free-solution conjugate CE at different temperatures, to assess effects on primer-dimer formation. When less than 30 out of 30 basepairs were bonded, dimerization was inversely correlated to temperature. Dimerization occurred when more than 15 consecutive basepairs formed, yet non-consecutive basepairs did not create stable dimers even when 20 out of 30 possible basepairs bonded. The use of free-solution electrophoresis in combination with a peptoid drag-tag and different fluorophores enabled precise separation of short DNA fragments to establish a new mobility shift assay for detection of primer-dimer formation. PMID:22331820

Structural Characterization of Amyloid β17-42 Dimer by Potential of Mean Force Analysis: Insights from Molecular Dynamics Simulations.

PubMed

Dutta, Mary; Chutia, Rajkalyan; Mattaparthi, Venkata Satish Kumar

2017-01-01

Recent experiments with Amyloid β1-42 peptide have indicated that the initial dimerization of Aβ1-42 monomers to form amyloid dimers stand out as a key event in the generation of toxic oligomers. However, the structural characterization of Aβ1-42 dimer at the atomistic level and the dimerization mechanism by which Aβ1-42 peptides co-aggregate still remains not clear. In the present study, the process of Aβ17-42 peptide dimerization which is known to play an important role in the plaque formation in Alzheimer's disease was evaluated in terms of potential of mean force. The Aβ17-42 dimer was constructed using PatchDock server. We have used molecular dynamics (MD) simulation with the umbrella sampling methodology to compute the Potential of Mean Force for the dimerization of Aβ17-42. The global minima structure at the minimum distance of separation was isolated from the calculated free energy profile and the interactions involved in the formation of the dimer structure were examined. Protein-protein interfaces and the residueresidue interactions vital for generation of the dimer complexes were also evaluated. The simulation results elucidated the interaction between the monomeric units to be governed primarily by the hydrophobic and hydrogen bonds. The resultant Aβ17-42 dimer was found to have an increased β-strands propensity at the hydrophobic regions encompassing the CHC region. Furthermore, specific hydrophobic residues were found to play a vital role in the formation of the dimer complex. From the results we may therefore conclude hydrophobic region encompassing the CHC region to be crucial in dimerization process. The findings from this study provide detailed information for the complex process of early events of Aβ aggregation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Dipole Approximation to Predict the Resonances of Dimers Composed of Dielectric Resonators for Directional Emission: Dielectric Dimers Dipole Approximation

DOE PAGES

Campione, Salvatore; Warne, Larry K.; Basilio, Lorena I.

2017-09-29

In this paper we develop a fully-retarded, dipole approximation model to estimate the effective polarizabilities of a dimer made of dielectric resonators. They are computed from the polarizabilities of the two resonators composing the dimer. We analyze the situation of full-cubes as well as split-cubes, which have been shown to exhibit overlapping electric and magnetic resonances. We compare the effective dimer polarizabilities to ones retrieved via full-wave simulations as well as ones computed via a quasi-static, dipole approximation. We observe good agreement between the fully-retarded solution and the full-wave results, whereas the quasi-static approximation is less accurate for the problemmore » at hand. The developed model can be used to predict the electric and magnetic resonances of a dimer under parallel or orthogonal (to the dimer axis) excitation. This is particularly helpful when interested in locating frequencies at which the dimer will emit directional radiation.« less

Ligand-induced type II interleukin-4 receptor dimers are sustained by rapid re-association within plasma membrane microcompartments

NASA Astrophysics Data System (ADS)

Richter, David; Moraga, Ignacio; Winkelmann, Hauke; Birkholz, Oliver; Wilmes, Stephan; Schulte, Markos; Kraich, Michael; Kenneweg, Hella; Beutel, Oliver; Selenschik, Philipp; Paterok, Dirk; Gavutis, Martynas; Schmidt, Thomas; Garcia, K. Christopher; Müller, Thomas D.; Piehler, Jacob

2017-07-01

The spatiotemporal organization of cytokine receptors in the plasma membrane is still debated with models ranging from ligand-independent receptor pre-dimerization to ligand-induced receptor dimerization occurring only after receptor uptake into endosomes. Here, we explore the molecular and cellular determinants governing the assembly of the type II interleukin-4 receptor, taking advantage of various agonists binding the receptor subunits with different affinities and rate constants. Quantitative kinetic studies using artificial membranes confirm that receptor dimerization is governed by the two-dimensional ligand-receptor interactions and identify a critical role of the transmembrane domain in receptor dimerization. Single molecule localization microscopy at physiological cell surface expression levels, however, reveals efficient ligand-induced receptor dimerization by all ligands, largely independent of receptor binding affinities, in line with the similar STAT6 activation potencies observed for all IL-4 variants. Detailed spatiotemporal analyses suggest that kinetic trapping of receptor dimers in actin-dependent microcompartments sustains robust receptor dimerization and signalling.

Domain-Swapped Dimers of Intracellular Lipid-Binding Proteins: Evidence for Ordered Folding Intermediates.

PubMed

Assar, Zahra; Nossoni, Zahra; Wang, Wenjing; Santos, Elizabeth M; Kramer, Kevin; McCornack, Colin; Vasileiou, Chrysoula; Borhan, Babak; Geiger, James H

2016-09-06

Human Cellular Retinol Binding Protein II (hCRBPII), a member of the intracellular lipid-binding protein family, is a monomeric protein responsible for the intracellular transport of retinol and retinal. Herein we report that hCRBPII forms an extensive domain-swapped dimer during bacterial expression. The domain-swapped region encompasses almost half of the protein. The dimer represents a novel structural architecture with the mouths of the two binding cavities facing each other, producing a new binding cavity that spans the length of the protein complex. Although wild-type hCRBPII forms the dimer, the propensity for dimerization can be substantially increased via mutation at Tyr60. The monomeric form of the wild-type protein represents the thermodynamically more stable species, making the domain-swapped dimer a kinetically trapped entity. Hypothetically, the wild-type protein has evolved to minimize dimerization of the folding intermediate through a critical hydrogen bond (Tyr60-Glu72) that disfavors the dimeric form. Copyright © 2016 Elsevier Ltd. All rights reserved.

Kosterlitz-Thouless transitions and phase diagrams of the interacting monomer-dimer model on a checkerboard lattice

NASA Astrophysics Data System (ADS)

Li, Sazi; Li, Wei; Chen, Ziyu

2014-11-01

Using the tensor network approach, we investigate the monomer-dimer models on a checkerboard lattice, in which there are interactions (with strength v ) between the parallel dimers on half of the plaquettes. For the fully packed interacting dimer model, we observe a Kosterlitz-Thouless (KT) transition between the low-temperature symmetry breaking and the high-temperature critical phases; for the doped monomer-dimer case with finite chemical potential μ , we also find an order-disorder phase transition which is of second order instead. We use the boundary matrix product state approach to detect the KT and second-order phase transitions and obtain the phase diagrams v -T and μ -T . Moreover, for the noninteracting monomer-dimer model (setting μ =ν =0 ), we get an extraordinarily accurate determination of the free energy per site (negative of the monomer-dimer constant h2) as f =-0.662 798 972 833 746 with the dimer density n =0.638 123 109 228 547 , both of 15 correct digits.

Detectability of Noble Gases in Jovian Atmospheres Utilizing Dimer Spectral Structures

NASA Astrophysics Data System (ADS)

Kim, S. J.; Min, Y.; Kim, Y.; Lee, Y.; Trafton, L.; Miller, S.; McKellar, A. R. W.

1997-07-01

The detection of jovian hydrogen-hydrogen dimers through the clear telluric 2-micron window (Kim et al. 1995; Trafton et al. 1997) suggests possibility to detect noble gases in the form of dimer with hydrogen in jovian atmospheres. Since noble gases do not have spectral structures in the infrared, it has been difficult to derive their abundances in the atmospheres of jovian planets. If there is a significant component of noble gases other than helium in the jovian atmospheres, it might be detected through its dimer spectrum with hydrogen molecule. The relatively sharp spectral structures of hydrogen-argon and hydrogen-neon dimers compared with those of hydrogen-hydrogen dimers are useful for the detection, if adequate S/N is obtained. However, these dimer structures should be much weaker than the nearby hydrogen-hydrogen features because noble gases are expected to be minor constituents of these atmospheres. We will discuss the detectability of these dimers based on laboratory measurements (McKellar, 1994; 1996), and current technology of infrared observations.

Dynamic Cholesterol-Conditioned Dimerization of the G Protein Coupled Chemokine Receptor Type 4

PubMed Central

Kranz, Franziska

2016-01-01

G protein coupled receptors (GPCRs) allow for the transmission of signals across biological membranes. For a number of GPCRs, this signaling was shown to be coupled to prior dimerization of the receptor. The chemokine receptor type 4 (CXCR4) was reported before to form dimers and their functionality was shown to depend on membrane cholesterol. Here, we address the dimerization pattern of CXCR4 in pure phospholipid bilayers and in cholesterol-rich membranes. Using ensembles of molecular dynamics simulations, we show that CXCR4 dimerizes promiscuously in phospholipid membranes. Addition of cholesterol dramatically affects the dimerization pattern: cholesterol binding largely abolishes the preferred dimer motif observed for pure phospholipid bilayers formed mainly by transmembrane helices 1 and 7 (TM1/TM5-7) at the dimer interface. In turn, the symmetric TM3,4/TM3,4 interface is enabled first by intercalating cholesterol molecules. These data provide a molecular basis for the modulation of GPCR activity by its lipid environment. PMID:27812115

Dimerization of the voltage-sensing phosphatase controls its voltage-sensing and catalytic activity.

PubMed

Rayaprolu, Vamseedhar; Royal, Perrine; Stengel, Karen; Sandoz, Guillaume; Kohout, Susy C

2018-05-07

Multimerization is a key characteristic of most voltage-sensing proteins. The main exception was thought to be the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP). In this study, we show that multimerization is also critical for Ci-VSP function. Using coimmunoprecipitation and single-molecule pull-down, we find that Ci-VSP stoichiometry is flexible. It exists as both monomers and dimers, with dimers favored at higher concentrations. We show strong dimerization via the voltage-sensing domain (VSD) and weak dimerization via the phosphatase domain. Using voltage-clamp fluorometry, we also find that VSDs cooperate to lower the voltage dependence of activation, thus favoring the activation of Ci-VSP. Finally, using activity assays, we find that dimerization alters Ci-VSP substrate specificity such that only dimeric Ci-VSP is able to dephosphorylate the 3-phosphate from PI(3,4,5)P 3 or PI(3,4)P 2 Our results indicate that dimerization plays a significant role in Ci-VSP function. © 2018 Rayaprolu et al.

Inducible nitric oxide synthase (iNOS) drives mTOR pathway activation and proliferation of human melanoma by reversible nitrosylation of TSC2

PubMed Central

Lopez-Rivera, Esther; Jayaraman, Padmini; Parikh, Falguni; Davies, Michael A.; Ekmekcioglu, Suhendan; Izadmehr, Sudeh; Milton, Denái R.; Chipuk, Jerry E.; Grimm, Elizabeth A.; Estrada, Yeriel; Aguirre-Ghiso, Julio; Sikora, Andrew G.

2014-01-01

Melanoma is one of the cancers of fastest-rising incidence in the world. iNOS is overexpressed in melanoma and other cancers, and previous data suggest that iNOS and nitric oxide (NO) drive survival and proliferation of human melanoma cells. However, specific mechanisms through which this occurs are poorly defined. One candidate is the PI3K/AKT/mTOR pathway, which plays a major role in proliferation, angiogenesis, and metastasis of melanoma and other cancers. We used the chick embryo chorioallantoic membrane (CAM) assay to test the hypothesis that melanoma growth is regulated by iNOS-dependent mTOR pathway activation. Both pharmacologic inhibition and siRNA-mediated gene silencing of iNOS suppressed melanoma proliferation and in vivo growth on the CAM in human melanoma models. This was associated with strong downregulation of mTOR pathway activation by Western blot analysis of p-mTOR, p-P70S6K, p-S6RP, and p-4EBP1. iNOS expression and NO were associated with reversible nitrosylation of TSC2, and inhibited dimerization of TSC2 with its inhibitory partner TSC1, enhancing GTPase activity of its target Rheb, a critical activator of mTOR signaling. Immunohistochemical analysis of tumor specimens from stage III melanoma patients showed a significant correlation between iNOS expression levels and expression of mTOR pathway members. Exogenously-supplied NO was also sufficient to reverse mTOR pathway inhibition by the B-Raf inhibitor Vemurafenib. In summary, covalent modification of TSC2 by iNOS-derived NO is associated with impaired TSC2/TSC1 dimerization, mTOR pathway activation, and proliferation of human melanoma. This model is consistent with the known association of iNOS overexpression and poor prognosis in melanoma and other cancers. PMID:24398473

Common Gating of Both CLC Transporter Subunits Underlies Voltage-dependent Activation of the 2Cl−/1H+ Exchanger ClC-7/Ostm1*

PubMed Central

Ludwig, Carmen F.; Ullrich, Florian; Leisle, Lilia; Stauber, Tobias; Jentsch, Thomas J.

2013-01-01

CLC anion transporters form dimers that function either as Cl− channels or as electrogenic Cl−/H+ exchangers. CLC channels display two different types of “gates,” “protopore” gates that open and close the two pores of a CLC dimer independently of each other and common gates that act on both pores simultaneously. ClC-7/Ostm1 is a lysosomal 2Cl−/1H+ exchanger that is slowly activated by depolarization. This gating process is drastically accelerated by many CLCN7 mutations underlying human osteopetrosis. Making use of some of these mutants, we now investigate whether slow voltage activation of plasma membrane-targeted ClC-7/Ostm1 involves protopore or common gates. Voltage activation of wild-type ClC-7 subunits was accelerated by co-expressing an excess of ClC-7 subunits carrying an accelerating mutation together with a point mutation rendering these subunits transport-deficient. Conversely, voltage activation of a fast ClC-7 mutant could be slowed by co-expressing an excess of a transport-deficient mutant. These effects did not depend on whether the accelerating mutation localized to the transmembrane part or to cytoplasmic cystathionine-β-synthase (CBS) domains of ClC-7. Combining accelerating mutations in the same subunit did not speed up gating further. No currents were observed when ClC-7 was truncated after the last intramembrane helix. Currents and slow gating were restored when the C terminus was co-expressed by itself or fused to the C terminus of the β-subunit Ostm1. We conclude that common gating underlies the slow voltage activation of ClC-7. It depends on the CBS domain-containing C terminus that does not require covalent binding to the membrane domain of ClC-7. PMID:23983121

Effects of the dimeric PSD-95 inhibitor UCCB01-144 in mouse models of pain, cognition and motor function.

PubMed

Andreasen, Jesper T; Nasser, Arafat; Caballero-Puntiverio, Maitane; Sahlholt, Maj; Bach, Anders; Gynther, Mikko; Strømgaard, Kristian; Pickering, Darryl S

2016-06-05

NMDAR antagonism shows analgesic action in humans and animal pain models, but disrupts cognitive and motor functions. NMDAR-dependent NO production requires tethering of the NMDAR to neuronal NO synthase (nNOS) by the postsynaptic density protein-95 (PSD-95). Perturbing the NMDAR/PSD-95/nNOS interaction has therefore been proposed as an alternative analgesic mechanism. We recently reported that UCCB01-125, a dimeric PSD-95 inhibitor with limited blood-brain-barrier permeability, reduced mechanical hypersensitivity in the complete Freund's adjuvant (CFA) inflammatory pain model, without disrupting cognitive or motor functions. Here, we investigated the analgesic efficacy in the CFA model of UCCB01-144, a PSD-95 inhibitor with improved blood-brain-barrier permeability. To extend the comparison of UCCB01-125 and UCCB01-144, we also tested both compounds in the spared nerve injury (SNI) model of neuropathic pain. Potential cognitive effects of UCCB01-144 were examined using the social transmission of food preference (STFP) test and the V-maze test, and motor coordination was assessed with the rotarod test. UCCB01-144 (10mg/kg) reversed CFA-induced mechanical hypersensitivity after 1h, and completely normalised sensitivity after 24h. In the SNI model, UCCB01-144 (30mg/kg) partially reversed hypersensitivity after 1h, but no effect was observed after 24h. UCCB01-125 did not affect SNI-induced hypersensitivity. Rotarod performance was unaffected by UCCB01-144, but 30mg/kg UCCB01-144 impaired performance in the STFP test. Collectively, UCCB01-144 reversed both CFA and SNI-induced hypersensitivity, but the efficacy in the SNI model was only transient. This suggests that enhanced BBB permeability of PSD-95 inhibitors improves the analgesic action in neuropathic pain states. Copyright © 2016 Elsevier B.V. All rights reserved.

The protein inhibitor of nNOS (PIN/DLC1/LC8) binding does not inhibit the NADPH-dependent heme reduction in nNOS, a key step in NO synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Parhad, Swapnil S.; Jaiswal, Deepa; TIFR Centre for Interdisciplinary Sciences, 21 Brundavan Colony, Narsingi, Hyderabad 500075

The neuronal nitric oxide synthase (nNOS) is an essential enzyme involved in the synthesis of nitric oxide (NO), a potent neurotransmitter. Although previous studies have indicated that the dynein light chain 1 (DLC1) binding to nNOS could inhibit the NO synthesis, the claim is challenged by contradicting reports. Thus, the mechanism of nNOS regulation remained unclear. nNOS has a heme-bearing, Cytochrome P450 core, and the functional enzyme is a dimer. The electron flow from NADPH to Flavin, and finally to the heme of the paired nNOS subunit within a dimer, is facilitated upon calmodulin (CaM) binding. Here, we show thatmore » DLC1 binding to nNOS-CaM complex does not affect the electron transport from the reductase to the oxygenase domain. Therefore, it cannot inhibit the rate of NADPH-dependent heme reduction in nNOS, which results in L-Arginine oxidation. Also, the NO release activity does not decrease with increasing DLC1 concentration in the reaction mix, which further confirmed that DLC1 does not inhibit nNOS activity. These findings suggest that the DLC1 binding may have other implications for the nNOS function in the cell. - Highlights: • The effect of interaction of nNOS with DLC1 has been debatable with contradicting reports in literature. • Purified DLC1 has no effect on electron transport between reductase and oxygenase domain of purified nNOS-CaM. • The NO release activity of nNOS was not altered by DLC1, supporting that DLC1 does not inhibit the enzyme. • These findings suggest that the DLC1 binding may have other implications for the nNOS function in the cell.« less

The Dimeric Architecture of Checkpoint Kinases Mec1ATR and Tel1ATM Reveal a Common Structural Organization.

PubMed

Sawicka, Marta; Wanrooij, Paulina H; Darbari, Vidya C; Tannous, Elias; Hailemariam, Sarem; Bose, Daniel; Makarova, Alena V; Burgers, Peter M; Zhang, Xiaodong

2016-06-24

The phosphatidylinositol 3-kinase-related protein kinases are key regulators controlling a wide range of cellular events. The yeast Tel1 and Mec1·Ddc2 complex (ATM and ATR-ATRIP in humans) play pivotal roles in DNA replication, DNA damage signaling, and repair. Here, we present the first structural insight for dimers of Mec1·Ddc2 and Tel1 using single-particle electron microscopy. Both kinases reveal a head to head dimer with one major dimeric interface through the N-terminal HEAT (named after Huntingtin, elongation factor 3, protein phosphatase 2A, and yeast kinase TOR1) repeat. Their dimeric interface is significantly distinct from the interface of mTOR complex 1 dimer, which oligomerizes through two spatially separate interfaces. We also observe different structural organizations of kinase domains of Mec1 and Tel1. The kinase domains in the Mec1·Ddc2 dimer are located in close proximity to each other. However, in the Tel1 dimer they are fully separated, providing potential access of substrates to this kinase, even in its dimeric form. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Dimerization of the keto tautomer of acetohydroxamic acid—infrared matrix isolation and theoretical study

NASA Astrophysics Data System (ADS)

Sałdyka, Magdalena; Mielke, Zofia

2005-05-01

Dimerization of the keto tautomer of acetohydroxamic acid has been studied using FTIR matrix isolation spectroscopy and DFT(B3LYP)/6-31+G(d,p) calculations. Analysis of CH 3CONHOH/Ar matrix spectra indicates formation of two dimers in which two intramolecular CO···H sbnd ON bonds within two interacting acetohydroxamic acid molecules are retained. A chain dimer I is stabilized by the intermolecular CO···H sbnd N hydrogen bond, whereas the cyclic dimer II is stabilized by two intermolecular N sbnd H···O(H)N bonds. Twelve vibrations were identified for dimer I and six vibrations for dimer II; the observed frequency shifts show a good agreement with the calculated ones for the structures I and II. Both dimers have comparable binding energies ( ΔEZPECPI, II = -7.02, -6.34 kcal mol -1) being less stable than calculated structures III and IV ( ΔEZPECPIII, IV = -9.50, -8.87 kcal mol -1) in which one or two intramolecular hydrogen bonds are disrupted. In the most stable 10-membered cyclic dimer III, two intermolecular CO···H sbnd ON hydrogen bonds are formed at expense of intramolecular hydrogen bonds of the same type. The formation of the less stable (AHA) 2 dimers in the studied matrixes indicates that the formation of (AHA) 2 is kinetically and not thermodynamically controlled.

Economic evaluation of different screening alternatives for patients with clinically suspected acute deep vein thrombosis.

PubMed

Bogavac-Stanojević, Natasa; Dopsaj, Violeta; Jelić-Ivanović, Zorana; Lakić, Dragana; Vasić, Dragan; Petrova, Guenka

2013-01-01

We examined the cost-effectiveness of the three different D-dimer measurements in the screening of DVT in models with and without calculation of pre-test probability (PTP) score. Moreover, we calculated the minimal cost in DVT detection. In the group of 192 patients with clinically suspected acute DVT, we examined the three different D-dimer measurements (Innovance D-dimer, Hemosil D-dimer HS and Vidas D-dimer Exclusion II) in combination with and without PTP assessment. The diagnostic alternative employing Vidas D-dimer Exclusion II assay without and with PTP calculation gave lower incremental cost-effectiveness ratio (ICER) than the alternative employing Hemosil D-dimer HS assay (0.187 Euros vs. 0.998 Euros per one additional DVT positive patient selected for CUS in model without PTP assessment and 0.450 vs. 0.753 Euros per one DVT positive patient selected for CUS in model with PTP assessment). According to sensitivity analysis, the Hemosil D-dimer HS assay was the most cost effective alternative when one patient was admitted to the vascular ambulance per day. Vidas D-dimer Exclusion II assay was the most cost effective alternative when more than one patient were admitted to the vascular ambulance per day. Cost minimisation analysis indicated that selection of patients according to PTP score followed by D-dimer analysis decreases the cost of DVT diagnosis. ICER analysis enables laboratories to choose optimal laboratory tests according to number of patients admitted to laboratory. Results support the feasibility of using PTP scoring and D-dimer measurement before CUS examination in DVT screening.

Inorganic pyrophosphatases of Family II-two decades after their discovery.

PubMed

Baykov, Alexander A; Anashkin, Viktor A; Salminen, Anu; Lahti, Reijo

2017-10-01

Inorganic pyrophosphatases (PPases) convert pyrophosphate (PP i ) to phosphate and are present in all cell types. Soluble PPases belong to three nonhomologous families, of which Family II is found in approximately a quarter of prokaryotic organisms, often pathogenic ones. Each subunit of dimeric canonical Family II PPases is formed by two domains connected by a flexible linker, with the active site located between the domains. These enzymes require both magnesium and a transition metal ion (manganese or cobalt) for maximal activity and are the most active (k cat ≈ 10 4 s -1 ) among all PPase types. Catalysis by Family II PPases requires four metal ions per substrate molecule, three of which form a unique trimetal center that coordinates the nucleophilic water and converts it to a reactive hydroxide ion. A quarter of Family II PPases contain an autoinhibitory regulatory insert formed by two cystathionine β-synthase (CBS) domains and one DRTGG domain. Adenine nucleotide binding either activates or inhibits the CBS domain-containing PPases, thereby tuning their activity and, hence, PP i levels, in response to changes in cell energy status (ATP/ADP ratio). © 2017 Federation of European Biochemical Societies.

A Common Fungal Associate of the Spruce Bark Beetle Metabolizes the Stilbene Defenses of Norway Spruce1[C][W][OA

PubMed Central

Hammerbacher, Almuth; Schmidt, Axel; Wadke, Namita; Wright, Louwrance P.; Schneider, Bernd; Bohlmann, Joerg; Brand, Willi A.; Fenning, Trevor M.; Gershenzon, Jonathan; Paetz, Christian

2013-01-01

Norway spruce (Picea abies) forests suffer periodic fatal attacks by the bark beetle Ips typographus and its fungal associate, Ceratocystis polonica. Norway spruce protects itself against fungal and bark beetle invasion by the production of terpenoid resins, but it is unclear whether resins or other defenses are effective against the fungus. We investigated stilbenes, a group of phenolic compounds found in Norway spruce bark with a diaryl-ethene skeleton with known antifungal properties. During C. polonica infection, stilbene biosynthesis was up-regulated, as evidenced by elevated transcript levels of stilbene synthase genes. However, stilbene concentrations actually declined during infection, and this was due to fungal metabolism. C. polonica converted stilbenes to ring-opened, deglycosylated, and dimeric products. Chromatographic separation of C. polonica protein extracts confirmed that these metabolites arose from specific fungal enzyme activities. Comparison of C. polonica strains showed that rapid conversion of host phenolics is associated with higher virulence. C. polonica is so well adapted to its host’s chemical defenses that it is even able to use host phenolic compounds as its sole carbon source. PMID:23729780

Tualang honey protects keratinocytes from ultraviolet radiation-induced inflammation and DNA damage.

PubMed

Ahmad, Israr; Jimenez, Hugo; Yaacob, Nik Soriani; Yusuf, Nabiha

2012-01-01

Malaysian tualang honey possesses strong antioxidant and anti-inflammatory properties. Here, we evaluated the effect of tualang honey on early biomarkers of photocarcinogenesis employing PAM212 mouse keratinocyte cell line. Keratinocytes were treated with tualang honey (1.0%, v/v) before a single UVB (150 mJ cm(-2) ) irradiation. We found that the treatment of tualang honey inhibited UVB-induced DNA damage, and enhanced repair of UVB-mediated formation of cyclobutane pyrimidine dimers and 8-oxo-7,8-dihydro-2'-deoxyguanosine. Treatment of tualang honey inhibited UVB-induced nuclear translocation of NF-κB and degradation of IκBα in murine keratinocyte cell line. The treatment of tualang honey also inhibited UVB-induced inflammatory cytokines and inducible nitric oxide synthase protein expression. Furthermore, the treatment of tualang honey inhibited UVB-induced COX-2 expression and PGE2 production. Taken together, we provide evidence that the treatment of tualang honey to keratinocytes affords substantial protection from the adverse effects of UVB radiation via modulation in early biomarkers of photocarcinogenesis and provide suggestion for its photochemopreventive potential. © 2012 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2012 The American Society of Photobiology.

Natural Phenolic Inhibitors of Trichothecene Biosynthesis by the Wheat Fungal Pathogen Fusarium culmorum: A Computational Insight into the Structure-Activity Relationship

PubMed Central

Pani, Giovanna; Dessì, Alessandro; Dallocchio, Roberto; Scherm, Barbara; Azara, Emanuela; Delogu, Giovanna

2016-01-01

A model of the trichodiene synthase (TRI5) of the wheat fungal pathogen and type-B trichothecene producer Fusarium culmorum was developed based on homology modelling with the crystallized protein of F. sporotrichioides. Eight phenolic molecules, namely ferulic acid 1, apocynin 2, propyl gallate 3, eugenol 4, Me-dehydrozingerone 5, eugenol dimer 6, magnolol 7, and ellagic acid 8, were selected for their ability to inhibit trichothecene production and/or fungal vegetative growth in F. culmorum. The chemical structures of phenols were constructed and partially optimised based on Molecular Mechanics (MM) studies and energy minimisation by Density Functional Theory (DFT). Docking analysis of the phenolic molecules was run on the 3D model of F. culmorum TRI5. Experimental biological activity, molecular descriptors and interacting-structures obtained from computational analysis were compared. Besides the catalytic domain, three privileged sites in the interaction with the inhibitory molecules were identified on the protein surface. The TRI5-ligand interactions highlighted in this study represent a powerful tool to the identification of new Fusarium-targeted molecules with potential as trichothecene inhibitors. PMID:27294666

Isoprene synthase genes form a monophyletic clade of acyclic terpene synthases in the TPS-B terpene synthase family.

PubMed

Sharkey, Thomas D; Gray, Dennis W; Pell, Heather K; Breneman, Steven R; Topper, Lauren

2013-04-01

Many plants emit significant amounts of isoprene, which is hypothesized to help leaves tolerate short episodes of high temperature. Isoprene emission is found in all major groups of land plants including mosses, ferns, gymnosperms, and angiosperms; however, within these groups isoprene emission is variable. The patchy distribution of isoprene emission implies an evolutionary pattern characterized by many origins or many losses. To better understand the evolution of isoprene emission, we examine the phylogenetic relationships among isoprene synthase and monoterpene synthase genes in the angiosperms. In this study we identify nine new isoprene synthases within the rosid angiosperms. We also document the capacity of a myrcene synthase in Humulus lupulus to produce isoprene. Isoprene synthases and (E)-β-ocimene synthases form a monophyletic group within the Tps-b clade of terpene synthases. No asterid genes fall within this clade. The chemistry of isoprene synthase and ocimene synthase is similar and likely affects the apparent relationships among Tps-b enzymes. The chronology of rosid evolution suggests a Cretaceous origin followed by many losses of isoprene synthase over the course of evolutionary history. The phylogenetic pattern of Tps-b genes indicates that isoprene emission from non-rosid angiosperms likely arose independently. © 2012 The Author(s). Evolution© 2012 The Society for the Study of Evolution.

Photodissociable dimer reduction products of 2-thiopyrimidine derivatives.

PubMed Central

Wrona, M; Giziewicz, J; Shugar, D

1975-01-01

Both 4,6-dimethyl-2-thipyrimidine and its 1-methyl derivative undergo polarographic reduction in aqueous medium, via a 1e/1H+ reduction to a free radical which rapidly dimerizes to products isolates and identified as 4,4'-bis-(4,6-dimethyl-3,4-dihydropyrimidin-2-thione) and the corresponding 1-methyl dimer. The dimers may be oxidized electrolytically to regenerate the parent monomers. Both dimers also undergo photodissociation to quantitatively regenerate the parent monomers, in high quantum yield, 0.23 and 0.35 M/Einstein. The correlation between electrochemical and photochemical reductions of 2-thiopyrimidines are discussed, as well as the significance of the dimer photodissociation reactions in relation to nucleic acid photochemistry. PMID:28516

Ultrafast photodimerization dynamics in α-cyano-4-hydroxycinnamic and sinapinic acid crystals

NASA Astrophysics Data System (ADS)

Hoyer, Theo; Tuszynski, Wilfried; Lienau, Christoph

2007-07-01

We report a sub-picosecond time-resolved fluorescence spectroscopic study of different cinnamic acid crystals, model systems for solid-state photodimerization reactions. For α-cyano-4-hydroxycinnamic acid (α-CHC), we identify the emission spectra of both monomers and dimers, allowing us to directly probe the photoinduced dynamics of both species. The dimerization occurs on a timescale of 10 ps and results in a long-lived dimer product, stable for hours. For sinapinic acid, we find an extremely fast, sub-picosecond dimerization reaction and a short-lived dimer. This first sub-picosecond time-resolved dimerization study in cinnamic acid crystals provides a new basis for relating their structural properties and microscopic reaction dynamics.

A HeI photoelectron spectrum of the [Al(CH 3) 3] 2 dimer

NASA Astrophysics Data System (ADS)

Wang, Dianxun; Qian, Ximei; Zheng, Shijun; Shi, Yizhong

1997-10-01

The HeI photoelectron spectrum (PES) of the [Al(CH 3) 3] 2 dimer is recorded for the first time. To assign the PES bands, an ab initio SCF MO calculation for the dimer has also been performed. The four splitting peaks of the first PE band are respectively designated to electron ionization of the four frontier 8b u, 13a g, 7b g, and 7b u orbitals of the dimer. They originate from the recombination of the two HOMO (5e') of the two monomers in the forming of the dimer. That is to say, during the formation of the dimer from the two monomers, the reduction of molecular symmetry (from the C 3h symmetry of the monomer to the C 2h symmetry of the dimer) leads to the undegeneration of the degenerate orbitals.

Solitary waves in dimer binary collision model

NASA Astrophysics Data System (ADS)

Ahsan, Zaid; Jayaprakash, K. R.

2017-01-01

Solitary wave propagation in nonlinear diatomic (dimer) chains is a very interesting topic of research in the study of nonlinear lattices. Such waves were recently found to be supported by the essentially nonlinear granular lattice and Toda lattice. An interesting aspect of this discovery is attributed to the realization of a spectrum of the mass ratio (the only system parameter governing the dynamics) that supports the propagation of such waves corresponding to the considered interaction potential. The objective of this exposition is to explore solitary wave propagation in the dimer binary collision (BC) model. Interestingly, the dimer BC model supports solitary wave propagation at a discrete spectrum of mass ratios similar to those observed in granular and Toda dimers. Further, we report a qualitative and one-to-one correspondence between the spectrum of the mass ratio corresponding to the dimer BC model and those corresponding to granular and Toda dimer chains.

The Phylogenetic Signature Underlying ATP Synthase c-Ring Compliance

DOE PAGES

Pandini, Alessandro; Kleinjung, Jens; Taylor, Willie R.; ...

2015-09-01

The proton-driven ATP synthase (F OF 1) is comprised of two rotary, stepping motors (F O and F 1) coupled by an elastic power transmission. The elastic compliance resides in the rotor module that includes the membrane-embedded FO c-ring. Proton transport by FO is firmly coupled to the rotation of the c-ring relative to other F O subunits (ab 2). It drives ATP synthesis. We used a computational method to investigate the contribution of the c-ring to the total elastic compliance. We performed principal component analysis of conformational ensembles built using distance constraints from the bovine mitochondrial c-ring x-ray structure.more » Angular rotary twist, the dominant ring motion, was estimated to show that the c-ring accounted in part for the measured compliance. Ring rotation was entrained to rotation of the external helix within each hairpin-shaped c-subunit in the ring. Ensembles of monomer and dimers extracted from complete c-rings showed that the coupling between collective ring and the individual subunit motions was independent of the size of the c-ring, which varies between organisms. Molecular determinants were identified by covariance analysis of residue coevolution and structural-alphabet-based local dynamics correlations. The residue coevolution gave a readout of subunit architecture. The dynamic couplings revealed that the hinge for both ring and subunit helix rotations was constructed from the proton-binding site and the adjacent glycine motif (IB-GGGG) in the midmembrane plane. IB-GGGG motifs were linked by long-range couplings across the ring, while intrasubunit couplings connected the motif to the conserved cytoplasmic loop and adjacent segments. The correlation with principal collective motions shows that the couplings underlie both ring rotary and bending motions. Noncontact couplings between IB-GGGG motifs matched the coevolution signal as well as contact couplings. The residue coevolution reflects the physiological importance of the dynamics that may link proton transfer to ring compliance.« less

The Phylogenetic Signature Underlying ATP Synthase c-Ring Compliance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pandini, Alessandro; Kleinjung, Jens; Taylor, Willie R.

The proton-driven ATP synthase (F OF 1) is comprised of two rotary, stepping motors (F O and F 1) coupled by an elastic power transmission. The elastic compliance resides in the rotor module that includes the membrane-embedded FO c-ring. Proton transport by FO is firmly coupled to the rotation of the c-ring relative to other F O subunits (ab 2). It drives ATP synthesis. We used a computational method to investigate the contribution of the c-ring to the total elastic compliance. We performed principal component analysis of conformational ensembles built using distance constraints from the bovine mitochondrial c-ring x-ray structure.more » Angular rotary twist, the dominant ring motion, was estimated to show that the c-ring accounted in part for the measured compliance. Ring rotation was entrained to rotation of the external helix within each hairpin-shaped c-subunit in the ring. Ensembles of monomer and dimers extracted from complete c-rings showed that the coupling between collective ring and the individual subunit motions was independent of the size of the c-ring, which varies between organisms. Molecular determinants were identified by covariance analysis of residue coevolution and structural-alphabet-based local dynamics correlations. The residue coevolution gave a readout of subunit architecture. The dynamic couplings revealed that the hinge for both ring and subunit helix rotations was constructed from the proton-binding site and the adjacent glycine motif (IB-GGGG) in the midmembrane plane. IB-GGGG motifs were linked by long-range couplings across the ring, while intrasubunit couplings connected the motif to the conserved cytoplasmic loop and adjacent segments. The correlation with principal collective motions shows that the couplings underlie both ring rotary and bending motions. Noncontact couplings between IB-GGGG motifs matched the coevolution signal as well as contact couplings. The residue coevolution reflects the physiological importance of the dynamics that may link proton transfer to ring compliance.« less

Constitutive NOS uncoupling and NADPH oxidase upregulation in the penis of type 2 diabetic men with erectile dysfunction

PubMed Central

Musicki, Biljana; Burnett, Arthur L.

2016-01-01

Erectile dysfunction (ED) associated with type 2 diabetes mellitus (T2DM) involves dysfunctional nitric oxide (NO) signaling and increased oxidative stress in the penis. However, the mechanisms of endothelial NO synthase (eNOS) and neuronal NO synthase (nNOS) dysregulation, and the sources of oxidative stress, are not well defined, particularly at the human level. The objective of this study was to define whether uncoupled eNOS and nNOS, and NADPH oxidase upregulation, contribute to the pathogenesis of ED in T2DM men. Penile erectile tissue was obtained from 9 T2DM patients with ED who underwent penile prosthesis surgery for ED, and from 6 control patients without T2DM or ED who underwent penectomy for penile cancer. The dimer-to-monomer protein expression ratio, an indicator of uncoupling for both eNOS and nNOS, total protein expressions of eNOS and nNOS, as well as protein expressions of NADPH oxidase catalytic subunit gp91phox (an enzymatic source of oxidative stress) and 4-hydroxy-2-nonenal [4-HNE] and nitrotyrosine (markers of oxidative stress) were measured by Western blot in this tissue. In the erectile tissue of T2DM men, eNOS and nNOS uncoupling and protein expressions of NADPH oxidase subunit gp91phox, 4-HNE- and nitrotyrosine-modified proteins were significantly (p<0.05) increased compared to control values. Total eNOS and nNOS protein expressions were not significantly different between the groups. In conclusion, mechanisms of T2DM-associated ED in the human penis may involve uncoupled eNOS and nNOS and NADPH oxidase upregulation. Our description of molecular factors contributing to the pathogenesis of T2DM-associated ED at the human level is relevant for advancing clinically therapeutic approaches to restore erectile function in T2DM patients. PMID:28076881

Fatty acid synthase affects expression of ErbB receptors in epithelial to mesenchymal transition of breast cancer cells and invasive ductal carcinoma

PubMed Central

Chen, Tingting; Zhou, Lan; Li, Hua; Tian, Yuan; Li, Junqin; Dong, Lihua; Zhao, Yuhua; Wei, Dapeng

2017-01-01

The aim of the present study was to investigate changes in the expression of ErbBs during epithelial-mesenchymal transition (EMT) of breast cancer cells and its association with the expression of fatty acid synthase (FASN). MCF-7-MEK5 cells were used as the experimental model, while MCF-7 cells were used as a control. Tumor cells were implanted into nude mice for in vivo analysis. Cerulenin was used as a FASN inhibitor. Reverse transcription-polymerase chain reaction and western blot analysis were used to detect expression levels of FASN and ErbB1-4. Immunohistochemistry was used to detect the expression of FASN and ErbB1-4 in 58 invasive ductal carcinomas (IDC), as well as their association with clinicopathological characteristics. The expression of FASN and ErbB1-4 in MCF-7-MEK5 cells and tumor tissues increased significantly compared with controls (P<0.001). Inhibition of FASN by cerulenin resulted in a significant decrease in expression of ErbB1, 2 and 4 (P<0.001), whereas there was no evident change in ErbB3. In IDC samples, the expression of FASN and ErbB1-4 increased considerably in lymph node metastases compared with non-lymph node metastases (P<0.05). ErbB2 expression increased in advanced clinical stages (II, III and IV) of IDC and in tumors with larger diameters (P<0.05). The expression of ErbB3 increased in ER-positive tumors (P<0.05). Additionally, a positive association between the expression of FASN and ErbB1, 2 and 4 was observed (P<0.05). FASN activates ErbB1, 2 and 4, and their dimers, which are polymerized via the microstructural domain of the cell membrane. This may initiate EMT and consequentlyincrease the invasion and migration of cancer cells. However, ErbB3 may also affect tumor progression via a FASN-independent pathway. PMID:29113229

Ultraviolet Spectrum And Chemical Reactivity Of CIO Dimer

NASA Technical Reports Server (NTRS)

Demore, William B.; Tschuikow-Roux, E.

1992-01-01

Report describes experimental study of ultraviolet spectrum and chemical reactivity of dimer of chlorine monoxide (CIO). Objectives are to measure absorption cross sections of dimer at near-ultraviolet wavelengths; determine whether asymmetrical isomer (CIOCIO) exists at temperatures relevant to Antarctic stratosphere; and test for certain chemical reactions of dimer. Important in photochemistry of Antarctic stratosphere.

40 CFR 721.9484 - Dimer acid/rosin amidoamine reaction product (generic).

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Dimer acid/rosin amidoamine reaction... Specific Chemical Substances § 721.9484 Dimer acid/rosin amidoamine reaction product (generic). (a... generically as Dimer acid/rosin amidoamine reaction product (PMN P-99-0143) is subject to reporting under this...

40 CFR 721.9484 - Dimer acid/rosin amidoamine reaction product (generic).

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Dimer acid/rosin amidoamine reaction... Specific Chemical Substances § 721.9484 Dimer acid/rosin amidoamine reaction product (generic). (a... generically as Dimer acid/rosin amidoamine reaction product (PMN P-99-0143) is subject to reporting under this...

Polarization-selective optical resonance with extremely narrow linewidth in Si dimers array for application in ultra-sensitive refractive sensing

NASA Astrophysics Data System (ADS)

Fu, Dong; Zhang, Zuyin; Li, Jian; Wu, Haoyue; Wang, Wenbo; Wei, Xin

2017-05-01

By exploiting the radiative coupling between the electromagnetic field scattered by individual Si dimer and the collective wave diffracted (Rayleigh Anomalies) in the plane of Si dimers array, optical resonance with extremely narrow linewidth is achieved, accompanied with dramatic enhancement of electric field in the gap of the dimer. We analyze the optical properties of Si dimers array by decomposing it into three fundamental sub-systems. Theoretical investigation employing the coupled dipole approximation is complemented with numerical simulations. The result shows that polarization angle has significant influence on the orientation of the field scattered by individual Si dimer, which determines the efficiency of radiative coupling and further impacts on the electric field enhancement. Moreover, we explore the feasibility of application in refractive sensing. It is shown that the figure of merit value for the proposed system of Si dimers array is as high as 306. The Si dimers array that takes advantage of multiple coupling creates new possibility to implement field-enhanced spectroscopy and refractive sensing with ultra-high sensitivity.

The Aggregation Paths and Products of Aβ42 Dimers Are Distinct from Those of the Aβ42 Monomer.

PubMed

O'Malley, Tiernan T; Witbold, William M; Linse, Sara; Walsh, Dominic M

2016-11-08

Extracts of Alzheimer's disease (AD) brain that contain what appear to be sodium dodecyl sulfate-stable amyloid β-protein (Aβ) dimers potently block LTP and impair memory consolidation. Brain-derived dimers can be physically separated the Aβ monomer, consist primarily of Aβ42, and resist denaturation by chaotropic agents. In nature, covalently cross-linked Aβ dimers could be generated in two ways: by the formation of a dityrosine (DiY) or an isopeptide ε-(γ-glutamyl)-lysine (Q-K) bond. We enzymatically cross-linked recombinant Aβ42 monomer to produce DiY and Q-K dimers and then used a range of biophysical methods to study their aggregation. Both Q-K and DiY dimers aggregate to form soluble assemblies distinct from the fibrillar aggregates formed by the Aβ monomer. The results suggest that the cross-links disfavor fibril formation from Aβ dimers, thereby enhancing the concentration of soluble aggregates akin to those in aqueous extracts of AD brain. Thus, it seems that Aβ dimers may play an important role in determining the formation of soluble rather than insoluble aggregates.

The aggregation paths and products of Aβ42 dimers are distinct from Aβ42 monomer

PubMed Central

O'Malley, Tiernan T.; Witbold, William M.; Linse, Sara; Walsh, Dominic M.

2017-01-01

Extracts of Alzheimer's disease (AD) brain that contain what appear to be SDS-stable amyloid β-protein (Aβ) dimers potently block LTP and impair memory consolidation. Brain-derived dimers can be physically separated from Aβ monomer, consist primarily of Aβ42 and resist denaturation by powerful chaotropic agents. In nature, covalently cross-linked Aβ dimers could be generated in only one of two different ways - either by the formation of a dityrosine (DiY) or an isopeptide ε-(γ-glutamyl)-lysine (Q-K) bond. We enzymatically cross-linked recombinant Aβ42 monomer to produce DiY and Q-K dimers and then applied a range of biophysical methods to study their aggregation. Both Q-K and DiY dimers aggregate to form soluble assemblies distinct from the fibrillar aggregates formed by Aβ monomer. These results suggest that Aβ dimers allow the formation of soluble aggregates akin to those in aqueous extracts of AD brain. Thus it seems that Aβ dimers may play an important role in determining the formation of soluble rather than insoluble aggregates. PMID:27750419

STIM1 dimers undergo unimolecular coupling to activate Orai1 channels

NASA Astrophysics Data System (ADS)

Zhou, Yandong; Wang, Xizhuo; Wang, Xianming; Loktionova, Natalia A.; Cai, Xiangyu; Nwokonko, Robert M.; Vrana, Erin; Wang, Youjun; Rothberg, Brad S.; Gill, Donald L.

2015-09-01

The endoplasmic reticulum (ER) Ca2+ sensor, STIM1, becomes activated when ER-stored Ca2+ is depleted and translocates into ER-plasma membrane junctions where it tethers and activates Orai1 Ca2+ entry channels. The dimeric STIM1 protein contains a small STIM-Orai-activating region (SOAR)--the minimal sequence sufficient to activate Orai1 channels. Since SOAR itself is a dimer, we constructed SOAR concatemer-dimers and introduced mutations at F394, which is critical for Orai1 coupling and activation. The F394H mutation in both SOAR monomers completely blocks dimer function, but F394H introduced in only one of the dimeric SOAR monomers has no effect on Orai1 binding or activation. This reveals an unexpected unimolecular coupling between STIM1 and Orai1 and argues against recent evidence suggesting dimeric interaction between STIM1 and two adjacent Orai1 channel subunits. The model predicts that STIM1 dimers may be involved in crosslinking between Orai1 channels with implications for the kinetics and localization of Orai1 channel opening.

Naturally occurring disulfide-bound dimers of three-fingered toxins: a paradigm for biological activity diversification.

PubMed

Osipov, Alexey V; Kasheverov, Igor E; Makarova, Yana V; Starkov, Vladislav G; Vorontsova, Olga V; Ziganshin, Rustam Kh; Andreeva, Tatyana V; Serebryakova, Marina V; Benoit, Audrey; Hogg, Ronald C; Bertrand, Daniel; Tsetlin, Victor I; Utkin, Yuri N

2008-05-23

Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of alpha-cobratoxin (a long-chain alpha-neurotoxin) and heterodimers formed by alpha-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26-30 in polypeptide loop II of alpha-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the alpha-cobratoxin capacity to compete with alpha-bungarotoxin for binding to Torpedo and alpha7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that alpha-cobratoxin dimer not only interacts with alpha7 nAChR but, in contrast to alpha-cobratoxin monomer, also blocks alpha3beta2 nAChR. In the latter activity it resembles kappa-bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric alpha3beta2 nAChRs.

Evidence for involvement of the C-terminal domain in the dimerization of the CopY repressor protein from Enterococcus hirae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pazehoski, Kristina O., E-mail: pazehosk@pitt.edu; Cobine, Paul A., E-mail: pac0006@auburn.edu; Winzor, Donald J.

2011-03-11

Research highlights: {yields} A metal-binding protein domain is directly involved in protein dimerization. {yields} Fusing the metal-binding domain to a monomeric protein induces dimerization. {yields} Frontal size-exclusion chromatography measures the strength of dimer interaction. {yields} Ultracentrifugation studies confirm the influence of metal binding on dimerization. -- Abstract: Metal binding to the C-terminal region of the copper-responsive repressor protein CopY is responsible for homodimerization and the regulation of the copper homeostasis pathway in Enterococcus hirae. Specific involvement of the 38 C-terminal residues of CopY in dimerization is indicated by zonal and frontal (large zone) size-exclusion chromatography studies. The studies demonstrate thatmore » the attachment of these CopY residues to the immunoglobulin-binding domain of streptococcal protein G (GB1) promotes dimerization of the monomeric protein. Although sensitivity of dimerization to removal of metal from the fusion protein is smaller than that found for CopY (as measured by ultracentrifugation studies), the demonstration that an unrelated protein (GB1) can be induced to dimerize by extending its sequence with the C-terminal portion of CopY confirms the involvement of this region in CopY homodimerization.« less

Studies on the Dissociation and Urea-Induced Unfolding of FtsZ Support the Dimer Nucleus Polymerization Mechanism

PubMed Central

Montecinos-Franjola, Felipe; Ross, Justin A.; Sánchez, Susana A.; Brunet, Juan E.; Lagos, Rosalba; Jameson, David M.; Monasterio, Octavio

2012-01-01

FtsZ is a major protein in bacterial cytokinesis that polymerizes into single filaments. A dimer has been proposed to be the nucleating species in FtsZ polymerization. To investigate the influence of the self-assembly of FtsZ on its unfolding pathway, we characterized its oligomerization and unfolding thermodynamics. We studied the assembly using size-exclusion chromatography and fluorescence spectroscopy, and the unfolding using circular dichroism and two-photon fluorescence correlation spectroscopy. The chromatographic analysis demonstrated the presence of monomers, dimers, and tetramers with populations dependent on protein concentration. Dilution experiments using fluorescent conjugates revealed dimer-to-monomer and tetramer-to-dimer dissociation constants in the micromolar range. Measurements of fluorescence lifetimes and rotational correlation times of the conjugates supported the presence of tetramers at high protein concentrations and monomers at low protein concentrations. The unfolding study demonstrated that the three-state unfolding of FtsZ was due to the mainly dimeric state of the protein, and that the monomer unfolds through a two-state mechanism. The monomer-to-dimer equilibrium characterized here (Kd = 9 μM) indicates a significant fraction (∼10%) of stable dimers at the critical concentration for polymerization, supporting a role of the dimeric species in the first steps of FtsZ polymerization. PMID:22824282

Analysis of hepatitis C virus RNA dimerization and core-RNA interactions.

PubMed

Ivanyi-Nagy, Roland; Kanevsky, Igor; Gabus, Caroline; Lavergne, Jean-Pierre; Ficheux, Damien; Penin, François; Fossé, Philippe; Darlix, Jean-Luc

2006-01-01

The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3'-untranslated region (3'-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623-2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3'-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus.

Ligand-independent Dimer Formation of Epidermal Growth Factor Receptor (EGFR) Is a Step Separable from Ligand-induced EGFR Signaling

PubMed Central

Yu, Xiaochun; Sharma, Kailash D.; Takahashi, Tsuyoshi; Iwamoto, Ryo; Mekada, Eisuke

2002-01-01

Dimerization and phosphorylation of the epidermal growth factor (EGF) receptor (EGFR) are the initial and essential events of EGF-induced signal transduction. However, the mechanism by which EGFR ligands induce dimerization and phosphorylation is not fully understood. Here, we demonstrate that EGFRs can form dimers on the cell surface independent of ligand binding. However, a chimeric receptor, comprising the extracellular and transmembrane domains of EGFR and the cytoplasmic domain of the erythropoietin receptor (EpoR), did not form a dimer in the absence of ligands, suggesting that the cytoplasmic domain of EGFR is important for predimer formation. Analysis of deletion mutants of EGFR showed that the region between 835Ala and 918Asp of the EGFR cytoplasmic domain is required for EGFR predimer formation. In contrast to wild-type EGFR ligands, a mutant form of heparin-binding EGF-like growth factor (HB2) did not induce dimerization of the EGFR-EpoR chimeric receptor and therefore failed to activate the chimeric receptor. However, when the dimerization was induced by a monoclonal antibody to EGFR, HB2 could activate the chimeric receptor. These results indicate that EGFR can form a ligand-independent inactive dimer and that receptor dimerization and activation are mechanistically distinct and separable events. PMID:12134089

Dynamics and kinetics of reversible homo-molecular dimerization of polycyclic aromatic hydrocarbons

NASA Astrophysics Data System (ADS)

Mao, Qian; Ren, Yihua; Luo, K. H.; van Duin, Adri C. T.

2017-12-01

Physical dimerization of polycyclic aromatic hydrocarbons (PAHs) has been investigated via molecular dynamics (MD) simulation with the ReaxFF reactive force field that is developed to bridge the gap between the quantum mechanism and classical MD. Dynamics and kinetics of homo-molecular PAH collision under different temperatures, impact parameters, and orientations are studied at an atomic level, which is of great value to understand and model the PAH dimerization. In the collision process, the enhancement factors of homo-molecular dimerizations are quantified and found to be larger at lower temperatures or with smaller PAH instead of size independent. Within the capture radius, the lifetime of the formed PAH dimer decreases as the impact parameter increases. Temperature and PAH characteristic dependent forward and reverse rate constants of homo-molecular PAH dimerization are derived from MD simulations, on the basis of which a reversible model is developed. This model can predict the tendency of PAH dimerization as validated by pyrene dimerization experiments [H. Sabbah et al., J. Phys. Chem. Lett. 1(19), 2962 (2010)]. Results from this study indicate that the physical dimerization cannot be an important source under the typical flame temperatures and PAH concentrations, which implies a more significant role played by the chemical route.

A multimode vibronic treatment of absorption, resonance Raman, and hyper-Rayleigh scattering of excitonically coupled molecular dimers

NASA Astrophysics Data System (ADS)

Myers Kelley, Anne

2003-08-01

The linear absorption spectra, resonance Raman excitation profiles and depolarization dispersion curves, and hyper-Rayleigh scattering profiles are calculated for excitonically coupled homodimers of a model electron donor-acceptor "push-pull" conjugated chromophore as a function of dimer geometry. The vibronic eigenstates of the dimer are calculated by diagonalizing the matrix of transition dipole couplings among the vibronic transitions of the constituent monomers. The absorption spectra show the usual red- or blueshifted transitions for J-type or H-type dimers, respectively. When the electronic coupling is large compared with the vibronic width of the monomer spectrum, the dimer absorption spectra exhibit simple Franck-Condon progressions having reduced vibronic intensities compared with the monomer, and the resonance Raman excitation profiles are shifted but otherwise only weakly perturbed. When the coupling is comparable to the vibronic width, the H-dimer absorption spectra exhibit irregular vibronic frequency spacings and intensity patterns and the effects on the Raman excitation profiles are larger. There is strong dispersion in the Raman depolarization ratios for dimer geometries in which both transitions carry oscillator strength. The first hyperpolarizabilities are somewhat enhanced in J-dimers and considerably reduced in H-dimers. These effects on the molecular β will amplify the effects of dimerization on the ground-state dipole moment in electro-optic materials formed from chromophore-doped polymers that must be electric field poled to obtain the net alignment needed for a macroscopic χ(2).

Making structural sense of dimerization interfaces of delta opioid receptor homodimers.

PubMed

Johnston, Jennifer M; Aburi, Mahalaxmi; Provasi, Davide; Bortolato, Andrea; Urizar, Eneko; Lambert, Nevin A; Javitch, Jonathan A; Filizola, Marta

2011-03-15

Opioid receptors, like other members of the G protein-coupled receptor (GPCR) family, have been shown to associate to form dimers and/or oligomers at the plasma membrane. Whether this association is stable or transient is not known. Recent compelling evidence suggests that at least some GPCRs rapidly associate and dissociate. We have recently calculated binding affinities from free energy estimates to predict transient association between mouse delta opioid receptor (DOR) protomers at a symmetric interface involving the fourth transmembrane (TM4) helix (herein termed "4" dimer). Here we present disulfide cross-linking experiments with DOR constructs with cysteines substituted at the extracellular ends of TM4 or TM5 that confirm the formation of DOR complexes involving these helices. Our results are consistent with the involvement of TM4 and/or TM5 at the DOR homodimer interface, but possibly with differing association propensities. Coarse-grained (CG) well-tempered metadynamics simulations of two different dimeric arrangements of DOR involving TM4 alone or with TM5 (herein termed "4/5" dimer) in an explicit lipid-water environment confirmed the presence of two structurally and energetically similar configurations of the 4 dimer, as previously assessed by umbrella sampling calculations, and revealed a single energetic minimum of the 4/5 dimer. Additional CG umbrella sampling simulations of the 4/5 dimer indicated that the strength of association between DOR protomers varies depending on the protein region at the interface, with the 4 dimer being more stable than the 4/5 dimer.

Role of sulfhydryl-dependent dimerization of soluble guanylyl cyclase in relaxation of porcine coronary artery to nitric oxide.

PubMed

Zheng, Xiaoxu; Ying, Lei; Liu, Juan; Dou, Dou; He, Qiong; Leung, Susan Wai Sum; Man, Ricky Y K; Vanhoutte, Paul M; Gao, Yuansheng

2011-06-01

Soluble guanylyl cyclase (sGC) is a heterodimer. The dimerization of the enzyme is obligatory for its function in mediating actions caused by agents that elevate cyclic guanosine monophosphate (cGMP). The present study aimed to determine whether sGC dimerization is modulated by thiol-reducing agents and whether its dimerization influences relaxations in response to nitric oxide (NO). The dimers and monomers of sGC and cGMP-dependent protein kinase (PKG) were analysed by western blotting. The intracellular cGMP content was measured by enzyme-linked immunosorbent assay. Changes in isometric tension were determined in organ chambers. In isolated porcine coronary arteries, the protein levels of sGC dimer were decreased by the thiol reductants dithiothreitol, l-cysteine, reduced l-glutathione and tris(2-carboxyethyl) phosphine. The effect was associated with reduced cGMP elevation and attenuated relaxations in response to nitric oxide donors. The dimerization of sGC and activation of the enzyme were also decreased by dihydrolipoic acid, an endogenous thiol antioxidant. Dithiothreitol at concentrations markedly affecting the dimerization of sGC had no significant effect on the dimerization of PKG or relaxation in response to 8-Br-cGMP. Relaxation of the coronary artery in response to a NO donor was potentiated by hypoxia when sGC was partly inhibited, coincident with an increase in sGC dimer and enhanced cGMP production. These effects were prevented by dithiothreitol and tris(2-carboxyethyl) phosphine. These results demonstrate that the dimerization of sGC is exquisitely sensitive to thiol reductants compared with that of PKG, which may provide a novel mechanism for thiol-dependent modulation of NO-mediated vasodilatation in conditions such as hypoxia.

Rough energy landscapes in protein folding: dimeric E. coli Trp repressor folds through three parallel channels.

PubMed

Gloss, L M; Simler, B R; Matthews, C R

2001-10-05

The folding mechanism of the dimeric Escherichia coli Trp repressor (TR) is a kinetically complex process that involves three distinguishable stages of development. Following the formation of a partially folded, monomeric ensemble of species, within 5 ms, folding to the native dimer is controlled by three kinetic phases. The rate-limiting step in each phase is either a non-proline isomerization reaction or a dimerization reaction, depending on the final denaturant concentration. Two approaches have been employed to test the previously proposed folding mechanism of TR through three parallel channels: (1) unfolding double-jump experiments demonstrate that all three folding channels lead directly to native dimer; and (2) the differential stabilization of the transition state for the final step in folding and the native dimer, by the addition of salt, shows that all three channels involve isomerization of a dimeric species. A refined model for the folding of Trp repressor is presented, in which all three channels involve a rapid dimerization reaction between partially folded monomers followed by the isomerization of the dimeric intermediates to yield native dimer. The ensemble of partially folded monomers can be captured at equilibrium by low pH; one-dimensional proton NMR spectra at pH 2.5 demonstrate that monomers exist in two distinct, slowly interconverting conformations. These data provide a potential structural explanation for the three-channel folding mechanism of TR: random association of two different monomeric forms, which are distinguished by alternative packing modes of the core dimerization domain and the DNA-binding, helix-turn-helix, domain. One, perhaps both, of these packing modes contains non-native contacts. Copyright 2001 Academic Press.

Binding Energies of Proton-Bound Dimers of Imidazole and n-Acetylalanine Methyl Ester Obtained by Blackbody Infrared Radiative Dissociation

PubMed Central

Jockusch, Rebecca A.; Williams*, Evan R.

2005-01-01

The dissociation kinetics of protonated n-acetyl-L-alanine methyl ester dimer (AcAlaMEd), imidazole dimer, and their cross dimer were measured using blackbody infrared radiative dissociation (BIRD). Master equation modeling of these data was used to extract threshold dissociation energies (Eo) for the dimers. Values of 1.18 ± 0.06, 1.11 ± 0.04, and 1.12 ± 0.08 eV were obtained for AcAlaMEd, imidazole dimer, and the cross dimer, respectively. Assuming that the reverse activation barrier for dissociation of the ion–molecule complex is negligible, the value of Eo can be compared to the dissociation enthalpy (ΔHd°) from HPMS data. The Eo values obtained for the imidazole dimer and the cross dimer are in agreement with HPMS values; the value for AcAlaMEd is somewhat lower. Radiative rate constants used in the master equation modeling were determined using transition dipole moments calculated at the semiempirical (AM1) level for all dimers and compared to ab initio (RHF/3-21G*) calculations where possible. To reproduce the experimentally measured dissociation rates using master equation modeling, it was necessary to multiply semiempirical transition dipole moments by a factor between 2 and 3. Values for transition dipole moments from the ab initio calculations could be used for two of the dimers but appear to be too low for AcAlaMEd. These results demonstrate that BIRD, in combination with master equation modeling, can be used to determine threshold dissociation energies for intermediate size ions that are in neither the truncated Boltzmann nor the rapid energy exchange limit. PMID:16604163

Deuteration Effect on the Nh/nd Stretch Band of the Jet-Cooled 7-AZAINDOLE and its Tautomeric Dimers: Relation to the Ground-State Double Proton-Transfer Reaction

NASA Astrophysics Data System (ADS)

Ishikawa, Haruki; Nakano, Takumi; Takashima, Tsukiko; Yabuguchi, Hiroki; Fuke, Kiyokazu

2013-06-01

In order to investigate the deuteration effect on the vibrational dynamics of the NH and/or ND stretch excited levels of the 7-azaindole (7-AI) normal dimer and its tautomeric dimer, we have carried out infrared spectroscopy of three isotopic species for each dimers; undeuterated one (NH-NH) and one or two hydrogen atom(s) of the NH groups is deuterated ones (NH-ND and ND-ND, respectively). It is found that the ND stretch band profiles of the NH-ND and ND-ND tautomeric dimers are very similar with each other. This result is very distinct from the result of the comparison of the NH stretch band profiles of the NH-NH and NH-ND dimers in our previous study. For a further discussion, we have examined the deuteration effect in the case of the 7-AI normal dimer. It is found that the NH stretch band profiles of the NH-NH and the NH-ND dimers and also the ND stretch band profiles of the NH-ND and the ND-ND dimers exhibit similar patterns, respectively. These facts indicates that the vibrational relaxation from the NH/ND stretch level of the normal dimer basically proceed within a monomer unit. The large deuteration effect of the NH stretch band profile observed previously is found to be characteristic of the tautomeric dimer. This behavior is related to a large anharmonicity of the potential energy surface originating from an existence of the double-proton transfer reaction barrier. H. Ishikawa, H. Yabuguchi, Y. Yamada, A. Fujihara, K. Fuke, J. Phys. Chem. A 114, 3199 (2010).

A designed point mutant in Fis1 disrupts dimerization and mitochondrial fission

PubMed Central

Lees, Jonathan P. B.; Manlandro, Cara Marie; Picton, Lora K.; Ebie Tan, Alexandra Z.; Casares, Salvador; Flanagan, John M.; Fleming, Karen G.; Hill, R. Blake

2012-01-01

Mitochondrial and peroxisomal fission are essential processes with defects resulting in cardiomyopathy and neonatal lethality. Central to organelle fission is Fis1, a monomeric tetratricopeptide-like repeat (TPR) protein whose role in assembly of the fission machinery remains obscure. Two non-functional, Saccharomyces cerevisiae Fis1 mutants (L80P or E78D/I85T/Y88H) were previously identified in genetic screens. Here, we find that these two variants in the cytosolic domain of Fis1 (Fis1ΔTM) are unexpectedly dimeric. A truncation variant of Fis1ΔTM that lacks an N-terminal regulatory domain is also found to be dimeric. The ability to dimerize is a property innate to the native Fis1ΔTM amino acid sequence as we find this domain is dimeric after transient exposure to elevated temperature or chemical denaturants and is kinetically trapped at room temperature. This is the first demonstration of a specific self-association in solution for the Fis1 cytoplasmic domain. We propose a three-dimensional domain-swapped model for dimerization that is validated by a designed mutation, A72P, which potently disrupts dimerization of wild type Fis1. A72P also disrupts dimerization of non-functional variants indicating a common structural basis for dimerization. The obligate monomer variant A72P, like the dimer-promoting variants, is non-functional in fission consistent with a model in which Fis1 activity depends on its ability to interconvert between monomer and dimer species. These studies suggest a new functionally important manner in which TPR containing proteins may reversibly self-associate. PMID:22789569

Engineering of a novel Ca{sup 2+}-regulated kinesin molecular motor using a calmodulin dimer linker

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shishido, Hideki; Maruta, Shinsaku, E-mail: maruta@soka.ac.jp

Highlights: Black-Right-Pointing-Pointer Engineered kinesin-M13 and calmodulin involving single cysteine were prepared. Black-Right-Pointing-Pointer CaM mutant was cross-linked to dimer by bifunctional thiol reactive reagent. Black-Right-Pointing-Pointer Kinesin-M13 was dimerized via CaM dimer in the presence of calcium. Black-Right-Pointing-Pointer Function of the engineered kinesin was regulated by a Ca{sup 2+}-calmodulin dimer linker. -- Abstract: The kinesin-microtubule system holds great promise as a molecular shuttle device within biochips. However, one current barrier is that such shuttles do not have 'on-off' control of their movement. Here we report the development of a novel molecular motor powered by an accelerator and brake system, using a kinesinmore » monomer and a calmodulin (CaM) dimer. The kinesin monomer, K355, was fused with a CaM target peptide (M13 peptide) at the C-terminal part of the neck region (K355-M13). We also prepared CaM dimers using CaM mutants (Q3C), (R86C), or (A147C) and crosslinkers that react with cysteine residues. Following induction of K355-M13 dimerization with CaM dimers, we measured K355-M13 motility and found that it can be reversibly regulated in a Ca{sup 2+}-dependent manner. We also found that velocities of K355-M13 varied depending on the type and crosslink position of the CaM dimer used; crosslink length also had a moderate effect on motility. These results suggest Ca{sup 2+}-dependent dimerization of K355-M13 could be used as a novel molecular shuttle, equipped with an accelerator and brake system, for biochip applications.« less

Circulating D-dimer level correlates with disease characteristics in hepatoblastoma patients

PubMed Central

Zhang, BinBin; Liu, GongBao; Liu, XiangQi; Zheng, Shan; Dong, Kuiran; Dong, Rui

2017-01-01

Abstract Objectives: Hepatoblastoma (HB) is the most common pediatric liver malignancy, typically affecting children within the first 4 years of life. However, only a few validated blood biomarkers are used in clinical evaluation. The current study explored the clinical application and significance of D-dimer levels in patients with HB. Method: Forty-four patients with HB were included in this retrospective study. D-dimer and plasma fibrinogen levels were examined, and their correlation with other clinical features was analyzed. D-dimer and plasma fibrinogen levels were examined between HB and other benign hepatic tumors. Results: D-dimer levels were significantly associated with high-risk HB features, such as advanced stage and high α-fetoprotein (AFP) levels. Higher D-dimer levels were observed in stage 4 patients compared with stage 1/2/3 patients (P = .028). Higher D-dimer levels were also observed in patients with higher AFP levels before chemotherapy compared with patients with lower AFP levels after chemotherapy (P< 0.001). In addition, higher D-dimer levels were observed in HB compared with other benign hepatic tumors such as hepatic hemangioma and hepatocellular adenoma (P = .012). No significant difference in D-dimer levels was found between sex (P = .503) and age (≥12 vs <12 months, P = .424). There was no significant difference in plasma fibrinogen levels between sex or age and high-risk HB features, such as advanced stage and high AFP levels. Conclusions: Elevated D-dimer levels could be a useful determinant biomarker for high-risk features in patients with HB. This finding also supports the clinical application of D-dimer in HB. PMID:29381980

Can very high level of D-dimer exclusively predict the presence of thromboembolic diseases?

PubMed

Ho, Chao-Hung

2011-04-01

D-dimer quantitative test is mainly used to rule out the presence of thromboembolic diseases (TEDs). Whether very high D-dimer (100 times above the cutoff point) can exclusively indicate the presence of TED should be known. D-dimer was detected by a quantitative immunoturbidimetric assay. The normal value is 0.2-0.7 mg/L fibrinogen equivalent units (FEUs). During the year of 2009, 1,053 D-dimer tests were performed. We analyzed the results of these patients to find out the causes of very high D-dimer. The mean value of D-dimer in the 1,053 tests was 8.56 mg/L FEU, ranging from <0.2 mg/L to 563.2 mg/L FEU. Of them, 28 samples from 21 patients had very high D-dimer value: >50 mg/L FEU. Of the 21 patients, 9 (43%) had TED, 1 had suspected TED, but not proved by computed tomographic (CT) angiogram, 3 had massive gastrointestinal or other site bleeding, 3 patients had cardiac arrest with samples taken immediately after recovery from cardiopulmonary resuscitation (CPR), 2 had sepsis with disseminated intravascular coagulation (DIC), 1 had postpartum hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome with acute pulmonary edema and renal failure, 1 had multiple traumatic injury, and 1 received thrombolytic therapy. Although TED was the most frequently seen disorder in patients with very high D-dimer value, very high D-dimer was not necessary exclusively the marker of TED. Other disorders such as massive bleeding, status post CPR, sepsis with DIC, multiple traumatic injuries, hyperfibrinolysis and HELLP syndrome can also have very high D-dimer. Copyright © 2011. Published by Elsevier B.V.

Endoplasmic reticulum degradation-enhancing α-mannosidase-like protein 1 targets misfolded HLA-B27 dimers for endoplasmic reticulum-associated degradation.

PubMed

Guiliano, David B; Fussell, Helen; Lenart, Izabela; Tsao, Edward; Nesbeth, Darren; Fletcher, Adam J; Campbell, Elaine C; Yousaf, Nasim; Williams, Sarah; Santos, Susana; Cameron, Amy; Towers, Greg J; Kellam, Paul; Hebert, Daniel N; Gould, Keith G; Powis, Simon J; Antoniou, Antony N

2014-11-01

HLA-B27 forms misfolded heavy chain dimers, which may predispose individuals to inflammatory arthritis by inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). This study was undertaken to define the role of the UPR-induced ER-associated degradation (ERAD) pathway in the disposal of HLA-B27 dimeric conformers. HeLa cell lines expressing only 2 copies of a carboxy-terminally Sv5-tagged HLA-B27 were generated. The ER stress-induced protein ER degradation-enhancing α-mannosidase-like protein 1 (EDEM1) was overexpressed by transfection, and dimer levels were monitored by immunoblotting. EDEM1, the UPR-associated transcription factor X-box binding protein 1 (XBP-1), the E3 ubiquitin ligase hydroxymethylglutaryl-coenzyme A reductase degradation 1 (HRD1), and the degradation-associated proteins derlin 1 and derlin 2 were inhibited using either short hairpin RNA or dominant-negative mutants. The UPR-associated ERAD of HLA-B27 was confirmed using ER stress-inducing pharamacologic agents in kinetic and pulse chase assays. We demonstrated that UPR-induced machinery can target HLA-B27 dimers and that dimer formation can be controlled by alterations to expression levels of components of the UPR-induced ERAD pathway. HLA-B27 dimers and misfolded major histocompatibility complex class I monomeric molecules bound to EDEM1 were detected, and overexpression of EDEM1 led to inhibition of HLA-B27 dimer formation. EDEM1 inhibition resulted in up-regulation of HLA-B27 dimers, while UPR-induced ERAD of dimers was prevented in the absence of EDEM1. HLA-B27 dimer formation was also enhanced in the absence of XBP-1, HRD1, and derlins 1 and 2. The present findings indicate that the UPR ERAD pathway can dispose of HLA-B27 dimers, thus presenting a potential novel therapeutic target for modulation of HLA-B27-associated inflammatory disease. Copyright © 2014 by the American College of Rheumatology.

Bioluminescence Resonance Energy Transfer Studies Reveal Constitutive Dimerization of the Human Lutropin Receptor and a Lack of Correlation between Receptor Activation and the Propensity for Dimerization*

PubMed Central

Guan, Rongbin; Feng, Xiuyan; Wu, Xueqing; Zhang, Meilin; Zhang, Xuesen; Hébert, Terence E.; Segaloff, Deborah L.

2009-01-01

Previous studies from our laboratory using co-immunoprecipitation techniques suggested that the human lutropin receptor (hLHR) constitutively self-associates into dimers/oligomers and that agonist treatment of cells either increased hLHR dimerization/oligomerization and/or stabilized hLHR dimers/oligomers to detergent solubilization (Tao, Y. X., Johnson, N. B., and Segaloff, D. L. (2004) J. Biol. Chem. 279, 5904–5914). In this study, bioluminescence resonance energy transfer (BRET2) analyses confirmed that the hLHR constitutively self-associates in living cells. After subcellular fractionation, hLHR dimers/oligomers were detected in both the plasma membrane and endoplasmic reticulum (ER). Further evidence supporting the constitutive formation of hLHR dimer/oligomers in the ER is provided by data showing homodimerization of misfolded hLHR mutants that are retained in the ER. These mutants, when co-expressed with wild-type receptor, are shown by BRET2 to heterodimerize, accounting for their dominant-negative effects on cell surface receptor expression. Hormone desorption assays using intact cells demonstrate allosterism between hLHR protomers, indicating functional cell surface hLHR dimers. However, quantitative BRET2 analyses in intact cells indicate a lack of effect of agonist on the propensity of the hLHR to dimerize. Using purified plasma membranes, human chorionic gonadotropin was similarly observed to have no effect on the BRET2 signal. An examination of the propensity for constitutively active and signaling inactive hLHR mutants to dimerize further showed no correlation between dimerization and the activation state of the hLHR. Taken altogether, our data suggest that hLHR dimers/oligomers are formed early in the biosynthetic pathway in the ER, are constitutively expressed on the plasma membrane, and are not affected by the activation state of the hLHR. PMID:19147490

An ion mobility-mass spectrometry investigation of monocyte chemoattractant protein-1

NASA Astrophysics Data System (ADS)

Schenauer, Matthew R.; Leary, Julie A.

2009-10-01

In the present article we describe the gas-phase dissociation behavior of the dimeric form of monocyte chemoattractant protein-1 (MCP-1) using quadrupole-traveling wave ion mobility spectrometry-time of flight mass spectrometry (q-TWIMS-TOF MS) (Waters Synapt(TM)). Through investigation of the 9+ charge state of the dimer, we were able to monitor dissociation product ion (monomer) formation as a function of activation energy. Using ion mobility, we were able to observe precursor ion structural changes occurring throughout the activation process. Arrival time distributions (ATDs) for the 5+ monomeric MCP-1 product ions, derived from the gas-phase dissociation of the 9+ dimer, were then compared with ATDs obtained for the 5+ MCP-1 monomer isolated directly from solution. The results show that the dissociated monomer is as compact as the monomer arising from solution, regardless of the trap collision energy (CE) used in the dissociation. The solution-derived monomer, when collisionally activated, also resists significant unfolding within measure. Finally, we compared the collisional activation data for the MCP-1 dimer with an MCP-1 dimer non-covalently bound to a single molecule of the semi-synthetic glycosaminoglycan (GAG) analog Arixtra(TM); the latter a therapeutic anti-thrombin III-activating pentasaccharide. We observed that while dimeric MCP-1 dissociated at relatively low trap CEs, the Arixtra-bound dimer required much higher energies, which also induced covalent bond cleavage in the bound Arixtra molecule. Both the free and Arixtra-bound dimers became less compact and exhibited longer arrival times with increasing trap CEs, albeit the Arixtra-bound complex at slightly higher energies. That both dimers shifted to longer arrival times with increasing activation energy, while the dissociated MCP-1 monomers remained compact, suggests that the longer arrival times of the Arixtra-free and Arixtra-bound dimers may represent a partial breach of non-covalent interactions between the associated MCP-1 monomers, rather than extensive unfolding of individual subunits. The fact that Arixtra preferentially binds MCP-1 dimers and prevents dimer dissociation at comparable activation energies to the Arixtra-free dimer, may suggest that the drug interacts across the two monomers, thereby inhibiting their dissociation.

Two callose synthases, GSL1 and GSL5, play an essential and redundant role in plant and pollen development and in fertility.

PubMed

Enns, Linda C; Kanaoka, Masahiro M; Torii, Keiko U; Comai, Luca; Okada, Kiyotaka; Cleland, Robert E

2005-06-01

Callose, a beta-1,3-glucan that is widespread in plants, is synthesized by callose synthase. Arabidopsis thaliana contains a family of 12 putative callose synthase genes (GSL1-12). The role of callose and of the individual genes in plant development is still largely uncertain. We have now used TILLING and T-DNA insertion mutants (gsl1-1, gsl5-2 and gsl5-3) to study the role of two closely related and linked genes, GSL1 and GSL5, in sporophytic development and in reproduction. Both genes are expressed in all parts of the plant. Sporophytic development was nearly normal in gsl1-1 homozygotes and only moderately defective in homozygotes for either of the two gsl5 alleles. On the other hand, plants that were gsl1-1/+ gsl5/gsl5 were severely defective, with smaller leaves, shorter roots and bolts and smaller flowers. Plants were fertile when the sporophytes had either two wild-type GSL1 alleles, or one GSL5 allele in a gsl1-1 background, but gsl1-1/+ gsl5/gsl5 plants produced an extremely reduced number of viable seeds. A chromosome with mutations in both GSL1 and GSL5 rendered pollen infertile, although such a chromosome could be transmitted via the egg. As a result, it was not possible to obtain plants that were homozygous for mutations in both the GSL genes. Pollen grain development was severely affected in double mutant plants. Many pollen grains were collapsed and inviable in the gsl1-1/gsl1-1 gsl5/+ and gsl1-1/+ gsl5/gsl5 plants. In addition, gsl1-1/+ gsl5/gsl5 plants produced abnormally large pollen with unusual pore structures, and had problems with tetrad dissociation. In this particular genotype, while the callose wall formed around the pollen mother cells, no callose wall separated the resulting tetrads. We conclude that GSL1 and GSL5 play important, but at least partially redundant roles in both sporophytic development and in the development of pollen. They are responsible for the formation of the callose wall that separates the microspores of the tetrad, and also play a gametophytic role later in pollen grain maturation. Other GSL genes may control callose formation at different steps during pollen development.

Biochemical Characterization and Homology Modeling of Methylbutenol Synthase and Implications for Understanding Hemiterpene Synthase Evolution in Plants*

PubMed Central

Gray, Dennis W.; Breneman, Steven R.; Topper, Lauren A.; Sharkey, Thomas D.

2011-01-01

2-Methyl-3-buten-2-ol (MBO) is a five-carbon alcohol produced and emitted in large quantities by many species of pine native to western North America. MBO is structurally and biosynthetically related to isoprene and can have an important impact on regional atmospheric chemistry. The gene for MBO synthase was identified from Pinus sabiniana, and the protein encoded was functionally characterized. MBO synthase is a bifunctional enzyme that produces both MBO and isoprene in a ratio of ∼90:1. Divalent cations are required for activity, whereas monovalent cations are not. MBO production is enhanced by K+, whereas isoprene production is inhibited by K+ such that, at physiologically relevant [K+], little or no isoprene emission should be detected from MBO-emitting trees. The Km of MBO synthase for dimethylallyl diphosphate (20 mm) is comparable with that observed for angiosperm isoprene synthases and 3 orders of magnitude higher than that observed for monoterpene and sesquiterpene synthases. Phylogenetic analysis showed that MBO synthase falls into the TPS-d1 group (gymnosperm monoterpene synthases) and is most closely related to linalool synthase from Picea abies. Structural modeling showed that up to three phenylalanine residues restrict the size of the active site and may be responsible for making this a hemiterpene synthase rather than a monoterpene synthase. One of these residues is homologous to a Phe residue found in the active site of isoprene synthases. The remaining two Phe residues do not have homologs in isoprene synthases but occupy the same space as a second Phe residue that closes off the isoprene synthase active site. PMID:21504898

Phenylthiazole Antibacterial Agents Targeting Cell Wall Synthesis Exhibit Potent Activity in Vitro and in Vivo against Vancomycin-Resistant Enterococci.

PubMed

Mohammad, Haroon; Younis, Waleed; Chen, Lu; Peters, Christine E; Pogliano, Joe; Pogliano, Kit; Cooper, Bruce; Zhang, Jianan; Mayhoub, Abdelrahman; Oldfield, Eric; Cushman, Mark; Seleem, Mohamed N

2017-03-23

The emergence of antibiotic-resistant bacterial species, such as vancomycin-resistant enterococci (VRE), necessitates the development of new antimicrobials. Here, we investigate the spectrum of antibacterial activity of three phenylthiazole-substituted aminoguanidines. These compounds possess potent activity against VRE, inhibiting growth of clinical isolates at concentrations as low as 0.5 μg/mL. The compounds exerted a rapid bactericidal effect, targeting cell wall synthesis. Transposon mutagenesis suggested three possible targets: YubA, YubB (undecaprenyl diphosphate phosphatase (UPPP)), and YubD. Both UPPP as well as undecaprenyl diphosphate synthase were inhibited by compound 1. YubA and YubD are annotated as transporters and may also be targets because 1 collapsed the proton motive force in membrane vesicles. Using Caenorhabditis elegans, we demonstrate that two compounds (1, 3, at 20 μg/mL) retain potent activity in vivo, significantly reducing the burden of VRE in infected worms. Taken altogether, the results indicate that compounds 1 and 3 warrant further investigation as novel antibacterial agents against drug-resistant enterococci.

Design and Preparation of Nanoparticle Dimers for SERS Detection

DTIC Science & Technology

2012-09-10

sensitivity afforded by surface enhanced Raman spectroscopy (SERS). Metal nanoparticles dimers were synthesized that incorporate SERS reporters...and antigens, based on the remarkable sensitivity afforded by surface enhanced Raman spectroscopy (SERS). Metal nanoparticles dimers were...Potma, V. A._Apkarian. High Sensitivity Surface-Enhanced Raman Scattering in Solution Using Engineered Silver Nanosphere Dimers, The Journal of

40 CFR 721.6980 - Dimer acids, polymer with polyalkylene glycol, bisphenol A-diglycidyl ether, and alky-lenepolyols...

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Dimer acids, polymer with polyalkylene... Substances § 721.6980 Dimer acids, polymer with polyalkylene glycol, bisphenol A-diglycidyl ether, and alky... reporting. (1) The chemical substance dimer acids, polymer with polyalkylene glycol, bisphenol A-diglycidyl...

40 CFR 721.6980 - Dimer acids, polymer with polyalkylene glycol, bisphenol A-diglycidyl ether, and alky-lenepolyols...

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Dimer acids, polymer with polyalkylene... Substances § 721.6980 Dimer acids, polymer with polyalkylene glycol, bisphenol A-diglycidyl ether, and alky... reporting. (1) The chemical substance dimer acids, polymer with polyalkylene glycol, bisphenol A-diglycidyl...

The Rate of Vitamin A Dimerization in Lipofuscinogenesis, Fundus Autofluorescence, Retinal Senescence and Degeneration.

PubMed

Washington, Ilyas; Saad, Leonide

2016-01-01

One of the earliest events preceding several forms of retinal degeneration is the formation and accumulation of vitamin A dimers in the retinal pigment epithelium (RPE) and underlying Bruch's membrane (BM). Such degenerations include Stargardt disease, Best disease, forms of retinitis pigmentosa, and age-related macular degeneration (AMD). Since their discovery in the 1990's, dimers of vitamin A, have been postulated as chemical triggers driving retinal senescence and degeneration. There is evidence to suggest that the rate at which vitamin A dimerizes and the eye's response to the dimerization products may dictate the retina's lifespan. Here, we present outstanding questions, finding the answers to which may help to elucidate the role of vitamin A dimerization in retinal degeneration.

Statistical transmutation in doped quantum dimer models.

PubMed

Lamas, C A; Ralko, A; Cabra, D C; Poilblanc, D; Pujol, P

2012-07-06

We prove a "statistical transmutation" symmetry of doped quantum dimer models on the square, triangular, and kagome lattices: the energy spectrum is invariant under a simultaneous change of statistics (i.e., bosonic into fermionic or vice versa) of the holes and of the signs of all the dimer resonance loops. This exact transformation enables us to define the duality equivalence between doped quantum dimer Hamiltonians and provides the analytic framework to analyze dynamical statistical transmutations. We investigate numerically the doping of the triangular quantum dimer model with special focus on the topological Z(2) dimer liquid. Doping leads to four (instead of two for the square lattice) inequivalent families of Hamiltonians. Competition between phase separation, superfluidity, supersolidity, and fermionic phases is investigated in the four families.

Constitutive activation and uncoupling of the atrial natriuretic peptide receptor by mutations at the dimer interface. Role of the dimer structure in signalling.

PubMed

Qiu, Yue; Ogawa, Haruo; Miyagi, Masaru; Misono, Kunio S

2004-02-13

The crystal packing of the extracellular hormone binding domain of the atrial natriuretic peptide (ANP) receptor contains two possible dimer pairs, the head-to-head (hh) and tail-to-tail (tt) dimer pairs associated through the membrane-distal and membrane-proximal subdomains, respectively. The tt-dimer structure has been proposed previously (van den Akker, F., Zhang, X., Miyagi, M., Huo, X., Misono, K. S., and Yee, V. C. (2000) Nature 406, 101-104). However, no direct evidence is available to identify the physiological dimer form. Here we report site-directed mutagenesis studies of residues at the two alternative dimer interfaces in the full-length receptor expressed on COS cells. The Trp74 to Arg mutation (W74R) or D71R at the hh-dimer interface caused partial constitutive guanylate cyclase activation, whereas mutation F96D or H99D caused receptor uncoupling. In contrast, mutation Y196D or L225D at the tt-interface had no such effect. His99 modification at the hh-dimer interface by ethoxyformic anhydride abolished ANP binding. These results suggest that the hh-dimer represents the physiological structure. Recently, we determined the crystal structure of ANPR complexed with ANP and proposed a hormone-induced rotation mechanism mediating transmembrane signaling (H. Ogawa, Y. Qiu, C. M. Ogata, and K. S. Misono, submitted for publication). The observed effects of mutations are consistent with the ANP-induced structural change identified from the crystal structures with and without ANP and support the proposed rotation mechanism for ANP receptor signaling.

Low temperatures shear viscosity of a two-component dipolar Fermi gas with unequal population

NASA Astrophysics Data System (ADS)

Darsheshdar, E.; Yavari, H.; Zangeneh, Z.

2016-07-01

By using the Green's functions method and linear response theory we calculate the shear viscosity of a two-component dipolar Fermi gas with population imbalance (spin polarized) in the low temperatures limit. In the strong-coupling Bose-Einstein condensation (BEC) region where a Feshbach resonance gives rise to tightly bound dimer molecules, a spin-polarized Fermi superfluid reduces to a simple Bose-Fermi mixture of Bose-condensed dimers and the leftover unpaired fermions (atoms). The interactions between dimer-atom, dimer-dimer, and atom-atom take into account to the viscous relaxation time (τη) . By evaluating the self-energies in the ladder approximation we determine the relaxation times due to dimer-atom (τDA) , dimer-dimer (τcDD ,τdDD) , and atom-atom (τAA) interactions. We will show that relaxation rates due to these interactions τDA-1 ,τcDD-1, τdDD-1, and τAA-1 have T2, T4, e - E /kB T (E is the spectrum of the dimer atoms), and T 3 / 2 behavior respectively in the low temperature limit (T → 0) and consequently, the atom-atom interaction plays the dominant role in the shear viscosity in this rang of temperatures. For small polarization (τDA ,τAA ≫τcDD ,τdDD), the low temperatures shear viscosity is determined by contact interaction between dimers and the shear viscosity varies as T-5 which has the same behavior as the viscosity of other superfluid systems such as superfluid neutron stars, and liquid helium.

Making Structural Sense of Dimerization Interfaces of Delta Opioid Receptor Homodimers†

PubMed Central

2011-01-01

Opioid receptors, like other members of the G protein-coupled receptor (GPCR) family, have been shown to associate to form dimers and/or oligomers at the plasma membrane. Whether this association is stable or transient is not known. Recent compelling evidence suggests that at least some GPCRs rapidly associate and dissociate. We have recently calculated binding affinities from free energy estimates to predict transient association between mouse delta opioid receptor (DOR) protomers at a symmetric interface involving the fourth transmembrane (TM4) helix (herein termed “4” dimer). Here we present disulfide cross-linking experiments with DOR constructs with cysteines substituted at the extracellular ends of TM4 or TM5 that confirm the formation of DOR complexes involving these helices. Our results are consistent with the involvement of TM4 and/or TM5 at the DOR homodimer interface, but possibly with differing association propensities. Coarse-grained (CG) well-tempered metadynamics simulations of two different dimeric arrangements of DOR involving TM4 alone or with TM5 (herein termed “4/5” dimer) in an explicit lipid−water environment confirmed the presence of two structurally and energetically similar configurations of the 4 dimer, as previously assessed by umbrella sampling calculations, and revealed a single energetic minimum of the 4/5 dimer. Additional CG umbrella sampling simulations of the 4/5 dimer indicated that the strength of association between DOR protomers varies depending on the protein region at the interface, with the 4 dimer being more stable than the 4/5 dimer. PMID:21261298

Alternative dimerization interfaces in the glucocorticoid receptor-α ligand binding domain.

PubMed

Bianchetti, Laurent; Wassmer, Bianca; Defosset, Audrey; Smertina, Anna; Tiberti, Marion L; Stote, Roland H; Dejaegere, Annick

2018-04-30

Nuclear hormone receptors (NRs) constitute a large family of multi-domain ligand-activated transcription factors. Dimerization is essential for their regulation, and both DNA binding domain (DBD) and ligand binding domain (LBD) are implicated in dimerization. Intriguingly, the glucocorticoid receptor-α (GRα) presents a DBD dimeric architecture similar to that of the homologous estrogen receptor-α (ERα), but an atypical dimeric architecture for the LBD. The physiological relevance of the proposed GRα LBD dimer is a subject of debate. We analyzed all GRα LBD homodimers observed in crystals using an energetic analysis based on the PISA and on the MM/PBSA methods and a sequence conservation analysis, using the ERα LBD dimer as a reference point. Several dimeric assemblies were observed for GRα LBD. The assembly generally taken to be physiologically relevant showed weak binding free energy and no significant residue conservation at the contact interface, while an alternative homodimer mediated by both helix 9 and C-terminal residues showed significant binding free energy and residue conservation. However, none of the GRα LBD assemblies found in crystals are as stable or conserved as the canonical ERα LBD dimer. GRα C-terminal sequence (F-domain) forms a steric obstacle to the canonical dimer assembly in all available structures. Our analysis calls for a re-examination of the currently accepted GRα homodimer structure and experimental investigations of the alternative architectures. This work questions the validity of the currently accepted architecture. This has implications for interpreting physiological data and for therapeutic design pertaining to glucocorticoid research. Copyright © 2018. Published by Elsevier B.V.

Cis elements and trans-acting factors involved in the RNA dimerization of the human immunodeficiency virus HIV-1.

PubMed

Darlix, J L; Gabus, C; Nugeyre, M T; Clavel, F; Barré-Sinoussi, F

1990-12-05

The retroviral genome consists of two identical RNA molecules joined at their 5' ends by the Dimer Linkage Structure (DLS). To study the mechanism of dimerization and the DLS of HIV-1 RNA, large amounts of bona fide HIV-1 RNA and of mutants have been synthesized in vitro. We report that HIV-1 RNA forms dimeric molecules and that viral nucleocapsid (NC) protein NCp15 greatly activates dimerization. Deletion mutagenesis in the RNA 5' 1333 nucleotides indicated that a small domain of 100 nucleotides, located between positions 311 to 415 from the 5' end, is necessary and sufficient to promote HIV-1 RNA dimerization. This dimerization domain encompasses an encapsidation element located between the 5' splice donor site and initiator AUG of gag and shows little sequence variations in different strains of HIV-1. Furthermore, cross-linking analysis of the interactions between NC and HIV-1 RNA (311 to 415) locates a major contact site in the encapsidation element of HIV-1 RNA. The genomic RNA dimer is tightly associated with nucleocapsid protein molecules in avian and murine retroviruses, and this ribonucleoprotein structure is believed to be the template for reverse transcription. Genomic RNA-protein interactions have been analyzed in human immunodeficiency virus (HIV) virions and results showed that NC protein molecules are tightly bound to the genomic RNA dimer. Since retroviral RNA dimerization and packaging appear to be under the control of the same cis element, the encapsidation sequences, and trans-acting factor, the NC protein, they are probably related events in the course of virion assembly.

Atomic resolution crystal structure of VcLMWPTP-1 from Vibrio cholerae O395: Insights into a novel mode of dimerization in the low molecular weight protein tyrosine phosphatase family

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nath, Seema; Banerjee, Ramanuj; Sen, Udayaditya, E-mail: udayaditya.sen@saha.ac.in

Highlights: • VcLMWPTP-1 forms dimer in solution. • The dimer is catalytically active unlike other reported dimeric LMWPTPs. • The formation of extended dimeric surface excludes the active site pocket. • The surface bears closer resemblance to eukaryotic LMWPTPs. - Abstract: Low molecular weight protein tyrosine phosphatase (LMWPTP) is a group of phosphotyrosine phosphatase ubiquitously found in a wide range of organisms ranging from bacteria to mammals. Dimerization in the LMWPTP family has been reported earlier which follows a common mechanism involving active site residues leading to an enzymatically inactive species. Here we report a novel form of dimerization inmore » a LMWPTP from Vibrio cholera 0395 (VcLMWPTP-1). Studies in solution reveal the existence of the dimer in solution while kinetic study depicts the active form of the enzyme. This indicates that the mode of dimerization in VcLMWPTP-1 is different from others where active site residues are not involved in the process. A high resolution (1.45 Å) crystal structure of VcLMWPTP-1 confirms a different mode of dimerization where the active site is catalytically accessible as evident by a tightly bound substrate mimicking ligand, MOPS at the active site pocket. Although being a member of a prokaryotic protein family, VcLMWPTP-1 structure resembles very closely to LMWPTP from a eukaryote, Entamoeba histolytica. It also delineates the diverse surface properties around the active site of the enzyme.« less

Differential action of small molecule HER kinase inhibitors on receptor heterodimerization: therapeutic implications.

PubMed

Sánchez-Martín, M; Pandiella, A

2012-07-01

Deregulation of ErbB/HER receptor tyrosine kinases has been linked to several types of cancer. The mechanism of activation of these receptors includes establishment of receptor dimers. Here, we have analyzed the action of different small molecule HER tyrosine kinase inhibitors (TKIs) on HER receptor dimerization. Breast cancer cell lines were treated with distinct TKIs and the formation of HER2-HER3 dimers was analyzed by coimmunoprecipitation and western blot or by Förster resonance energy transfer assays. Antibody-dependent cellular cytotoxicity was analyzed by measuring the release of lactate dehydrogenase and cell viability. Lapatinib and neratinib interfered with ligand-induced dimerization of HER receptors; while pelitinib, gefitinib, canertinib or erlotinib did not. Moreover, lapatinib and neratinib were able to disrupt previously formed receptor dimers. Structural analyses allowed the elucidation of the mechanism by which some TKIs prevent the formation of HER receptor dimers, while others do not. Experiments aimed at defining the functional importance of dimerization indicated that TKIs that impeded dimerization prevented down-regulation of HER2 receptors, and favored the action of trastuzumab. We postulate that TKIs that prevent dimerization and down-regulation of HER2 may augment the antitumoral action of trastuzumab, and this mechanism of action should be considered in the treatment of HER2 positive tumors which combine TKIs with antireceptor antibodies. Copyright © 2011 UICC.

Cold-active alkaline phosphatase is irreversibly transformed into an inactive dimer by low urea concentrations.

PubMed

Hjörleifsson, Jens Guðmundur; Ásgeirsson, Bjarni

2016-07-01

Alkaline phosphatase is a homodimeric metallo-hydrolase where both Zn(2+) and Mg(2+) are important for catalysis and stability. Cold-adapted alkaline phosphatase variants have high activity at low temperatures and lower thermal stability compared with variants from mesophilic hosts. The instability, and thus inactivation, could be due to loose association of the dimers and/or loosely bound Mg(2)(+) in the active site, but this has not been studied in detail for the cold-adapted variants. Here, we focus on using the intrinsic fluorescence of Trp in alkaline phosphatase from the marine bacterium Vibrio splendidus (VAP) to probe for dimerization. Trp→Phe substitutions showed that two out of the five native Trp residues contributed mostly to the fluorescence emission. One residue, 15Å away from the active site (W460) and highly solvent excluded, was phosphorescent and had a distant role in substrate binding. An additional Trp residue was introduced to the dimer interface to act as a possible probe for dimerization. Urea denaturation curves indicated that an inactive dimer intermediate, structurally equivalent to the native state, was formed before dimer dissociation took place. This is the first example of the transition of a native dimer to an inactive dimer intermediate for alkaline phosphatase without using mutagenesis, ligands, or competitive inhibition. Copyright © 2016 Elsevier B.V. All rights reserved.

Proline Substitution of Dimer Interface β-strand Residues as a Strategy for the Design of Functional Monomeric Proteins

PubMed Central

Joseph, Prem Raj B.; Poluri, Krishna Mohan; Gangavarapu, Pavani; Rajagopalan, Lavanya; Raghuwanshi, Sandeep; Richardson, Ricardo M.; Garofalo, Roberto P.; Rajarathnam, Krishna

2013-01-01

Proteins that exist in monomer-dimer equilibrium can be found in all organisms ranging from bacteria to humans; this facilitates fine-tuning of activities from signaling to catalysis. However, studying the structural basis of monomer function that naturally exists in monomer-dimer equilibrium is challenging, and most studies to date on designing monomers have focused on disrupting packing or electrostatic interactions that stabilize the dimer interface. In this study, we show that disrupting backbone H-bonding interactions by substituting dimer interface β-strand residues with proline (Pro) results in fully folded and functional monomers, by exploiting proline’s unique feature, the lack of a backbone amide proton. In interleukin-8, we substituted Pro for each of the three residues that form H-bonds across the dimer interface β-strands. We characterized the structures, dynamics, stability, dimerization state, and activity using NMR, molecular dynamics simulations, fluorescence, and functional assays. Our studies show that a single Pro substitution at the middle of the dimer interface β-strand is sufficient to generate a fully functional monomer. Interestingly, double Pro substitutions, compared to single Pro substitution, resulted in higher stability without compromising native monomer fold or function. We propose that Pro substitution of interface β-strand residues is a viable strategy for generating functional monomers of dimeric, and potentially tetrameric and higher-order oligomeric proteins. PMID:24048001

Analysis of hepatitis C virus RNA dimerization and core–RNA interactions

PubMed Central

Ivanyi-Nagy, Roland; Kanevsky, Igor; Gabus, Caroline; Lavergne, Jean-Pierre; Ficheux, Damien; Penin, François; Fossé, Philippe; Darlix, Jean-Luc

2006-01-01

The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3′-untranslated region (3′-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623–2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3′-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus. PMID:16707664

Mass spectrometric characterization of human serum albumin dimer: A new potential biomarker in chronic liver diseases.

PubMed

Naldi, Marina; Baldassarre, Maurizio; Nati, Marina; Laggetta, Maristella; Giannone, Ferdinando Antonino; Domenicali, Marco; Bernardi, Mauro; Caraceni, Paolo; Bertucci, Carlo

2015-08-10

Human serum albumin (HSA) undergoes several structural alterations affecting its properties in pro-oxidant and pro-inflammatory environments, as it occurs during liver cirrhosis. These modifications include the formation of albumin dimers. Although HSA dimers were reported to be an oxidative stress biomarker, to date nothing is known about their role in liver cirrhosis and related complications. Additionally, no high sensitive analytical method was available for HSA dimers assessment in clinical settings. Thus the HSA dimeric form in human plasma was characterized by mass spectrometry using liquid chromatography tandem mass spectrometry (LC-ESI-Q-TOF) and matrix assisted laser desorption time of flight (MALDI-TOF) techniques. N-terminal and C-terminal truncated HSA, as well as the native HSA, undergo dimerization by binding another HSA molecule. This study demonstrated the presence of both homo- and hetero-dimeric forms of HSA. The dimerization site was proved to be at Cys-34, forming a disulphide bridge between two albumin molecules, as determined by LC-MS analysis after tryptic digestion. Interestingly, when plasma samples from cirrhotic subjects were analysed, the dimer/monomer ratio resulted significantly increased when compared to that of healthy subjects. These isoforms could represent promising biomarkers for liver disease. Additionally, this analytical approach leads to the relative quantification of the residual native HSA, with fully preserved structural integrity. Copyright © 2014 Elsevier B.V. All rights reserved.

The Far Infrared Vibration-Rotation Spectrum of the Ammonia Dimer.

NASA Astrophysics Data System (ADS)

Loeser, Jennifer Gertrud

1995-11-01

The ammonia dimer has been shown to exhibit unusual weak bonding properties relative to those of the other prototypical second row systems, the hydrogen fluoride dimer and the water dimer. The ultimate goal of the work initiated in this dissertation is to determine a complete intermolecular potential energy surface for the ammonia dimer. It is first necessary to observe its far infrared vibration-rotation-tunneling (VRT) spectrum and to develop a group theoretical model that explains this spectrum in terms of the internal dynamics of the ammonia dimer. These first steps are the subject of this dissertation. First, the current understanding of the ammonia dimer system is reviewed. Group theoretical descriptions of the nature of the ammonia dimer VRT states are explained in detail. An overview of the experimental and theoretical studies of the ammonia dimer made during the last decade is presented. Second, progress on the analysis of the microwave and far infrared spectrum of (ND_3)_2 below 13 cm^{-1} is reported. These spectra directly measure the 'donor -acceptor' interchange splittings in (ND_3) _2, and determine some of the monomer umbrella inversion tunneling splittings. Third, new 80-90 cm^{-1} far infrared spectra of (NH_3)_2 are presented and a preliminary analysis is proposed. Most of the new excited VRT states have been assigned as tunneling sublevels of an out-of-plane intermolecular vibration.

Geranyl diphosphate synthase large subunit, and methods of use

DOEpatents

Croteau, Rodney B.; Burke, Charles C.; Wildung, Mark R.

2001-10-16

A cDNA encoding geranyl diphosphate synthase large subunit from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase large subunit). In another aspect, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase large subunit. In yet another aspect, the present invention provides isolated, recombinant geranyl diphosphate synthase protein comprising an isolated, recombinant geranyl diphosphate synthase large subunit protein and an isolated, recombinant geranyl diphosphate synthase small subunit protein. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase.

Molecular architectures of benzoic acid-specific type III polyketide synthases

PubMed Central

Stewart, Charles; Woods, Kate; Macias, Greg; Allan, Andrew C.; Noel, Joseph P.

2017-01-01

Biphenyl synthase and benzophenone synthase constitute an evolutionarily distinct clade of type III polyketide synthases (PKSs) that use benzoic acid-derived substrates to produce defense metabolites in plants. The use of benzoyl-CoA as an endogenous substrate is unusual for type III PKSs. Moreover, sequence analyses indicate that the residues responsible for the functional diversification of type III PKSs are mutated in benzoic acid-specific type III PKSs. In order to gain a better understanding of structure–function relationships within the type III PKS family, the crystal structures of biphenyl synthase from Malus × domestica and benzophenone synthase from Hypericum androsaemum were compared with the structure of an archetypal type III PKS: chalcone synthase from Malus × domestica. Both biphenyl synthase and benzophenone synthase contain mutations that reshape their active-site cavities to prevent the binding of 4-coumaroyl-CoA and to favor the binding of small hydrophobic substrates. The active-site cavities of biphenyl synthase and benzophenone synthase also contain a novel pocket associated with their chain-elongation and cyclization reactions. Collectively, these results illuminate structural determinants of benzoic acid-specific type III PKSs and expand the understanding of the evolution of specialized metabolic pathways in plants. PMID:29199980

Suites of Terpene Synthases Explain Differential Terpenoid Production in Ginger and Turmeric Tissues

PubMed Central

Koo, Hyun Jo; Gang, David R.

2012-01-01

The essential oils of ginger (Zingiber officinale) and turmeric (Curcuma longa) contain a large variety of terpenoids, some of which possess anticancer, antiulcer, and antioxidant properties. Despite their importance, only four terpene synthases have been identified from the Zingiberaceae family: (+)-germacrene D synthase and (S)-β-bisabolene synthase from ginger rhizome, and α-humulene synthase and β-eudesmol synthase from shampoo ginger (Zingiber zerumbet) rhizome. We report the identification of 25 mono- and 18 sesquiterpene synthases from ginger and turmeric, with 13 and 11, respectively, being functionally characterized. Novel terpene synthases, (−)-caryolan-1-ol synthase and α-zingiberene/β-sesquiphellandrene synthase, which is responsible for formation of the major sesquiterpenoids in ginger and turmeric rhizomes, were also discovered. These suites of enzymes are responsible for formation of the majority of the terpenoids present in these two plants. Structures of several were modeled, and a comparison of sets of paralogs suggests how the terpene synthases in ginger and turmeric evolved. The most abundant and most important sesquiterpenoids in turmeric rhizomes, (+)-α-turmerone and (+)-β-turmerone, are produced from (−)-α-zingiberene and (−)-β-sesquiphellandrene, respectively, via α-zingiberene/β-sesquiphellandrene oxidase and a still unidentified dehydrogenase. PMID:23272109

Geranyl diphosphate synthase from mint

DOEpatents

Croteau, Rodney Bruce; Wildung, Mark Raymond; Burke, Charles Cullen; Gershenzon, Jonathan

1999-01-01

A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate.

Geranyl diphosphate synthase from mint

DOEpatents

Croteau, R.B.; Wildung, M.R.; Burke, C.C.; Gershenzon, J.

1999-03-02

A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate. 5 figs.

Structural features of the KPI domain control APP dimerization, trafficking, and processing.

PubMed

Ben Khalifa, Naouel; Tyteca, Donatienne; Marinangeli, Claudia; Depuydt, Mathieu; Collet, Jean-François; Courtoy, Pierre J; Renauld, Jean-Christophe; Constantinescu, Stefan; Octave, Jean-Noël; Kienlen-Campard, Pascal

2012-02-01

The two major isoforms of human APP, APP695 and APP751, differ by the presence of a Kunitz-type protease inhibitor (KPI) domain in the extracellular region. APP processing and function is thought to be regulated by homodimerization. We used bimolecular fluorescence complementation (BiFC) to study dimerization of different APP isoforms and mutants. APP751 was found to form significantly more homodimers than APP695. Mutation of dimerization motifs in the TM domain did not affect fluorescence complementation, but native folding of KPI is critical for APP751 homodimerization. APP751 and APP695 dimers were mostly localized at steady state in the Golgi region, suggesting that most of the APP751 and 695 dimers are in the secretory pathway. Mutation of the KPI led to the retention of the APP homodimers in the endoplasmic reticulum. We finally showed that APP751 is more efficiently processed through the nonamyloidogenic pathway than APP695. These findings provide new insight on the particular role of KPI domain in APP dimerization. The correlation observed between dimerization, subcellular localization, and processing suggests that dimerization acts as an efficient regulator of APP trafficking in the secretory compartments that has major consequences on its processing.

The Dimer Interface of the Membrane Type 1 Matrix Metalloproteinase Hemopexin Domain

PubMed Central

Tochowicz, Anna; Goettig, Peter; Evans, Richard; Visse, Robert; Shitomi, Yasuyuki; Palmisano, Ralf; Ito, Noriko; Richter, Klaus; Maskos, Klaus; Franke, Daniel; Svergun, Dmitri; Nagase, Hideaki; Bode, Wolfram; Itoh, Yoshifumi

2011-01-01

Homodimerization is an essential step for membrane type 1 matrix metalloproteinase (MT1-MMP) to activate proMMP-2 and to degrade collagen on the cell surface. To uncover the molecular basis of the hemopexin (Hpx) domain-driven dimerization of MT1-MMP, a crystal structure of the Hpx domain was solved at 1.7 Å resolution. Two interactions were identified as potential biological dimer interfaces in the crystal structure, and mutagenesis studies revealed that the biological dimer possesses a symmetrical interaction where blades II and III of molecule A interact with blades III and II of molecule B. The mutations of amino acids involved in the interaction weakened the dimer interaction of Hpx domains in solution, and incorporation of these mutations into the full-length enzyme significantly inhibited dimer-dependent functions on the cell surface, including proMMP-2 activation, collagen degradation, and invasion into the three-dimensional collagen matrix, whereas dimer-independent functions, including gelatin film degradation and two-dimensional cell migration, were not affected. These results shed light on the structural basis of MT1-MMP dimerization that is crucial to promote cellular invasion. PMID:21193411

Effect of thermal sterilization on ferulic, coumaric and cinnamic acids: dimerization and antioxidant activity.

PubMed

Arrieta-Baez, Daniel; Dorantes-Álvarez, Lidia; Martinez-Torres, Rocio; Zepeda-Vallejo, Gerardo; Jaramillo-Flores, Maria Eugenia; Ortiz-Moreno, Alicia; Aparicio-Ozores, Gerardo

2012-10-01

Some phenolic compounds, such as ferulic acid and p-coumaric acid, exist in the form of free acids, in fruits, rice, corn and other grains. Thermal treatment (121 °C at 15-17 psi) for different times on ferulic, p-coumaric and cinnamic acids as well as equimolar mixtures of these acids was investigated. Ferulic and p-coumaric acids underwent decarboxylation, yielding dimeric products formed through their corresponding radical intermediates, while cinnamic acid was recovered unreacted. High-performance liquid chromatography analysis showed no cross-dimerization when equimolar mixtures of pairs of hydroxycinnamic acids were treated under the same conditions. Dimers were characterized as (E)-4',4″-(but-1-ene-1,3-diyl)bis(2'-methoxyphenol)) (dimer of 4-vinylguaiacol) and (E)-4,4'-(but-1-ene-1,3-diyl)diphenol) (dimer of 4-vinylphenol) by nuclear magnetic resonance and mass spectrometry. Sterilization by thermal processing produced dimers of ferulic and coumaric acid. The antioxidant activity of these dimers was greater than that of the respective hydroxycinnamic acids. These results may be relevant for fruits and grains that contain hydroxycinnamic acids and undergo sterilization processes such as canning. Copyright © 2012 Society of Chemical Industry.

The dimer interface of the membrane type 1 matrix metalloproteinase hemopexin domain: crystal structure and biological functions.

PubMed

Tochowicz, Anna; Goettig, Peter; Evans, Richard; Visse, Robert; Shitomi, Yasuyuki; Palmisano, Ralf; Ito, Noriko; Richter, Klaus; Maskos, Klaus; Franke, Daniel; Svergun, Dmitri; Nagase, Hideaki; Bode, Wolfram; Itoh, Yoshifumi

2011-03-04

Homodimerization is an essential step for membrane type 1 matrix metalloproteinase (MT1-MMP) to activate proMMP-2 and to degrade collagen on the cell surface. To uncover the molecular basis of the hemopexin (Hpx) domain-driven dimerization of MT1-MMP, a crystal structure of the Hpx domain was solved at 1.7 Å resolution. Two interactions were identified as potential biological dimer interfaces in the crystal structure, and mutagenesis studies revealed that the biological dimer possesses a symmetrical interaction where blades II and III of molecule A interact with blades III and II of molecule B. The mutations of amino acids involved in the interaction weakened the dimer interaction of Hpx domains in solution, and incorporation of these mutations into the full-length enzyme significantly inhibited dimer-dependent functions on the cell surface, including proMMP-2 activation, collagen degradation, and invasion into the three-dimensional collagen matrix, whereas dimer-independent functions, including gelatin film degradation and two-dimensional cell migration, were not affected. These results shed light on the structural basis of MT1-MMP dimerization that is crucial to promote cellular invasion.

Single-molecule photobleaching reveals increased MET receptor dimerization upon ligand binding in intact cells

PubMed Central

2013-01-01

Background The human receptor tyrosine kinase MET and its ligand hepatocyte growth factor/scatter factor are essential during embryonic development and play an important role during cancer metastasis and tissue regeneration. In addition, it was found that MET is also relevant for infectious diseases and is the target of different bacteria, amongst them Listeria monocytogenes that induces bacterial uptake through the surface protein internalin B. Binding of ligand to the MET receptor is proposed to lead to receptor dimerization. However, it is also discussed whether preformed MET dimers exist on the cell membrane. Results To address these issues we used single-molecule fluorescence microscopy techniques. Our photobleaching experiments show that MET exists in dimers on the membrane of cells in the absence of ligand and that the proportion of MET dimers increases significantly upon ligand binding. Conclusions Our results indicate that partially preformed MET dimers may play a role in ligand binding or MET signaling. The addition of the bacterial ligand internalin B leads to an increase of MET dimers which is in agreement with the model of ligand-induced dimerization of receptor tyrosine kinases. PMID:23731667

Fano-like resonance in symmetry-broken gold nanotube dimer.

PubMed

Wu, DaJian; Jiang, ShuMin; Cheng, Ying; Liu, XiaoJun

2012-11-19

The influences of the symmetry-breaking on the plasmon resonance couplings in the isolated gold nanotube and the gold nanotube dimer have been investigated by means of the finite element method. It is found that the core offset of gold nanotubes leads to the red-shifts of the low energy modes and the enhanced near-field on the thin shell side of the symmetry-broken gold nanotube (SBGNT). In the weak coupling model of the SBGNT dimer, the interference of the bonding octupole mode of the dimer with the dipole modes causes a strong Fano-like resonance in scattering spectrum. The Fano dip shows a red-shift and becomes deep with the increase of the offset-value. In the strong coupling model of the SBGNT dimer, the coupling between two SBGNTs induces giant electric field enhancement at the gap of the dimer, which is much larger than that in the symmetry gold nanotube dimer. The SBGNT with larger offset-value exhibits stronger near-field at the "hot spot".

The Reach of Linear Protein-DNA Dimerizers

PubMed Central

Stafford, Ryan L.; Dervan, Peter B.

2008-01-01

A protein-DNA dimerizer constructed from a DNA-binding pyrrole-imidazole polyamide and the peptide FYPWMK facilitates binding of the natural transcription factor Exd to an adjacent DNA site. Previous dimerizers have been constructed with the peptide attached to an internal pyrrole monomer in an overall branched oligomer. Linear oligomers constructed by attaching the peptide to the polyamide C-terminus expand the range of protein-DNA dimerization to six additional DNA sites. Replacing the FYPWMK hexapeptide with a WM dipeptide, which was previously functional in branched compounds, does not lead to a functional linear dimerizer. Instead, inserting an additional lysine generates a minimal, linear WMK tripeptide conjugate that maintains the activity of the larger FYPWMK dimerizers in a single DNA-binding site orientation. These studies provide insight into the importance of linker length and composition, binding site spacing and orientation, and the protein-binding domain content that are important for the optimization of protein DNA-dimerizers suitable for biological experiments. PMID:17949089

Stability of ALS-related Superoxide Dismutase Protein variants

NASA Astrophysics Data System (ADS)

Lusebrink, Daniel; Plotkin, Steven

Superoxide dismutase (SOD1) is a metal binding, homodimeric protein, whose misfolding is implicated in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Monomerization is believed to be a key step in the propagation of the disease. The dimer stability is often difficult to measure experimentally however, because it is entangled with protein unfolding and metal loss. We thus computationally investigate the dimer stability of mutants of SOD1 known to be associated with ALS. We report on systematic trends in dimer stability, as well as intriguing allosteric communication between mutations and the dimer interface. We study the dimer stabilities in molecular dynamics simulations and obtain the binding free energies of the dimers from pulling essays. Mutations are applied in silicoand we compare the differences of binding free energies compared to the wild type.

Heterologous Quaternary Structure of CXCL12 and its Relationship to the CC Chemokine Family

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murphy, J.; Yuan, H; Kong, Y

2010-01-01

X-ray crystallographic studies reveal that CXCL12 is able to form multiple dimer types, a traditional CXC dimer and a 'CC-like' form. Phylogenetic analysis of all known human chemokines demonstrates CXCL12 is more closely related to the CC chemokine class than other CXC chemokines. These observations indicate that CXCL12 contains genomic and structural elements characteristic of both CXC and CC chemokines.Chemokines are members of a superfamily of proteins involved in the migration of cells to the proper anatomical position during embryonic development or in response to infection or stress during an immune response. There are two major (CC and CXC) andmore » two minor (CX3C and XC) families based on the sequence around the first conserved cysteine. The topology of all structures is essentially identical with a flexible N-terminal region of 3-8 amino acids, a 10-20 residue N-terminal loop, a short 3{sub 10}-helix, three {beta}-strands, and a {alpha}-helix. The major consequence of the subtle difference between the families occurs at the oligomeric level. Monomers of the CC, CXC, and CX3C families form dimers in a family-specific manner. The XCL1 chemokine is a monomer that can interconvert between two folded states. All chemokines activate GPCRs according to family-specificity, however there are a few examples of chemokines crossing the family boundary to function as antagonists. A two-stage mechanism for chemokine activation of GPCRs has been proposed. The N-terminal region of the receptor interacts with the chemokine, followed by receptor activation by the chemokine N-terminal region. Monomeric chemokines have been demonstrated to be the active form for receptor function. There are numerous examples of both chemokines and their receptors forming dimers. While family-specific dimerization may be an attractive explanation for why specific chemokines only activate GPCRs within their own family, the role of dimers in the function of chemokines has not been resolved. Given that CXCL12 is in the CXC family, the CXC dimer is considered the physiologic dimer in all previous studies based on crystallographic evidence. NMR and mutational studies agree with the CXC dimer form in solution. The CXC form of the dimer is seen in recent structures of CXCL12 bound to a heparin disaccharide and several CXCR4 peptides. In one case, crystals of the CXC-type dimer were soaked in a heparin disaccharide solution to determine the interactions between this dimer and bound disaccharide. In another case, in order to overcome NMR chemical shift line broadening when CXCR4 peptides are added, a 'locked' dimer was constructed by introducing a cysteine mutant that linked subunits as a CXC dimer through an inter-subunit disulfide bond. The solution structures of the locked CXC dimer with CXCR4 peptides were determined. The locked CXC dimer retained Ca{sup 2+} mobilization yet lost chemotaxis activity, presumably because the monomer is the active form. In addition to existing as a monomer and CXC dimer, CXCL12 is now demonstrated to have the capacity to form CC type dimers in the presence of a CXCR4 peptide.« less

Oligomerization of deoxynucleoside-biphosphate dimers - Template and linkage specificity

NASA Technical Reports Server (NTRS)

Visscher, J.; Van Der Woerd, R.; Bakker, C. G.; Schwartz, Alan W.

1989-01-01

The oligomerization of the activated 3-prime-5-prime pyrophosphate-linked dimer, pdAppdAp, is presently noted to be selectively favored by a poly(U) template over the 3-prime-3-prime and 5-prime-5-prime linked dimers. Both overall yields and the production of the longest oligomers were markedly stimulated by poly(U)'s presence; in its absence, the 5-prime-5-prime linked dimer became the most reactive, yielding chains of the order of 60 monomer-unit lengths. Remarkable self-organization properties are noted for the 5-prime-5-prime dimer of pdAp.

Mutational Analysis of the Stability of the H2A and H2B Histone Monomers

PubMed Central

Stump, Matthew R.; Gloss, Lisa M.

2008-01-01

The eukaryotic histone heterodimer H2A-H2B folds through an obligatory dimeric intermediate that forms in a nearly diffusion-limited association reaction in the stopped-flow dead time. It is unclear whether there is partial folding of the isolated monomers before association. To address the possible contributions of structure in the monomers to the rapid association, we characterized H2A and H2B monomers in the absence of their heterodimeric partner. By far-UV circular dichroism, the H2A and H2B monomers are 15% and 31% helical, respectively—significantly less than observed in X-ray crystal structures. Acrylamide quenching of the intrinsic Tyr fluorescence was indicative of tertiary structure. The H2A and H2B monomers exhibit free energies of unfolding of 2.5 and 2.9 kcal mol−1, respectively; at 10 μM, the sum of the stability of the monomers is ~60% of the stability of the native dimer. The helical content, stability and m values indicate that H2B has a more stable, compact structure than H2A. The monomer m values are larger than expected for the extended histone fold motif, suggesting that the monomers adopt an overly-collapsed structure. Stopped-flow refolding—initiated from urea-denatured monomers or the partially folded monomers populated at low denaturant concentrations—yielded essentially identical rates, indicating that monomer folding is productive in the rapid association and folding of the heterodimer. A series of Ala and Gly mutations were introduced into H2A and H2B to probe the importance of helix propensity on the structure and stability of the monomers. The mutational studies show that the central α-helix of the histone fold, which makes extensive inter-monomer contacts, is structured in H2B but only partially folded in H2A. PMID:18976667

Protein folding, misfolding and aggregation: The importance of two-electron stabilizing interactions

PubMed Central

2017-01-01

Proteins associated with neurodegenerative diseases are highly pleiomorphic and may adopt an all-α-helical fold in one environment, assemble into all-β-sheet or collapse into a coil in another, and rapidly polymerize in yet another one via divergent aggregation pathways that yield broad diversity of aggregates’ morphology. A thorough understanding of this behaviour may be necessary to develop a treatment for Alzheimer’s and related disorders. Unfortunately, our present comprehension of folding and misfolding is limited for want of a physicochemical theory of protein secondary and tertiary structure. Here we demonstrate that electronic configuration and hyperconjugation of the peptide amide bonds ought to be taken into account to advance such a theory. To capture the effect of polarization of peptide linkages on conformational and H-bonding propensity of the polypeptide backbone, we introduce a function of shielding tensors of the Cα atoms. Carrying no information about side chain-side chain interactions, this function nonetheless identifies basic features of the secondary and tertiary structure, establishes sequence correlates of the metamorphic and pH-driven equilibria, relates binding affinities and folding rate constants to secondary structure preferences, and manifests common patterns of backbone density distribution in amyloidogenic regions of Alzheimer’s amyloid β and tau, Parkinson’s α-synuclein and prions. Based on those findings, a split-intein like mechanism of molecular recognition is proposed to underlie dimerization of Aβ, tau, αS and PrPC, and divergent pathways for subsequent association of dimers are outlined; a related mechanism is proposed to underlie formation of PrPSc fibrils. The model does account for: (i) structural features of paranuclei, off-pathway oligomers, non-fibrillar aggregates and fibrils; (ii) effects of incubation conditions, point mutations, isoform lengths, small-molecule assembly modulators and chirality of solid-liquid interface on the rate and morphology of aggregation; (iii) fibril-surface catalysis of secondary nucleation; and (iv) self-propagation of infectious strains of mammalian prions. PMID:28922400

Inhibition of glycogen-synthase kinase 3 stimulates glycogen synthase and glucose transport by distinct mechanisms in 3T3-L1 adipocytes.

PubMed

Oreña, S J; Torchia, A J; Garofalo, R S

2000-05-26

The role of glycogen-synthase kinase 3 (GSK3) in insulin-stimulated glucose transport and glycogen synthase activation was investigated in 3T3-L1 adipocytes. GSK3 protein was clearly present in adipocytes and was found to be more abundant than in muscle and liver cell lines. The selective GSK3 inhibitor, LiCl, stimulated glucose transport and glycogen synthase activity (20 and 65%, respectively, of the maximal (1 microm) insulin response) and potentiated the responses to a submaximal concentration (1 nm) of insulin. LiCl- and insulin-stimulated glucose transport were abolished by the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor, wortmannin; however, LiCl stimulation of glycogen synthase was not. In contrast to the rapid stimulation of glucose transport by insulin, transport stimulated by LiCl increased gradually over 3-5 h reaching 40% of the maximal insulin-stimulated level. Both LiCl- and insulin-stimulated glycogen synthase activity were maximal at 25 min. However, insulin-stimulated glycogen synthase activity returned to basal after 2 h, coincident with reactivation of GSK3. After a 2-h exposure to insulin, glycogen synthase was refractory to restimulation with insulin, indicating selective desensitization of this pathway. However, LiCl could partially stimulate glycogen synthase in desensitized cells. Furthermore, coincubation with LiCl during the 2 h exposure to insulin completely blocked desensitization of glycogen synthase activity. In summary, inhibition of GSK3 by LiCl: 1) stimulated glycogen synthase activity directly and independently of PI3-kinase, 2) stimulated glucose transport at a point upstream of PI3-kinase, 3) stimulated glycogen synthase activity in desensitized cells, and 4) prevented desensitization of glycogen synthase due to chronic insulin treatment. These data are consistent with GSK3 playing a central role in the regulation of glycogen synthase activity and a contributing factor in the regulation of glucose transport in 3T3-L1 adipocytes.

Unique and Highly Selective Anticytomegalovirus Activities of Artemisinin-Derived Dimer Diphenyl Phosphate Stem from Combination of Dimer Unit and a Diphenyl Phosphate Moiety

PubMed Central

He, Ran; Forman, Michael; Mott, Bryan T.; Venkatadri, Rajkumar; Posner, Gary H.

2013-01-01

We report that the artemisinin-derived dimer diphenyl phosphate (DPP; dimer 838) is the most selective inhibitor of human cytomegalovirus (CMV) replication among a series of artemisinin-derived monomers and dimers. Dimer 838 was also unique in being an irreversible CMV inhibitor. The peroxide unit within artemisinins' chemical structures is critical to their activities, and its absence results in loss of anti-CMV activities. Surprisingly, the deoxy dimer of 838 retained modest anti-CMV activity, suggesting that the DPP moiety of dimer 838 contributes to its anti-CMV activities. DPP alone did not inhibit CMV replication, but triphenyl phosphate (TPP) had modest CMV inhibition, although its selectivity index was low. Artemisinin DPP derivatives dimer 838 and monomer diphenyl phosphate (compound 558) showed stronger CMV inhibition and a higher selectivity index than their analogs lacking the DPP unit. An add-on and removal assay revealed that removing DPP derivatives (compounds 558 and 838) but not the non-DPP backbones (artesunate and compound 606) at 24 h postinfection (hpi) already resulted in dominant CMV inhibition. CMV inhibition was fully irreversible with 838 and partially irreversible with 558, while non-DPP artemisinins were reversible inhibitors. While all artemisinin derivatives and TPP reduced the expression of the CMV immediate early 2 (IE2), UL44, and pp65 proteins at or after 48 hpi, only TPP inhibited the expression of both IE1 and IE2. Combination of a non-DPP dimer (compound 606) with TPP was synergistic in CMV inhibition, while ganciclovir and TPP were additive. Although TPP shared structural similarity with monomer DPP (compound 558) and dimer DPP (compound 838), its pattern of CMV inhibition was significantly different from the patterns of the artemisinins. These findings demonstrate that the DPP group contributes to the unique activities of compound 838. PMID:23774439

D-Dimer in African Americans: Whole Genome Sequence Analysis and Relationship to Cardiovascular Disease Risk in the Jackson Heart Study.

PubMed

Raffield, Laura M; Zakai, Neil A; Duan, Qing; Laurie, Cecelia; Smith, Joshua D; Irvin, Marguerite R; Doyle, Margaret F; Naik, Rakhi P; Song, Ci; Manichaikul, Ani W; Liu, Yongmei; Durda, Peter; Rotter, Jerome I; Jenny, Nancy S; Rich, Stephen S; Wilson, James G; Johnson, Andrew D; Correa, Adolfo; Li, Yun; Nickerson, Deborah A; Rice, Kenneth; Lange, Ethan M; Cushman, Mary; Lange, Leslie A; Reiner, Alex P

2017-11-01

Plasma levels of the fibrinogen degradation product D-dimer are higher among African Americans (AAs) compared with those of European ancestry and higher among women compared with men. Among AAs, little is known of the genetic architecture of D-dimer or the relationship of D-dimer to incident cardiovascular disease. We measured baseline D-dimer in 4163 AAs aged 21 to 93 years from the prospective JHS (Jackson Heart Study) cohort and assessed association with incident cardiovascular disease events. In participants with whole genome sequencing data (n=2980), we evaluated common and rare genetic variants for association with D-dimer. Each standard deviation higher baseline D-dimer was associated with a 20% to 30% increased hazard for incident coronary heart disease, stroke, and all-cause mortality. Genetic variation near F3 was associated with higher D-dimer (rs2022030, β=0.284, P =3.24×10 -11 ). The rs2022030 effect size was nearly 3× larger among women (β=0.373, P =9.06×10 -13 ) than among men (β=0.135, P =0.06; P interaction =0.009). The sex by rs2022030 interaction was replicated in an independent sample of 10 808 multiethnic men and women ( P interaction =0.001). Finally, the African ancestral sickle cell variant ( HBB rs334) was significantly associated with higher D-dimer in JHS (β=0.507, P =1.41×10 -14 ), and this association was successfully replicated in 1933 AAs ( P =2.3×10 -5 ). These results highlight D-dimer as an important predictor of cardiovascular disease risk in AAs and suggest that sex-specific and African ancestral genetic effects of the F3 and HBB loci contribute to the higher levels of D-dimer among women and AAs. © 2017 American Heart Association, Inc.

Molecular determinants and thermodynamics of the amyloid precursor protein transmembrane domain implicated in Alzheimer's disease

PubMed Central

Wang, Hao; Barreyro, Laura; Provasi, Davide; Djemil, Imane; Torres-Arancivia, Celia; Filizola, Marta; Ubarretxena-Belandia, Iban

2011-01-01

The deposition of toxic amyloid-β peptide (Aβ) aggregates in the brain is a hallmark of Alzheimer's disease. The intramembrane proteolysis by γ-secretase of the amyloid precursor protein carboxy-terminal fragment (APP-βCTF) constitutes the final step in the production of Aβs. Mounting evidence suggests that APP-βCTF is a transmembrane domain (TMD) dimer, and that dimerization might modulate the production of Aβ species that are prone to aggregation, and therefore most toxic. We combined experimental and computational approaches to study the molecular determinants and thermodynamics of APP-βCTF dimerization, and produced a unifying structural model that reconciles much of the published data. Using a cell assay, which exploits a dimerization-dependent activator of transcription, we identified specific dimerization-disrupting mutations located mostly at the N-terminus of the TMD of APP-βCTF. The ability of selected mutants to disrupt the dimerization of full length APP-βCTF was confirmed by fluorescence resonance energy transfer experiments. Free-energy estimates of wild-type (WT) and mutants of the TMD of APP-βCTF derived from enhanced molecular dynamics simulations showed that the dimeric state is comprised of different arrangements, in which either 709GXXXA713 or 700GXXXG704GXXXG708 interaction motifs can engage in symmetric or asymmetric associations. Mutations along the TMD of APP-βCTF were found to modulate the relative free energy of the dimeric configurations, and to differently affect the distribution of interfaces within the dimeric state. This observation might have important biological implications, since dimers with a different arrangement of the transmembrane helices are likely to be recognized differently by γ-secretase and lead to a variation of Aβ levels. PMID:21440556

Base Pair Opening in a Deoxynucleotide Duplex Containing a cis-syn Thymine Cyclobutane Dimer Lesion

PubMed Central

Wenke, Belinda B.; Huiting, Leah N.; Frankel, Elisa B.; Lane, Benjamin F.; Núñez, Megan E.

2014-01-01

The cis-syn thymine cyclobutane dimer is a DNA photoproduct implicated in skin cancer. We compared the stability of individual base pairs in thymine dimer-containing duplexes to undamaged parent 10-mer duplexes. UV melting thermodynamic measurements, CD spectroscopy, and 2D NOESY NMR spectroscopy confirm that the thymine dimer lesion is locally and moderately destabilizing within an overall B-form duplex conformation. We measured the rates of exchange of individual imino protons by NMR using magnetization transfer from water and determined the equilibrium constant for the opening of each base pair Kop. In the normal duplex Kop decreases from the frayed ends of the duplex toward the center, such that the central TA pair is the most stable with a Kop of 8×10−7. In contrast, base pair opening at the 5’T of the thymine dimer is facile. The 5’T of the dimer has the largest equilibrium constant (Kop =3×10−4) in its duplex, considerably larger than even the frayed penultimate base pairs. Notably, base pairing by the 3’T of the dimer is much more stable than by the 5’T, indicating that the predominant opening mechanism for the thymine dimer lesion is not likely to be flipping out into solution as a single unit. The dimer asymmetrically affects the stability of the duplex in its vicinity, destabilizing base pairing on its 5’ side more than on the 3’ side. The striking differences in base pair opening between parent and dimer duplexes occur independently of the duplex-single strand melting transitions. PMID:24328089

Structural insight into GRIP1-PDZ6 in Alzheimer's disease: study from protein expression data to molecular dynamics simulations.

PubMed

Chatterjee, Paulami; Roy, Debjani

2017-08-01

Protein-protein interaction domain, PDZ, plays a critical role in efficient synaptic transmission in brain. Dysfunction of synaptic transmission is thought to be the underlying basis of many neuropsychiatric and neurodegenerative disorders including Alzheimer's disease (AD). In this study, Glutamate Receptor Interacting Protein1 (GRIP1) was identified as one of the most important differentially expressed, topologically significant proteins in the protein-protein interaction network. To date, very few studies have analyzed the detailed structural basis of PDZ-mediated protein interaction of GRIP1. In order to gain better understanding of structural and dynamic basis of these interactions, we employed molecular dynamics (MD) simulations of GRIP1-PDZ6 dimer bound with Liprin-alpha and GRIP1-PDZ6 dimer alone each with 100 ns simulations. The analyses of MD simulations of Liprin-alpha bound GRIP1-PDZ6 dimer show considerable conformational differences than that of peptide-free dimer in terms of SASA, hydrogen bonding patterns, and along principal component 1 (PC1). Our study also furnishes insight into the structural attunement of the PDZ6 domains of Liprin-alpha bound GRIP1 that is attributed by significant shift of the Liprin-alpha recognition helix in the simulated peptide-bound dimer compared to the crystal structure and simulated peptide-free dimer. It is evident that PDZ6 domains of peptide-bound dimer show differential movements along PC1 than that of peptide-free dimers. Thus, Liprin-alpha also serves an important role in conferring conformational changes along the dimeric interface of the peptide-bound dimer. Results reported here provide information that may lead to novel therapeutic approaches in AD.

Membrane association and localization dynamics of the Ebola virus matrix protein VP40.

PubMed

Gc, Jeevan B; Gerstman, Bernard S; Chapagain, Prem P

2017-10-01

The Ebola virus matrix protein VP40 is a major structural protein that provides the scaffolding for new Ebola virus particles. For this, VP40 is first trafficked to the lower leaflet of the plasma membrane (PM) in its dimeric form. Once associated with the PM, the VP40 dimers undergo structural rearrangements and oligomerize into hexamers and filaments that make up the virus matrix. Therefore, association of the VP40 dimers and their stabilization at the PM is a crucial step in the Ebola life-cycle. To understand the molecular details of the VP40 dimer-PM interactions, we investigated the dimer association with the inner leaflet of the PM using detailed all-atom molecular dynamics (MD) simulations. The formation of the dimer-PM complex is facilitated by the interactions of the VP40 lysine residues and the anionic lipids POPS, POPI, and PIP 2 in the PM. In contrast, the dimer fails to associate with a membrane without POPS, POPI, or PIP 2 lipids. We explored the mechanisms of the association and identified important residues and lipids involved in localization and stabilization of VP40 dimers at the PM. MD simulations elucidate the role of a C-terminal α-helix alignment parallel to the lipid bilayer surface as well as the creation of membrane defects that allow partial insertion of the hydrophobic residue V276 into the membrane to further stabilize the VP40 dimer-PM complex. Understanding the mechanisms of the VP40 dimer-PM association that facilitate oligomerization can be important for potentially targeting the VP40 for small molecules that can interfere with the virus life-cycle. Copyright © 2017 Elsevier B.V. All rights reserved.

Laser desorption single-conformation UV and IR spectroscopy of the sulfonamide drug sulfanilamide, the sulfanilamide-water complex, and the sulfanilamide dimer.

PubMed

Uhlemann, Thomas; Seidel, Sebastian; Müller, Christian W

2017-06-07

We have studied the conformational preferences of the sulfonamide drug sulfanilamide, its dimer, and its monohydrated complex through laser desorption single-conformation UV and IR spectroscopy in a molecular beam. Based on potential energy curves for the inversion of the anilinic and the sulfonamide NH 2 groups calculated at DFT level, we suggest that the zero-point level wave function of the sulfanilamide monomer is appreciably delocalized over all four conformer wells. The sulfanilamide dimer, and the monohydrated complex each exhibit a single isomer in the molecular beam. The isomeric structures of the sulfanilamide dimer and the monohydrated sulfanilamide complex were assigned based on their conformer-specific IR spectra in the NH and OH stretch region. Quantum Theory of Atoms in Molecules (QTAIM) analysis of the calculated electron density in the water complex suggests that the water molecule is bound side-on in a hydrogen bonding pocket, donating one O-HO[double bond, length as m-dash]S hydrogen bond and accepting two hydrogen bonds, a NHO and a CHO hydrogen bond. QTAIM analysis of the dimer electron density suggests that the C i symmetry dimer structure exhibits two dominating N-HO[double bond, length as m-dash]S hydrogen bonds, and three weaker types of interactions: two CHO bonds, two CHN bonds, and a chalcogen OO interaction. Most interestingly, the molecular beam dimer structure closely resembles the R dimer unit - the dimer unit with the greatest interaction energy - of the α, γ, and δ crystal polymorphs. Interacting Quantum Atoms analysis provides evidence that the total intermolecular interaction in the dimer is dominated by the short-range exchange-correlation contribution.

Interaction between dimer interface residues of native and mutated SOD1 protein: a theoretical study.

PubMed

Keerthana, S P; Kolandaivel, P

2015-04-01

Cu-Zn superoxide dismutase 1 (SOD1) is a highly conserved bimetallic protein enzyme, used for the scavenging the superoxide radicals (O2 (-)) produced due to aerobic metabolism in the mitochondrial respiratory chain. Over 100 mutations have been identified and found to be in the homodimeric structure of SOD1. The enzyme has to be maintained in its dimeric state for the structural stability and enzymatic activity. From our investigation, we found that the mutations apart from the dimer interface residues are found to affect the dimer stability of protein and hence enhancing the aggregation and misfolding tendency of mutated protein. The homodimeric state of SOD1 is found to be held together by the non-covalent interactions. The molecular dynamics simulation has been used to study the hydrogen bond interactions between the dimer interface residues of the monomers in native and mutated forms of SOD1 in apo- and holo-states. The results obtained by this analysis reveal the fact that the loss of hydrogen bond interactions between the monomers of the dimer is responsible for the reduced stability of the apo- and holo-mutant forms of SOD1. The conformers with dimer interface residues in native and mutated protein obtained by the molecular dynamics simulation is subjected to quantum mechanical study using M052X/6-31G(d) level of theory. The charge transfer between N-H···O interactions in the dimer interface residues were studied. The weak interaction between the monomers of the dimer accounts for the reduced dimerization and enhanced deformation energy in the mutated SOD1 protein.

Interaction investigations of HipA binding to HipB dimer and HipB dimer + DNA complex: a molecular dynamics simulation study.

PubMed

Li, Chaoqun; Wang, Yaru; Wang, Yan; Chen, Guangju

2013-11-01

We carried out molecular dynamics simulations and free energy calculations for a series of ternary and diplex models for the HipA protein, HipB dimer, and DNA molecule to address the mechanism of HipA sequestration and the binding order of events from apo HipB/HipA to 2HipA + HipB dimer + DNA complex. The results revealed that the combination of DNA with the HipB dimer is energetically favorable for the combination of HipB dimer with HipA protein. The binding of DNA to HipB dimer induces a long-range allosteric communication from the HipB2 -DNA interface to the HipA-HipB2 interface, which involves the closeness of α1 helices of HipB dimer to HipA protein and formations of extra hydrogen bonds in the HipA-HipB2 interface through the extension of α2/3 helices in the HipB dimer. These simulated results suggested that the DNA molecule, as a regulative media, modulates the HipB dimer conformation, consequently increasing the interactions of HipB dimer with the HipA proteins, which explains the mechanism of HipA sequestration reported by the previous experiment. Simultaneously, these simulations also explored that the thermodynamic binding order in a simulated physiological environment, that is, the HipB dimer first bind to DNA to form HipB dimer + DNA complex, then capturing strongly the HipA proteins to form a ternary complex, 2HipA + HipB dimer + DNA, for sequestrating HipA in the nucleoid. Copyright © 2013 John Wiley & Sons, Ltd.

Plaquette order in a dimerized frustrated spin ladder

NASA Astrophysics Data System (ADS)

Shlagman, Ofer; Shimshoni, Efrat

2014-11-01

We study the effect of dimerization (due to, e.g., spin-Peierls instability) on the phase diagram of a frustrated antiferromagnetic spin-1/2 ladder, with weak transverse and diagonal rung coupling. Our analysis focuses on a one-dimensional version of the model (i.e., a single two-leg ladder) where we consider two forms of dimerization on the legs: columnar dimers (CDs) and staggered dimers (SDs). We examine in particular the regime of parameters (corresponding to an intermediate X X Z anisotropy) in which the leg dimerization and the rung coupling terms are equally relevant. In both the CD and SD cases, we find that the effective field theory describing the system is a self-dual sine-Gordon model, which favors ordering and the opening of a gap to excitations. The order parameter, which reflects the interplay between the leg and rung dimerization interactions, represents a crystal of 4-spin plaquettes on which longitudinal and transverse dimers are in a coherent superposition. Depending on the leg dimerization mode, these plaquettes are closed or open, however both types spontaneously break reflection symmetry across the ladder. The closed plaquettes are stable, while the open plaquette order is relatively fragile and the corresponding gap may be tuned to zero under extreme conditions. We further find that a first-order transition occurs from the plaquette order to a valence bond crystal (VBC) of dimers on the legs. This suggests that in a higher-dimensional version of this system, this variety of distinct VBC states with comparable energies leads to the formation of domains. Effectively one-dimensional gapless spinon modes on domain boundaries may account for the experimental observation of spin-liquid behavior in a physical realization of the model.

Targeting cysteine-mediated dimerization of the MUC1-C oncoprotein in human cancer cells

PubMed Central

RAINA, DEEPAK; AHMAD, REHAN; RAJABI, HASAN; PANCHAMOORTHY, GOVIND; KHARBANDA, SURENDER; KUFE, DONALD

2012-01-01

The MUC1 heterodimeric protein is aberrantly overexpressed in diverse human carcinomas and contributes to the malignant phenotype. The MUC1-C transmembrane subunit contains a CQC motif in the cytoplasmic domain that has been implicated in the formation of dimers and in its oncogenic function. The present study demonstrates that MUC1-C forms dimers in human breast and lung cancer cells. MUC1-C dimerization was detectable in the cytoplasm and was independent of MUC1-N, the N-terminal mucin subunit that extends outside the cell. We show that the MUC1-C cytoplasmic domain forms dimers in vitro that are disrupted by reducing agents. Moreover, dimerization of the MUC1-C subunit in cancer cells was blocked by reducing agents and increased by oxidative stress, supporting involvement of the CQC motif in forming disulfide bonds. In support of these observations, mutation of the MUC1-C CQC motif to AQA completely blocked MUC1-C dimerization. Importantly, this study was performed with MUC1-C devoid of fluorescent proteins, such as GFP, CFP and YFP. In this regard, we show that GFP, CFP and YFP themselves form dimers that are readily detectable with cross-linking agents. The present results further demonstrate that a cell-penetrating peptide that targets the MUC1-C CQC cysteines blocks MUC1-C dimerization in cancer cells. These findings provide definitive evidence that: i) the MUC1-C cytoplasmic domain cysteines are necessary and sufficient for MUC1-C dimerization, and ii) these CQC motif cysteines represent an Achilles’ heel for targeting MUC1-C function. PMID:22200620

Predictors of 30-day mortality and the risk of recurrent systemic thromboembolism in cancer patients suffering acute ischemic stroke.

PubMed

Nam, Ki-Woong; Kim, Chi Kyung; Kim, Tae Jung; An, Sang Joon; Oh, Kyungmi; Mo, Heejung; Kang, Min Kyoung; Han, Moon-Ku; Demchuk, Andrew M; Ko, Sang-Bae; Yoon, Byung-Woo

2017-01-01

Stroke in cancer patients is not rare but is a devastating event with high mortality. However, the predictors of mortality in stroke patients with cancer have not been well addressed. D-dimer could be a useful predictor because it can reflect both thromboembolic events and advanced stages of cancer. In this study, we evaluate the possibility of D-dimer as a predictor of 30-day mortality in stroke patients with active cancer. We included 210 ischemic stroke patients with active cancer. The 30-day mortality data were collected by reviewing medical records. We also collected follow-up D-dimer levels in 106 (50%) participants to evaluate the effects of treatment response on D-dimer levels. Of the 210 participants, 30-day mortality occurred in 28 (13%) patients. Higher initial NIHSS scores, D-dimer levels, and CRP levels as well as frequent cryptogenic mechanism, systemic metastasis, multiple vascular territory lesion, hemorrhagic transformation, and larger infarct volume were related to 30-day mortality. In the multivariate analysis, D-dimer [adjusted OR (aOR) = 2.19; 95% CI, 1.46-3.28, P < 0.001] predicted 30-day mortality after adjusting for confounders. The initial NIHSS score (aOR = 1.07; 95% CI, 1.00-1.14, P = 0.043) and hemorrhagic transformation (aOR = 3.02; 95% CI, 1.10-8.29, P = 0.032) were also significant independent of D-dimer levels. In the analysis of D-dimer changes after treatment, the mortality group showed no significant decrease in D-dimer levels, despite treatment, while the survivor group showed the opposite response. D-dimer levels may predict 30-day mortality in acute ischemic stroke patients with active cancer.

Predictors of 30-day mortality and the risk of recurrent systemic thromboembolism in cancer patients suffering acute ischemic stroke

PubMed Central

Kim, Tae Jung; An, Sang Joon; Oh, Kyungmi; Mo, Heejung; Kang, Min Kyoung; Han, Moon-Ku; Demchuk, Andrew M.; Ko, Sang-Bae; Yoon, Byung-Woo

2017-01-01

Background Stroke in cancer patients is not rare but is a devastating event with high mortality. However, the predictors of mortality in stroke patients with cancer have not been well addressed. D-dimer could be a useful predictor because it can reflect both thromboembolic events and advanced stages of cancer. Aim In this study, we evaluate the possibility of D-dimer as a predictor of 30-day mortality in stroke patients with active cancer. Methods We included 210 ischemic stroke patients with active cancer. The 30-day mortality data were collected by reviewing medical records. We also collected follow-up D-dimer levels in 106 (50%) participants to evaluate the effects of treatment response on D-dimer levels. Results Of the 210 participants, 30-day mortality occurred in 28 (13%) patients. Higher initial NIHSS scores, D-dimer levels, and CRP levels as well as frequent cryptogenic mechanism, systemic metastasis, multiple vascular territory lesion, hemorrhagic transformation, and larger infarct volume were related to 30-day mortality. In the multivariate analysis, D-dimer [adjusted OR (aOR) = 2.19; 95% CI, 1.46–3.28, P < 0.001] predicted 30-day mortality after adjusting for confounders. The initial NIHSS score (aOR = 1.07; 95% CI, 1.00–1.14, P = 0.043) and hemorrhagic transformation (aOR = 3.02; 95% CI, 1.10–8.29, P = 0.032) were also significant independent of D-dimer levels. In the analysis of D-dimer changes after treatment, the mortality group showed no significant decrease in D-dimer levels, despite treatment, while the survivor group showed the opposite response. Conclusions D-dimer levels may predict 30-day mortality in acute ischemic stroke patients with active cancer. PMID:28282388

Preparation of A-type proanthocyanidin dimers from peanut skins and persimmon pulp and comparison of the antioxidant activity of A-type and B-type dimers.

PubMed

Dong, Xiao-qian; Zou, Bo; Zhang, Ying; Ge, Zhen-zhen; Du, Jing; Li, Chun-mei

2013-12-01

We have established a simple method for preparing large quantities of A-type dimers from peanut skin and persimmon for further structure-activity relationship study. Peanut skins were defatted with hexane and oligomeric proanthocyanidins were extracted from it with 20% of methanol, and the extract was fractionated with ethyl acetate. Persimmon tannin was extracted from persimmon with methanol acidified with 1% hydrochloric acid, after removing the sugar and small phenols, the high molecular weight persimmon tannin was partially cleaved with 6.25% hydrochloric acid in methanol. The ethyl acetate fraction from peanut skins and persimmon tannin cleaved products was chromatographed on AB-8 macroporous resin followed by Toyopearl HW-50F resin to yield about 378.3mg of A-type (epi)catechin (EC) dimer from 1 kg dry peanut skins and 34.3mg of A-type (epi)catechin-3-O-gallate (ECG) dimer and 37.7 mg of A-type (epi)gallocatechin-3-O-gallate (EGCG) dimer from 1 kg fresh persimmon fruit. The antioxidant properties of the A-type and B-type dimers were compared in five different assays, namely, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, 2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical, hydroxyl radical, lipid peroxidation in mice liver homogenate and erythrocyte hemolysis in rat blood. Our results showed that both A-type and B-type dimers showed high antioxidant potency in a dose-dependent manner. In general, B-type dimers showed higher radical scavenging potency than A-type ones with the same subunits in aqueous systems. But in tissue or lipid systems, A-type dimers showed similar or even higher antioxidant potency than B-type ones. © 2013.

Sandalwood Fragrance Biosynthesis Involves Sesquiterpene Synthases of Both the Terpene Synthase (TPS)-a and TPS-b Subfamilies, including Santalene Synthases*

PubMed Central

Jones, Christopher G.; Moniodis, Jessie; Zulak, Katherine G.; Scaffidi, Adrian; Plummer, Julie A.; Ghisalberti, Emilio L.; Barbour, Elizabeth L.; Bohlmann, Jörg

2011-01-01

Sandalwood oil is one of the worlds most highly prized fragrances. To identify the genes and encoded enzymes responsible for santalene biosynthesis, we cloned and characterized three orthologous terpene synthase (TPS) genes SaSSy, SauSSy, and SspiSSy from three divergent sandalwood species; Santalum album, S. austrocaledonicum, and S. spicatum, respectively. The encoded enzymes catalyze the formation of α-, β-, epi-β-santalene, and α-exo-bergamotene from (E,E)-farnesyl diphosphate (E,E-FPP). Recombinant SaSSy was additionally tested with (Z,Z)-farnesyl diphosphate (Z,Z-FPP) and remarkably, found to produce a mixture of α-endo-bergamotene, α-santalene, (Z)-β-farnesene, epi-β-santalene, and β-santalene. Additional cDNAs that encode bisabolene/bisabolol synthases were also cloned and functionally characterized from these three species. Both the santalene synthases and the bisabolene/bisabolol synthases reside in the TPS-b phylogenetic clade, which is more commonly associated with angiosperm monoterpene synthases. An orthologous set of TPS-a synthases responsible for formation of macrocyclic and bicyclic sesquiterpenes were characterized. Strict functionality and limited sequence divergence in the santalene and bisabolene synthases are in contrast to the TPS-a synthases, suggesting these compounds have played a significant role in the evolution of the Santalum genus. PMID:21454632

[BIOINFORMATIC SEARCH AND PHYLOGENETIC ANALYSIS OF THE CELLULOSE SYNTHASE GENES OF FLAX (LINUM USITATISSIMUM)].

PubMed

Pydiura, N A; Bayer, G Ya; Galinousky, D V; Yemets, A I; Pirko, Ya V; Podvitski, T A; Anisimova, N V; Khotyleva, L V; Kilchevsky, A V; Blume, Ya B

2015-01-01

A bioinformatic search of sequences encoding cellulose synthase genes in the flax genome, and their comparison to dicots orthologs was carried out. The analysis revealed 32 cellulose synthase gene candidates, 16 of which are highly likely to encode cellulose synthases, and the remaining 16--cellulose synthase-like proteins (Csl). Phylogenetic analysis of gene products of cellulose synthase genes allowed distinguishing 6 groups of cellulose synthase genes of different classes: CesA1/10, CesA3, CesA4, CesA5/6/2/9, CesA7 and CesA8. Paralogous sequences within classes CesA1/10 and CesA5/6/2/9 which are associated with the primary cell wall formation are characterized by a greater similarity within these classes than orthologous sequences. Whereas the genes controlling the biosynthesis of secondary cell wall cellulose form distinct clades: CesA4, CesA7, and CesA8. The analysis of 16 identified flax cellulose synthase gene candidates shows the presence of at least 12 different cellulose synthase gene variants in flax genome which are represented in all six clades of cellulose synthase genes. Thus, at this point genes of all ten known cellulose synthase classes are identify in flax genome, but their correct classification requires additional research.

Direct Assessment of the Effect of the Gly380Arg Achondroplasia Mutation on FGFR3 Dimerization Using Quantitative Imaging FRET

PubMed Central

Placone, Jesse; Hristova, Kalina

2012-01-01

The Gly380Arg mutation in FGFR3 is the genetic cause for achondroplasia (ACH), the most common form of human dwarfism. The mutation has been proposed to increase FGFR3 dimerization, but the dimerization propensities of wild-type and mutant FGFR3 have not been compared. Here we use quantitative imaging FRET to characterize the dimerization of wild-type FGFR3 and the ACH mutant in plasma membrane-derived vesicles from HEK293T cells. We demonstrate a small, but statistically significant increase in FGFR3 dimerization due to the ACH mutation. The data are consistent with the idea that the ACH mutation causes a structural change which affects both the stability and the activity of FGFR3 dimers in the absence of ligand. PMID:23056398

Stabilization of the dimeric birch pollen allergen Bet v 1 impacts its immunological properties.

PubMed

Kofler, Stefan; Ackaert, Chloé; Samonig, Martin; Asam, Claudia; Briza, Peter; Horejs-Hoeck, Jutta; Cabrele, Chiara; Ferreira, Fatima; Duschl, Albert; Huber, Christian; Brandstetter, Hans

2014-01-03

Many allergens share several biophysical characteristics, including the capability to undergo oligomerization. The dimerization mechanism in Bet v 1 and its allergenic properties are so far poorly understood. Here, we report crystal structures of dimeric Bet v 1, revealing a noncanonical incorporation of cysteine at position 5 instead of genetically encoded tyrosine. Cysteine polysulfide bridging stabilized different dimeric assemblies, depending on the polysulfide linker length. These dimers represent quaternary arrangements that are frequently observed in related proteins, reflecting their prevalence in unmodified Bet v 1. These conclusions were corroborated by characteristic immunologic properties of monomeric and dimeric allergen variants. Hereby, residue 5 could be identified as an allergenic hot spot in Bet v 1. The presented results refine fundamental principles in protein chemistry and emphasize the importance of protein modifications in understanding the molecular basis of allergenicity.

Visualization of multipolar longitudinal and transversal surface plasmon modes in nanowire dimers.

PubMed

Alber, Ina; Sigle, Wilfried; Müller, Sven; Neumann, Reinhard; Picht, Oliver; Rauber, Markus; van Aken, Peter A; Toimil-Molares, Maria Eugenia

2011-12-27

We study the transversal and longitudinal localized surface plasmon resonances in single nanowires and nanowire dimers excited by the fast traveling electron beam in a transmission electron microscope equipped with high-resolution electron energy-loss spectroscopy. Bright and dark longitudinal modes up to the fifth order are resolved on individual metallic nanowires. On nanowire dimers, mode splitting into bonding and antibonding is measured up to the third order for several dimers with various aspect ratio and controlled gap size. We observe that the electric field maxima of the bonding modes are shifted toward the gap, while the electric field maxima of the antibonding modes are shifted toward the dimer ends. Finally, we observe that the transversal mode is not detected in the region of the dimer gap and decays away from the rod more rapidly than the longitudinal modes.

Anion Photoelectron Spectroscopy of the Homogenous 2-Hydroxypyridine Dimer Electron Induced Proton Transfer System

NASA Astrophysics Data System (ADS)

Vlk, Alexandra; Stokes, Sarah; Wang, Yi; Hicks, Zachary; Zhang, Xinxing; Blando, Nicolas; Frock, Andrew; Marquez, Sara; Bowen, Kit; Bowen Lab JHU Team

Anion photoelectron spectroscopic (PES) and density functional theory (DFT) studies on the dimer anion of (2-hydroxypyridine)2-are reported. The experimentally measured vertical detachment energy (VDE) of 1.21eV compares well with the theoretically predicted values. The 2-hydroxypyridine anionic dimer system was investigated because of its resemblance to the nitrogenous heterocyclic pyrimidine nucleobases. Experimental and theoretical results show electron induced proton transfer (EIPT) in both the lactim and lactam homogeneous dimers. Upon electron attachment, the anion can serve as the intermediate between the two neutral dimers. A possible double proton transfer process can occur from the neutral (2-hydroxypyridine)2 to (2-pyridone)2 through the dimer anion. This potentially suggests an electron catalyzed double proton transfer mechanism of tautomerization. Research supported by the NSF Grant No. CHE-1360692.

Study of structural stability and damaging effect on membrane for four Aβ42 dimers

PubMed Central

Feng, Wei; Lei, Huimin; Si, Jiarui; Zhang, Tao

2017-01-01

Increasing evidence shows that Aβ oligomers are key pathogenic molecules in Alzheimer’s disease. Among Aβ oligomers, dimer is the smallest aggregate and toxic unit. Therefore, understanding its structural and dynamic properties is quite useful to prevent the formation and toxicity of the Aβ oligomers. In this study, we performed molecular dynamic simulations on four Aβ42 dimers, 2NCb, CNNC, NCNC and NCCN, within the hydrated DPPC membrane. Four Aβ42 dimers differ in the arrangements of two Aβ42 peptides. This study aims to investigate the impact of aggregation pattern of two Aβ peptides on the structural stability of the Aβ42 dimer and its disruption to the biological membrane. The MD results demonstrate that the NCCN, CNNC and NCNC have the larger structural fluctuation at the N-terminus of Aβ42 peptide, where the β-strand structure converts into the coil structure. The loss of the N-terminal β-strand further impairs the aggregate ability of Aβ42 dimer. In addition, inserting Aβ42 dimer into the membrane can considerably decrease the average APL of DPPC membrane. Moreover this decrease effect is largely dependent on the distance to the location of Aβ42 dimer and its secondary structure forms. Based on the results, the 2NCb is considered as a stable dimeric unit for aggregating the larger Aβ42 oligomer, and has a potent ability to disrupt the membrane. PMID:28594887

Skin sensitization potency of isoeugenol and its dimers evaluated by a non-radioisotopic modification of the local lymph node assay and guinea pig maximization test.

PubMed

Takeyoshi, Masahiro; Iida, Kenji; Suzuki, Keiko; Yamazaki, Shunsuke

2008-05-01

Allergic contact dermatitis is the serious unwanted effect arising from the use of consumer products such as cosmetics. Isoeugenol is a fragrance chemical with spicy, carnation-like scent, is used in many kinds of cosmetics and is a well-known moderate human sensitizer. It was previously reported that the dimerization of eugenol yielded two types of dimer possessing different sensitization potencies. This study reports the differences in skin sensitization potencies for isoeugenol and two types of dimer, beta-O-4-dilignol and dehydrodiisoeugenol (DIEG), as evaluated by the non-radioisotopic local lymph node assay (non-RI LLNA) and guinea pig maximization test. In the guinea pig maximization test, isoeugenol, beta-O-4-dilignol and DIEG were classified as extreme, weak and moderate sensitizers, respectively. As for the results of non-RI LLNA, the EC3 for isoeugenol, beta-O-4-dilignol and DIEG were calculated as 12.7%, >30% and 9.4%, respectively. The two types of isoeugenol dimer showed different sensitizing activities similar to the case for eugenol dimers. A reduction of sensitization potency achieved by dimerization may lead to developing safer cosmetic ingredients. Isoeugenol dimers are not currently used for fragrance chemicals. However, the dimerization of isoeugenol may yield a promising candidate as a cosmetic ingredient with low sensitization risk. The data may also provide useful information for the structure-activity relationship (SAR) in skin sensitization. Copyright (c) 2007 John Wiley & Sons, Ltd.

Proline substitution of dimer interface β-strand residues as a strategy for the design of functional monomeric proteins.

PubMed

Joseph, Prem Raj B; Poluri, Krishna Mohan; Gangavarapu, Pavani; Rajagopalan, Lavanya; Raghuwanshi, Sandeep; Richardson, Ricardo M; Garofalo, Roberto P; Rajarathnam, Krishna

2013-09-17

Proteins that exist in monomer-dimer equilibrium can be found in all organisms ranging from bacteria to humans; this facilitates fine-tuning of activities from signaling to catalysis. However, studying the structural basis of monomer function that naturally exists in monomer-dimer equilibrium is challenging, and most studies to date on designing monomers have focused on disrupting packing or electrostatic interactions that stabilize the dimer interface. In this study, we show that disrupting backbone H-bonding interactions by substituting dimer interface β-strand residues with proline (Pro) results in fully folded and functional monomers, by exploiting proline's unique feature, the lack of a backbone amide proton. In interleukin-8, we substituted Pro for each of the three residues that form H-bonds across the dimer interface β-strands. We characterized the structures, dynamics, stability, dimerization state, and activity using NMR, molecular dynamics simulations, fluorescence, and functional assays. Our studies show that a single Pro substitution at the middle of the dimer interface β-strand is sufficient to generate a fully functional monomer. Interestingly, double Pro substitutions, compared to single Pro substitution, resulted in higher stability without compromising native monomer fold or function. We propose that Pro substitution of interface β-strand residues is a viable strategy for generating functional monomers of dimeric, and potentially tetrameric and higher-order oligomeric proteins. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

High D-dimer levels after stopping anticoagulants in pulmonary embolism with sleep apnoea.

PubMed

García Suquia, Angela; Alonso-Fernández, Alberto; de la Peña, Mónica; Romero, David; Piérola, Javier; Carrera, Miguel; Barceló, Antonia; Soriano, Joan B; Arque, Meritxell; Fernández-Capitán, Carmen; Lorenzo, Alicia; García-Río, Francisco

2015-12-01

Obstructive sleep apnoea is a risk factor for pulmonary embolism. Elevated D-dimer levels and other biomarkers are associated with recurrent pulmonary embolism. The objectives were to compare the frequency of elevated D-dimer levels (>500 ng·mL(-1)) and further coagulation biomarkers after oral anticoagulation withdrawal in pulmonary embolism patients, with and without obstructive sleep apnoea, including two control groups without pulmonary embolism.We performed home respiratory polygraphy. We also measured basic biochemical profile and haemogram, and coagulation biomarkers (D-dimer, prothrombin fragment 1+2, thrombin-antithrombin complex, plasminogen activator inhibitor 1, and soluble P-selectin).64 (74.4%) of the pulmonary embolism cases and 41 (46.11%) of the controls without pulmonary embolism had obstructive sleep apnoea. Plasmatic D-dimer was higher in PE patients with OSA than in those without obstructive sleep apnoea. D-dimer levels were significantly correlated with apnoea-hypopnoea index, and nocturnal hypoxia. There were more patients with high D-dimer after stopping anticoagulants in those with pulmonary embolism and obstructive sleep apnoea compared with PE without obstructive sleep apnoea (35.4% versus 19.0%, p=0.003). Apnoea-hypopnoea index was independently associated with high D-dimer.Pulmonary embolism patients with obstructive sleep apnoea had higher rates of elevated D-dimer levels after anticoagulation discontinuation for pulmonary embolism than in patients without obstructive sleep apnoea and, therefore, higher procoagulant state that might increase the risk of pulmonary embolism recurrence. Copyright ©ERS 2015.

The polar 2e/12c bond in phenalenyl-azaphenalenyl hetero-dimers: Stronger stacking interaction and fascinating interlayer charge transfer.

PubMed

Zhong, Rong-Lin; Xu, Hong-Liang; Li, Zhi-Ru

2016-08-07

An increasing number of chemists have focused on the two-electron/multicenter bond (2e/mc) that was first introduced to interpret the bonding mechanism of radical dimers. Herein, we report the polar two-electron/twelve center (2e/12c) bonding character in a series of phenalenyl-azaphenalenyl radical hetero-dimers. Interestingly, the bonding energy of weaker polar hetero-dimer (P-TAP) is dominated by the overlap of the two different singly occupied molecular orbital of radicals, while that of stronger polar hetero-dimer (P-HAP) is dominated by the electrostatic attraction. Results show that the difference between the electronegativity of the monomers plays a prominent role in the essential attribution of the polar 2e/12c bond. Correspondingly, a stronger stacking interaction in the hetero-dimer could be effectively achieved by increasing the difference of nitrogen atoms number between the monomers. It is worthy of note that an interesting interlayer charge transfer character is induced in the polar hetero-dimers, which is dependent on the difference between the electronegativity of the monomers. It is our expectation that the new knowledge about the bonding nature of radical hetero-dimers might provide important information for designing radical based functional materials with various applications.

STEM-EELS analysis of multipole surface plasmon modes in symmetry-broken AuAg nanowire dimers

NASA Astrophysics Data System (ADS)

Schubert, Ina; Sigle, Wilfried; van Aken, Peter A.; Trautmann, Christina; Toimil-Molares, Maria Eugenia

2015-03-01

Surface plasmon coupling in nanowires separated by small gaps generates high field enhancements at the position of the gap and is thus of great interest for sensing applications. It is known that the nanowire dimensions and in particular the symmetry of the structures has strong influence on the plasmonic properties of the dimer structure. Here, we report on multipole surface plasmon coupling in symmetry-broken AuAg nanowire dimers. Our dimers, consisting of two nanowires with different lengths and separated by gaps of only 10 to 30 nm, were synthesized by pulsed electrochemical deposition in ion track-etched polymer templates. Electron energy-loss spectroscopy in scanning transmission electron microscopy allows us to resolve up to nine multipole order surface plasmon modes of these dimers spectrally separated from each other. The spectra evidence plasmon coupling between resonances of different multipole order, resulting in the generation of additional plasmonic modes. Since such complex structures require elaborated synthesis techniques, dimer structures with complex composition, morphology and shape are created. We demonstrate that finite element simulations on pure Au dimers can predict the generated resonances in the fabricated structures. The excellent agreement of our experiment on AuAg dimers with finite integration simulations using CST microwave studio manifests great potential to design complex structures for sensing applications.

Rapid detection of D-Dimers with mLabs® whole blood method for venous thromboembolism exclusion. Comparison with Vidas® D-Dimers assay.

PubMed

Gerotziafas, Grigoris T; Ray, Patrick; Gkalea, Vasiliki; Benzarti, Ahlem; Khaterchi, Amir; Cast, Claire; Pernet, Julie; Lefkou, Eleftheria; Elalamy, Ismail

2016-12-01

Easy to use point of care assays for D-Dimers measurement in whole blood from patients with clinical suspicion of venous thromboembolism (VTE) will facilitate the diagnostic strategy in the Emergency Department (ED) setting. We prospectively evaluated the diagnostic performance of the point-of-care mLabs® Whole Blood D-Dimers test and we compared it with the Vidas® D-Dimers assay. As part of the diagnostic algorithm applied in patients with clinical suspicion of VTE, the VIDAS® D-Dimers Test was prescribed by the emergency physician in charge. The mLabs® Whole Blood D-Dimers Test was used on the same samples. All patients had undergone exploration with the recommended imaging techniques for VTE diagnosis. Both assays were performed, on 99 emergency patients (mean age was 65 years) with clinical suspicion of VTE. In 3% of patients, VTE was documented with a reference imaging technique. The Bland and Altman test showed significant agreement between the two methods. Both assays showed equal sensitivity and negative predictive value for VTE. The mLabs whole blood assay is a promising point of care method for measurement of D-Dimers and exclusion of VTE diagnosis in the emergency setting which should be validated in a larger prospective study.

The polar 2e/12c bond in phenalenyl-azaphenalenyl hetero-dimers: Stronger stacking interaction and fascinating interlayer charge transfer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhong, Rong-Lin; Li, Zhi-Ru, E-mail: hlxu@nenu.edu.cn, E-mail: lzr@jlu.edu.cn; Xu, Hong-Liang, E-mail: hlxu@nenu.edu.cn, E-mail: lzr@jlu.edu.cn

An increasing number of chemists have focused on the two-electron/multicenter bond (2e/mc) that was first introduced to interpret the bonding mechanism of radical dimers. Herein, we report the polar two-electron/twelve center (2e/12c) bonding character in a series of phenalenyl-azaphenalenyl radical hetero-dimers. Interestingly, the bonding energy of weaker polar hetero-dimer (P-TAP) is dominated by the overlap of the two different singly occupied molecular orbital of radicals, while that of stronger polar hetero-dimer (P-HAP) is dominated by the electrostatic attraction. Results show that the difference between the electronegativity of the monomers plays a prominent role in the essential attribution of the polarmore » 2e/12c bond. Correspondingly, a stronger stacking interaction in the hetero-dimer could be effectively achieved by increasing the difference of nitrogen atoms number between the monomers. It is worthy of note that an interesting interlayer charge transfer character is induced in the polar hetero-dimers, which is dependent on the difference between the electronegativity of the monomers. It is our expectation that the new knowledge about the bonding nature of radical hetero-dimers might provide important information for designing radical based functional materials with various applications.« less

Chloroplast Preproteins Bind to the Dimer Interface of the Toc159 Receptor during Import1[OPEN

PubMed Central

Chen, Lih-Jen; Yeh, Yi-Hung; Hsiao, Chwan-Deng

2017-01-01

Most chloroplast proteins are synthesized in the cytosol as higher molecular weight preproteins and imported via the translocons in the outer (TOC) and inner (TIC) envelope membranes of chloroplasts. Toc159 functions as a primary receptor and directly binds preproteins through its dimeric GTPase domain. As a first step toward a molecular understanding of how Toc159 mediates preprotein import, we mapped the preprotein-binding regions on the Toc159 GTPase domain (Toc159G) of pea (Pisum sativum) using cleavage by bound preproteins conjugated with the artificial protease FeBABE and cysteine-cysteine cross-linking. Our results show that residues at the dimer interface and the switch II region of Toc159G are in close proximity to preproteins. The mature portion of preproteins was observed preferentially at the dimer interface, whereas the transit peptide was found at both regions equally. Chloroplasts from transgenic plants expressing engineered Toc159 with a cysteine placed at the dimer interface showed increased cross-linking to bound preproteins. Our data suggest that, during preprotein import, the Toc159G dimer disengages and the dimer interface contacts translocating preproteins, which is consistent with a model in which conformational changes induced by dimer-monomer conversion in Toc159 play a direct role in facilitating preprotein import. PMID:28250068

Hydrolysis at One of the Two Nucleotide-binding Sites Drives the Dissociation of ATP-binding Cassette Nucleotide-binding Domain Dimers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zoghbi, M. E.; Altenberg, G. A.

The functional unit of ATP-binding cassette (ABC) transporters consists of two transmembrane domains and two nucleotide-binding domains (NBDs). ATP binding elicits association of the two NBDs, forming a dimer in a head-to-tail arrangement, with two nucleotides “sandwiched” at the dimer interface. Each of the two nucleotide-binding sites is formed by residues from the two NBDs. We recently found that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii dimerizes in response to ATP binding and dissociates completely following ATP hydrolysis. However, it is still unknown whether dissociation of NBD dimers follows ATP hydrolysis at one or both nucleotide-binding sites. Here, we usedmore » luminescence resonance energy transfer to study heterodimers formed by one active (donor-labeled) and one catalytically defective (acceptor-labeled) NBD. Rapid mixing experiments in a stop-flow chamber showed that NBD heterodimers with one functional and one inactive site dissociated at a rate indistinguishable from that of dimers with two hydrolysis-competent sites. Comparison of the rates of NBD dimer dissociation and ATP hydrolysis indicated that dissociation followed hydrolysis of one ATP. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dimer dissociation.« less

Study of DNA Origami Dimerization and Dimer Dissociation Dynamics and of the Factors that Limit Dimerization.

PubMed

Liber, Miran; Tomov, Toma E; Tsukanov, Roman; Berger, Yaron; Popov, Mary; Khara, Dinesh C; Nir, Eyal

2018-06-01

Organizing DNA origami building blocks into higher order structures is essential for fabrication of large structurally and functionally diverse devices and molecular machines. Unfortunately, the yields of origami building block attachment reactions are typically not sufficient to allow programed assembly of DNA devices made from more than a few origami building blocks. To investigate possible reasons for these low yields, a detailed single-molecule fluorescence study of the dynamics of rectangular origami dimerization and origami dimer dissociation reactions is conducted. Reactions kinetics and yields are investigated at different origami and ion concentrations, for different ion types, for different lengths of bridging strands, and for the "sticky end" and "weaving welding" attachment techniques. Dimerization yields are never higher than 86%, which is typical for such systems. Analysis of the dynamic data shows that the low yield cannot be explained by thermodynamic instability or structural imperfections of the origami constructs. Atomic force microscopy and gel electrophoresis evidence reveal self-dimerization of the origami monomers, likely via blunt-end interactions made possible by the presence of bridging strands. It is suggested that this mechanism is the major factor that inhibits correct dimerization and means to overcome it are discussed. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fibulin 5 Forms a Compact Dimer in Physiological Solutions*

PubMed Central

Jones, Richard P. O.; Wang, Ming-Chuan; Jowitt, Thomas A.; Ridley, Caroline; Mellody, Kieran T.; Howard, Marjorie; Wang, Tao; Bishop, Paul N.; Lotery, Andrew J.; Kielty, Cay M.; Baldock, Clair; Trump, Dorothy

2009-01-01

Fibulin 5 is a 52-kDa calcium-binding epidermal growth factor (cbEGF)-rich extracellular matrix protein that is essential for the formation of elastic tissues. Missense mutations in fibulin 5 cause the elastin disorder cutis laxa and have been associated with age-related macular degeneration, a leading cause of blindness. We investigated the structure, hydrodynamics, and oligomerization of fibulin 5 using small angle x-ray scattering, EM, light scattering, circular dichroism, and sedimentation. Compact structures for the monomer were determined by small angle x-ray scattering and EM, and are supported by close agreement between the theoretical sedimentation of the structures and the experimental sedimentation of the monomer in solution. EM showed that monomers associate around a central cavity to form a dimer. Light scattering and equilibrium sedimentation demonstrated that the equilibrium between the monomer and the dimer is dependent upon NaCl and Ca2+ concentrations and that the dimer is dominant under physiological conditions. The dimerization of fragments containing just the cbEGF domains suggests that intermolecular interactions between cbEGFs cause dimerization of fibulin 5. It is possible that fibulin 5 functions as a dimer during elastinogenesis or that dimerization may provide a method for limiting interactions with binding partners such as tropoelastin. PMID:19617354

Appearance of Sodium Dodecyl Sulfate-Stable Amyloid β-Protein (Aβ) Dimer in the Cortex During Aging

PubMed Central

Enya, Miho; Morishima-Kawashima, Maho; Yoshimura, Masahiro; Shinkai, Yasuhisa; Kusui, Kaoru; Khan, Karen; Games, Dora; Schenk, Dale; Sugihara, Shiro; Yamaguchi, Haruyasu; Ihara, Yasuo

1999-01-01

We previously noted that some aged human cortical specimens containing very low or negligible levels of amyloid β-protein (Aβ) by enzyme immunoassay (EIA) provided prominent signals at 6∼8 kd on the Western blot, probably representing sodium dodecyl sulfate (SDS)-stable Aβ dimer. Re-examination of the specificity of the EIA revealed that BAN50- and BNT77-based EIA, most commonly used for the quantitation of Aβ, capture SDS-dissociable Aβ but not SDS-stable Aβ dimer. Thus, all cortical specimens in which the levels of Aβ were below the detection limits of EIA were subjected to Western blot analysis. A fraction of such specimens contained SDS-stable dimer at 6∼8 kd, but not SDS-dissociable Aβ monomer at ∼4 kd, as judged from the blot. This Aβ dimer is unlikely to be generated after death, because (i) specimens with very short postmortem delay contained the Aβ dimer, and (ii) until 12 hours postmortem, such SDS-stable Aβ dimer is detected only faintly in PDAPP transgenic mice. The presence of Aβ dimer in the cortex may characterize the accumulation of Aβ in the human brain, which takes much longer than that in PDAPP transgenic mice. PMID:9916941

Computational and biochemical characterization of two partially overlapping interfaces and multiple weak-affinity K-Ras dimers

NASA Astrophysics Data System (ADS)

Prakash, Priyanka; Sayyed-Ahmad, Abdallah; Cho, Kwang-Jin; Dolino, Drew M.; Chen, Wei; Li, Hongyang; Grant, Barry J.; Hancock, John F.; Gorfe, Alemayehu A.

2017-01-01

Recent studies found that membrane-bound K-Ras dimers are important for biological function. However, the structure and thermodynamic stability of these complexes remained unknown because they are hard to probe by conventional approaches. Combining data from a wide range of computational and experimental approaches, here we describe the structure, dynamics, energetics and mechanism of assembly of multiple K-Ras dimers. Utilizing a range of techniques for the detection of reactive surfaces, protein-protein docking and molecular simulations, we found that two largely polar and partially overlapping surfaces underlie the formation of multiple K-Ras dimers. For validation we used mutagenesis, electron microscopy and biochemical assays under non-denaturing conditions. We show that partial disruption of a predicted interface through charge reversal mutation of apposed residues reduces oligomerization while introduction of cysteines at these positions enhanced dimerization likely through the formation of an intermolecular disulfide bond. Free energy calculations indicated that K-Ras dimerization involves direct but weak protein-protein interactions in solution, consistent with the notion that dimerization is facilitated by membrane binding. Taken together, our atomically detailed analyses provide unique mechanistic insights into K-Ras dimer formation and membrane organization as well as the conformational fluctuations and equilibrium thermodynamics underlying these processes.

C-terminal Lysine-Linked Magainin 2 with Increased Activity Against Multidrug-Resistant Bacteria.

PubMed

Lorenzón, Esteban N; Santos-Filho, Norival A; Ramos, Matheus A S; Bauab, Tais M; Camargo, Ilana L B C; Cilli, Eduardo M

2016-01-01

Due to the growing problem of antibiotic-resistant microorganisms, the development of novel antimicrobial agents is a very important challenge. Dimerization of cationic antimicrobial peptides (cAMPs) is a potential strategy for enhancing antimicrobial activity. Here, we studied the effects of magainin 2 (MG2) dimerization on its structure and biological activity. Lysine and glutamic acid were used to synthesize the C- and N-terminal dimers of MG2, respectively, in order to evaluate the impact of linker position used to obtain the dimers. Both MG2 and its dimeric versions showed a random coil structure in aqueous solution. However, in the presence of a structure-inducing solvent or a membrane mimetic, all peptides acquired helical structure. N-terminal dimerization did not affect the biological activity of the peptide. On the other hand, the C-terminal dimer, (MG2)2K, showed antimicrobial activity 8-16 times higher than that of MG2, and the time required to kill Escherichia coli was lower. The enhanced antimicrobial activity was related to membrane permeabilization. (MG2)2K was also more active against multidrug-resistant bacteria of clinical origin. Overall, the results presented here demonstrate that C-terminal lysine-linked dimerization improve the activity of MG2, and (MG2)2K can be considered as a potential antimicrobial agent.

Elucidation of the active conformation of vancomycin dimers with antibacterial activity against vancomycin-resistant bacteria.

PubMed

Nakamura, Jun; Yamashiro, Hidenori; Hayashi, Sayaka; Yamamoto, Mami; Miura, Kenji; Xu, Shu; Doi, Takayuki; Maki, Hideki; Yoshida, Osamu; Arimoto, Hirokazu

2012-10-01

Covalently linked vancomycin dimers have attracted a great deal of attention among researchers because of their enhanced antibacterial activity against vancomycin-resistant strains. However, the lack of a clear insight into the mechanisms of action of these dimers hampers rational optimization of their antibacterial potency. Here, we describe the synthesis and antibacterial activity of novel vancomycin dimers with a constrained molecular conformation achieved by two tethers between vancomycin units. Conformational restriction is a useful strategy for studying the relationship between the molecular topology and biological activity of compounds. In this study, two vancomycin units were linked at three distinct positions of the glycopeptide (vancosamine residue (V), C terminus (C), and N terminus (N)) to form two types of novel vancomycin cyclic dimers. Active NC-VV-linked dimers with a stable conformation as indicated by molecular mechanics calculations selectively suppressed the peptidoglycan polymerization reaction of vancomycin-resistant Staphylococcus aureus in vitro. In addition, double-disk diffusion tests indicated that the antibacterial activity of these dimers against vancomycin-resistant enterococci might arise from the inhibition of enzymes responsible for peptidoglycan polymerization. These findings provide a new insight into the biological targets of vancomycin dimers and the conformational requirements for efficient antibacterial activity against vancomycin-resistant strains. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular cloning and functional expression of geranylgeranyl pyrophosphate synthase from Coleus forskohlii Briq

PubMed Central

Engprasert, Surang; Taura, Futoshi; Kawamukai, Makoto; Shoyama, Yukihiro

2004-01-01

Background Isopentenyl diphosphate (IPP), a common biosynthetic precursor to the labdane diterpene forskolin, has been biosynthesised via a non-mevalonate pathway. Geranylgeranyl diphosphate (GGPP) synthase is an important branch point enzyme in terpenoid biosynthesis. Therefore, GGPP synthase is thought to be a key enzyme in biosynthesis of forskolin. Herein we report the first confirmation of the GGPP synthase gene in Coleus forskohlii Briq. Results The open reading frame for full-length GGPP synthase encodes a protein of 359 amino acids, in which 1,077 nucleotides long with calculated molecular mass of 39.3 kDa. Alignments of C. forskohlii GGPP synthase amino acid sequences revealed high homologies with other plant GGPP synthases. Several highly conserved regions, including two aspartate-rich motifs were identified. Transient expression of the N-terminal region of C. forskohlii GGPP synthase-GFP fusion protein in tobacco cells demonstrated subcellular localization in the chloroplast. Carotenoid production was observed in Escherichia coli harboring pACCAR25ΔcrtE from Erwinia uredovora and plasmid carrying C. forskohlii GGPP synthase. These results suggested that cDNA encoded functional GGPP synthase. Furthermore, C. forskohlii GGPP synthase expression was strong in leaves, decreased in stems and very little expression was observed in roots. Conclusion This investigation proposed that forskolin was synthesised via a non-mevalonate pathway. GGPP synthase is thought to be involved in the biosynthesis of forskolin, which is primarily synthesised in the leaves and subsequently accumulates in the stems and roots. PMID:15550168

21 CFR 176.120 - Alkyl ketene dimers.

Code of Federal Regulations, 2011 CFR

2011-04-01

... section. (a) The alkyl ketene dimers are manufactured by the dehydrohalogenation of the acyl halides derived from the fatty acids of animal or vegetable fats and oils. (b) The alkyl ketene dimers are used as...

[Advances in isoprene synthase research].

PubMed

Gou, Yan; Liu, Zhongchuan; Wang, Ganggang

2017-11-25

Isoprene emission can lead to significant consequence for atmospheric chemistry. In addition, isoprene is a chemical compound for various industrial applications. In the organisms, isoprene is produced by isoprene synthase that eliminates the pyrophosphate from the dimethylallyl diphosphate. As a key enzyme of isoprene formation, isoprene synthase plays an important role in the process of natural emission and artificial synthesis of isoprene. So far, isoprene synthase has been found in various plants. Isoprene synthases from different sources are of conservative structural and similar biochemical properties. In this review, the biochemical and structural characteristics of isoprene synthases from different sources were compared, the catalytic mechanism of isoprene synthase was discussed, and the perspective application of the enzyme in bioengineering was proposed.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Herman, Michael F.; Currier, Robert P.; Peery, Travis B.

Intermolecular coupling of dipole moments is studied for a model system consisting of two diatomic molecules (AB monomers) arranged co-linearly and which can form non-covalently bound dimers. The dipolar coupling is a function of the bond length in each molecule as well as of the distance between the centers-of-mass of the two molecules. The calculations show that intermolecular coupling of the vibrations results in an isotope-dependent modification of the AB-AB intermolecular potential. This in turn alters the energies of the low-lying bound states of the dimers, producing isotope-dependent changes in the AB-AB dimer partition function. Explicit inclusion of intermolecular vibrationalmore » coupling then changes the predicted gas-dimer isotopic fractionation. In addition, a mass dependence in the intermolecular potential can also result in changes in the number of bound dimer states in an equilibrium mixture. This in turn leads to a significant dimer population shift in the model monomer-dimer equilibrium system considered here. Finally, the results suggest that intermolecular coupling terms should be considered when probing the origins of isotopic fractionation.« less

Multiple hydrogen-bonded complexes based on 2-ureido-4[1H]-pyrimidinone: a theoretical study.

PubMed

Sun, Hao; Lee, Hui Hui; Blakey, Idriss; Dargaville, Bronwin; Chirila, Traian V; Whittaker, Andrew K; Smith, Sean C

2011-09-29

In the present work, the electronic structures and properties of a series of 2-ureido-4[1H]-pyrimidinone(UPy)-based monomers and dimers in various environments (vacuum, chloroform, and water) are studied by density functional theoretical methods. Most dimers prefer to form a DDAA-AADD (D, H-bond donor; A, H-bond acceptor) array in both vacuum and solvents. Topological analysis proved that intramolecular and intermolecular hydrogen bonds coexist in the dimers. Frequency and NBO calculations show that all the hydrogen bonds exhibit an obvious red shift in their stretching vibrational frequencies. Larger substituents at position 6 of the pyrimidinone ring with stronger electron-donating ability favor the total binding energy and free energy of dimerization. Calculations on the solvent effect show that dimerization is discouraged by the stronger polarity of the solvent. Further computations show that Dimer-1 may be formed in chloroform, but water molecules may interact with the donor or acceptor sites and hence disrupt the hydrogen bonds of Dimer-1. © 2011 American Chemical Society

X-ray induced dimerization of cinnamic acid: Time-resolved inelastic X-ray scattering study

NASA Astrophysics Data System (ADS)

Inkinen, Juho; Niskanen, Johannes; Talka, Tuomas; Sahle, Christoph J.; Müller, Harald; Khriachtchev, Leonid; Hashemi, Javad; Akbari, Ali; Hakala, Mikko; Huotari, Simo

2015-11-01

A classic example of solid-state topochemical reactions is the ultraviolet-light induced photodimerization of α-trans-cinnamic acid (CA). Here, we report the first observation of an X-ray-induced dimerization of CA and monitor it in situ using nonresonant inelastic X-ray scattering spectroscopy (NRIXS). The time-evolution of the carbon core-electron excitation spectra shows the effects of two X-ray induced reactions: dimerization on a short time-scale and disintegration on a long time-scale. We used spectrum simulations of CA and its dimerization product, α-truxillic acid (TA), to gain insight into the dimerization effects. From the time-resolved spectra, we extracted component spectra and time-dependent weights corresponding to CA and TA. The results suggest that the X-ray induced dimerization proceeds homogeneously in contrast to the dimerization induced by ultraviolet light. We also utilized the ability of NRIXS for direct tomography with chemical-bond contrast to image the spatial progress of the reactions in the sample crystal. Our work paves the way for other time-resolved studies on chemical reactions using inelastic X-ray scattering.

X-ray induced dimerization of cinnamic acid: Time-resolved inelastic X-ray scattering study

PubMed Central

Inkinen, Juho; Niskanen, Johannes; Talka, Tuomas; Sahle, Christoph J.; Müller, Harald; Khriachtchev, Leonid; Hashemi, Javad; Akbari, Ali; Hakala, Mikko; Huotari, Simo

2015-01-01

A classic example of solid-state topochemical reactions is the ultraviolet-light induced photodimerization of α-trans-cinnamic acid (CA). Here, we report the first observation of an X-ray-induced dimerization of CA and monitor it in situ using nonresonant inelastic X-ray scattering spectroscopy (NRIXS). The time-evolution of the carbon core-electron excitation spectra shows the effects of two X-ray induced reactions: dimerization on a short time-scale and disintegration on a long time-scale. We used spectrum simulations of CA and its dimerization product, α-truxillic acid (TA), to gain insight into the dimerization effects. From the time-resolved spectra, we extracted component spectra and time-dependent weights corresponding to CA and TA. The results suggest that the X-ray induced dimerization proceeds homogeneously in contrast to the dimerization induced by ultraviolet light. We also utilized the ability of NRIXS for direct tomography with chemical-bond contrast to image the spatial progress of the reactions in the sample crystal. Our work paves the way for other time-resolved studies on chemical reactions using inelastic X-ray scattering. PMID:26568420

A test of the significance of intermolecular vibrational coupling in isotopic fractionation

DOE PAGES

Herman, Michael F.; Currier, Robert P.; Peery, Travis B.; ...

2017-07-15

Intermolecular coupling of dipole moments is studied for a model system consisting of two diatomic molecules (AB monomers) arranged co-linearly and which can form non-covalently bound dimers. The dipolar coupling is a function of the bond length in each molecule as well as of the distance between the centers-of-mass of the two molecules. The calculations show that intermolecular coupling of the vibrations results in an isotope-dependent modification of the AB-AB intermolecular potential. This in turn alters the energies of the low-lying bound states of the dimers, producing isotope-dependent changes in the AB-AB dimer partition function. Explicit inclusion of intermolecular vibrationalmore » coupling then changes the predicted gas-dimer isotopic fractionation. In addition, a mass dependence in the intermolecular potential can also result in changes in the number of bound dimer states in an equilibrium mixture. This in turn leads to a significant dimer population shift in the model monomer-dimer equilibrium system considered here. Finally, the results suggest that intermolecular coupling terms should be considered when probing the origins of isotopic fractionation.« less

The dimerization equilibrium of a ClC Cl−/H+ antiporter in lipid bilayers

PubMed Central

Chadda, Rahul; Krishnamani, Venkatramanan; Mersch, Kacey; Wong, Jason; Brimberry, Marley; Chadda, Ankita; Kolmakova-Partensky, Ludmila; Friedman, Larry J; Gelles, Jeff; Robertson, Janice L

2016-01-01

Interactions between membrane protein interfaces in lipid bilayers play an important role in membrane protein folding but quantification of the strength of these interactions has been challenging. Studying dimerization of ClC-type transporters offers a new approach to the problem, as individual subunits adopt a stable and functionally verifiable fold that constrains the system to two states – monomer or dimer. Here, we use single-molecule photobleaching analysis to measure the probability of ClC-ec1 subunit capture into liposomes during extrusion of large, multilamellar membranes. The capture statistics describe a monomer to dimer transition that is dependent on the subunit/lipid mole fraction density and follows an equilibrium dimerization isotherm. This allows for the measurement of the free energy of ClC-ec1 dimerization in lipid bilayers, revealing that it is one of the strongest membrane protein complexes measured so far, and introduces it as new type of dimerization model to investigate the physical forces that drive membrane protein association in membranes. DOI: http://dx.doi.org/10.7554/eLife.17438.001 PMID:27484630

Hsa-miR-1587 G-quadruplex formation and dimerization induced by NH4+, molecular crowding environment and jatrorrhizine derivatives.

PubMed

Tan, Wei; Yi, Long; Zhu, Zhentao; Zhang, Lulu; Zhou, Jiang; Yuan, Gu

2018-03-01

A guanine-rich human mature microRNA, miR-1587, was discovered to form stable intramolecular G-quadruplexes in the presence of K + , Na + and low concentration of NH 4 + (25mM) by electrospray ionization mass spectrometry (ESI-MS) combined with circular dichroism (CD) spectroscopy. Furthermore, under high concentration of NH 4 + (100mM) or molecular crowding environments, miR-1587 formed a dimeric G-quadruplex through 3'-to-3' stacking of two monomeric G-quadruplex subunits with one ammonium ion sandwiched between the interfaces. Specifically, two synthesized jatrorrhizine derivatives with terminal amine groups could also induce the dimerization of miR-1587 G-quadruplex and formed 1:1 and 2:1 complexes with the dimeric G-quadruplex. In contrast, jatrorrhizine could bind with the dimeric miR-1587 G-quadruplex, but could not induce dimerization of miR-1587 G-quadruplex. These results provide a new strategy to regulate the functions of miR-1587 through induction of G-quadruplex formation and dimerization. Copyright © 2017 Elsevier B.V. All rights reserved.

Infrared spectroscopy and structure of (NO) n clusters

DOE PAGES

Hoshina, Hiromichi; Slipchenko, Mikhail; Prozument, Kirill; ...

2016-01-12

Nitrogen oxide clusters (NO) n have been studied in He droplets via infrared depletion spectroscopy and by quantum chemical calculations. The ν 1 and ν 5 bands of cis-ON-NO dimer have been observed at 1868.2 and 1786.5 cm –1, respectively. Furthermore, spectral bands of the trimer and tetramer have been located in the vicinity of the corresponding dimer bands in accord with computed frequencies that place NO-stretch bands of dimer, trimer, and tetramer within a few wavenumbers of each other. In addition, a new line at 1878.1 cm –1 close to the band origin of single molecules was assigned tomore » van der Waals bound dimers of (NO) 2, which are stabilized due to the rapid cooling in He droplets. Spectra of larger clusters (n > 5), have broad unresolved features in the vicinity of the dimer bands. As a result, experiments and calculations indicate that trimers consist of a dimer and a loosely bound third molecule, whereas the tetramer consists of two weakly bound dimers.« less

D-dimer as an applicable test for detection of posttraumatic deep vein thrombosis in lower limb fracture.

PubMed

Bakhshi, Hooman; Alavi-Moghaddam, Mostafa; Wu, Karin C; Imami, Mohammad; Banasiri, Mohammad

2012-06-01

Measuring the plasma levels of D-dimer is an accurate and easy modality to detect deep vein thrombosis (DVT) in nontraumatic settings. However, the diagnostic reliability of D-dimer assays in detecting posttraumatic DVT among patients with lower limb fracture undergoing orthopedic surgery is not validated. In this study, 141 patients with lower limb fracture admitted through the emergency department and undergoing orthopedic surgery were enrolled. Postoperative venous blood samples for D-dimer assay were taken on the 1st, 7th, and 28th postoperative days. Color Doppler sonography examination of both lower limbs was performed at the same time as a standard test. Eight out of the 141 patients (6%) had acute DVT based on Color Doppler sonography. Mean D-dimer was 2160 ng/mL in DVT positive patients and 864 in DVT negative patients. D-dimer levels greater than 1000 ng/mL were 100% sensitive and 71% specific for detecting postoperative DVT. D-dimer assay is a useful and sensitive test for detecting posttraumatic DVT.

ESR studies on reactivity of protein-derived tyrosyl radicals formed by prostaglandin H synthase and ribonucleotide reductase.

PubMed

Lassmann, G; Curtis, J; Liermann, B; Mason, R P; Eling, T E

1993-01-01

Using ESR spectroscopy, the ability of enzyme inhibitors to quench protein-derived tyrosyl radicals was studied in two different enzymes, prostaglandin H synthase and ribonucleotide reductase. The prostaglandin H synthase inhibitors indomethacin, eugenol, and MK-410 effectively prevent the formation of tyrosyl radicals during the oxidation of arachidonic acid by prostaglandin H synthase from ram seminal vesicles. A direct reaction with preformed tyrosyl radicals was observed only with eugenol. The other prostaglandin H synthase inhibitors were ineffective. The ribonucleotide reductase inhibitors hydroxyurea and 4-hydroxyanisole, which effectively inactivate the tyrosyl radical in the active site of ribonucleotide reductase present in tumor cells, exhibit a different reactivity with tyrosyl radicals formed by prostaglandin H synthase. Hydroxyurea quenches preformed tyrosyl radicals in prostaglandin H synthase weakly, whereas 4-hydroxyanisole does not quench tyrosyl radicals in prostaglandin H synthase at all. Eugenol, which quenches preformed prostaglandin H synthase-derived tyrosyl radicals, also quenches the tyrosyl radical in ribonucleotide reductase. The results suggest that the reactivity of protein-linked tyrosyl radicals in ribonucleotide reductase and those formed during prostaglandin H synthase catalysis are very different and have unrelated roles in enzyme catalysis.

Dimers in α-pinene secondary organic aerosol: effect of hydroxyl radical, ozone, relative humidity and aerosol acidity

NASA Astrophysics Data System (ADS)

Kristensen, K.; Cui, T.; Zhang, H.; Gold, A.; Glasius, M.; Surratt, J. D.

2014-04-01

The formation of secondary organic aerosol (SOA) from both ozonolysis and hydroxyl radical (OH)-initiated oxidation of α-pinene under conditions of high nitric oxide (NO) concentrations with varying relative humidity (RH) and aerosol acidity was investigated in the University of North Carolina dual outdoor smog chamber facility. SOA formation from ozonolysis of α-pinene was enhanced relative to that from OH-initiated oxidation in the presence of initially high-NO conditions. However, no effect of RH on SOA mass was evident. Ozone (O3)-initiated oxidation of α-pinene in the presence of ammonium sulfate (AS) seed coated with organic aerosol from OH-initiated oxidation of α-pinene showed reduced nucleation compared to ozonolysis in the presence of pure AS seed aerosol. The chemical composition of α-pinene SOA was investigated by ultra-performance liquid chromatography/electrospray ionization high-resolution quadrupole time-of-flight mass spectrometry (UPLC/ESI-HR-Q-TOFMS), with a focus on the formation of carboxylic acids and high-molecular weight dimers. A total of eight carboxylic acids and four dimers were identified, constituting between 8 and 12% of the total α-pinene SOA mass. OH-initiated oxidation of α-pinene in the presence of nitrogen oxides (NOx) resulted in the formation of highly oxidized carboxylic acids, such as 3-methyl-1,2,3-butanetricarboxylic acid (MBTCA) and diaterpenylic acid acetate (DTAA). The formation of dimers was observed only in SOA produced from the ozonolysis of α-pinene in the absence of NOx, with increased concentrations by a factor of two at higher RH (50-90%) relative to lower RH (30-50%). The increased formation of dimers correlates with an observed increase in new particle formation at higher RH due to nucleation. Increased aerosol acidity was found to have a negligible effect on the formation of the dimers. SOA mass yield did not influence the chemical composition of SOA formed from α-pinene ozonolysis with respect to carboxylic acids and dimers. The results support the formation of the high-molecular weight dimers through gas-phase reactions of the stabilized Criegee Intermediate (sCI) formed from the ozonolysis of α-pinene. The high molecular weight and polar nature of dimers formed in the gas phase may explain increased particle number concentration as a result of homogenous nucleation. Since three of these dimers (i.e. pinyl-diaterpenyl dimer (MW 358), pinyl-diaterebyl dimer (MW 344) and pinonyl-pinyl dimer (MW 368)) have been observed in both laboratory-generated and ambient fine organic aerosol samples, we conclude that the dimers observed in this study can be used as tracers for the O3-initiated oxidation of α-pinene, and are therefore indicative of enhanced anthropogenic activities, and that the high molecular weight and low volatility dimers result in homogenous nucleation under laboratory conditions, increasing the particle number concentration.

Sandalwood fragrance biosynthesis involves sesquiterpene synthases of both the terpene synthase (TPS)-a and TPS-b subfamilies, including santalene synthases.

PubMed

Jones, Christopher G; Moniodis, Jessie; Zulak, Katherine G; Scaffidi, Adrian; Plummer, Julie A; Ghisalberti, Emilio L; Barbour, Elizabeth L; Bohlmann, Jörg

2011-05-20

Sandalwood oil is one of the worlds most highly prized fragrances. To identify the genes and encoded enzymes responsible for santalene biosynthesis, we cloned and characterized three orthologous terpene synthase (TPS) genes SaSSy, SauSSy, and SspiSSy from three divergent sandalwood species; Santalum album, S. austrocaledonicum, and S. spicatum, respectively. The encoded enzymes catalyze the formation of α-, β-, epi-β-santalene, and α-exo-bergamotene from (E,E)-farnesyl diphosphate (E,E-FPP). Recombinant SaSSy was additionally tested with (Z,Z)-farnesyl diphosphate (Z,Z-FPP) and remarkably, found to produce a mixture of α-endo-bergamotene, α-santalene, (Z)-β-farnesene, epi-β-santalene, and β-santalene. Additional cDNAs that encode bisabolene/bisabolol synthases were also cloned and functionally characterized from these three species. Both the santalene synthases and the bisabolene/bisabolol synthases reside in the TPS-b phylogenetic clade, which is more commonly associated with angiosperm monoterpene synthases. An orthologous set of TPS-a synthases responsible for formation of macrocyclic and bicyclic sesquiterpenes were characterized. Strict functionality and limited sequence divergence in the santalene and bisabolene synthases are in contrast to the TPS-a synthases, suggesting these compounds have played a significant role in the evolution of the Santalum genus. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Structure of an electric double layer containing a 2:2 valency dimer electrolyte

DOE PAGES

Silvestre-Alcantara, Whasington; Henderson, Douglas; Wu, Jianzhong; ...

2014-12-05

In this study, the structure of a planar electric double layer formed by a 2:2 valency dimer electrolyte in the vicinity of a uniformly charged planar hard electrode is investigated using density functional theory and Monte Carlo simulations. The dimer electrolyte consists of a mixture of charged divalent dimers and charged divalent monomers in a dielectric continuum. A dimer is constructed by two tangentially tethered rigid spheres, one of which is divalent and positively charged and the other neutral, whereas the monomer is a divalent and negatively charged rigid sphere. The density functional theory reproduces well the simulation results formore » (i) the singlet distributions of the various ion species with respect to the electrode, and (ii) the mean electrostatic potential. Lastly, comparison with earlier results for a 2:1/1:2 dimer electrolyte shows that the double layer structure is similar when the counterion has the same valency.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Campione, Salvatore; Warne, Larry K.; Basilio, Lorena I.

In this paper we develop a fully-retarded, dipole approximation model to estimate the effective polarizabilities of a dimer made of dielectric resonators. They are computed from the polarizabilities of the two resonators composing the dimer. We analyze the situation of full-cubes as well as split-cubes, which have been shown to exhibit overlapping electric and magnetic resonances. We compare the effective dimer polarizabilities to ones retrieved via full-wave simulations as well as ones computed via a quasi-static, dipole approximation. We observe good agreement between the fully-retarded solution and the full-wave results, whereas the quasi-static approximation is less accurate for the problemmore » at hand. The developed model can be used to predict the electric and magnetic resonances of a dimer under parallel or orthogonal (to the dimer axis) excitation. This is particularly helpful when interested in locating frequencies at which the dimer will emit directional radiation.« less

Magnetic anisotropy of heteronuclear dimers in the gas phase and supported on graphene: relativistic density-functional calculations.

PubMed

Błoński, Piotr; Hafner, Jürgen

2014-04-09

The structural and magnetic properties of mixed PtCo, PtFe, and IrCo dimers in the gas phase and supported on a free-standing graphene layer have been calculated using density-functional theory, both in the scalar-relativistic limit and self-consistently including spin-orbit coupling. The influence of the strong magnetic moments of the 3d atoms on the spin and orbital moments of the 5d atoms, and the influence of the strong spin-orbit coupling contributed by the 5d atom on the orbital moments of the 3d atoms have been studied in detail. The magnetic anisotropy energy is found to depend very sensitively on the nature of the eigenstates in the vicinity of the Fermi level, as determined by band filling, exchange splitting and spin-orbit coupling. The large magnetic anisotropy energy of free PtCo and IrCo dimers relative to the easy direction parallel to the dimer axis is coupled to a strong anisotropy of the orbital magnetic moments of the Co atom for both dimers, and also on the Ir atom in IrCo. In contrast the PtFe dimer shows a weak perpendicular anisotropy and only small spin and orbital anisotropies of opposite sign on the two atoms. For dimers supported on graphene, the strong binding within the dimer and the stronger interaction of the 3d atom with the substrate stabilizes an upright geometry. Spin and orbital moments on the 3d atom are strongly quenched, but due to the weaker binding within the dimer the properties of the 5d atom are more free-atom-like with increased spin and orbital moments. The changes in the magnetic moment are reflected in the structure of the electronic eigenstates near the Fermi level, for all three dimers the easy magnetic direction is now parallel to the dimer axis and perpendicular to the graphene layer. The already very large magnetic anisotropy energy (MAE) of IrCo is further enhanced by the interaction with the support, the MAE of PtFe changes sign, and that of the PtCo dimer is reduced. These changes are discussed in relation to the relativistic electronic structure of free and supported dimers and it is demonstrated that the existence of a partially occupied quasi-degenerate state at the Fermi level favors the formation of a large magnetic anisotropy.

Aromatic Polyketide Synthases (Purification, Characterization, and Antibody Development to Benzalacetone Synthase from Raspberry Fruits).

PubMed Central

Borejsza-Wysocki, W.; Hrazdina, G.

1996-01-01

p-Hydroxyphenylbutan-2-one, the characteristic aroma compound of raspberries (Rubus idaeus L.), is synthesized from p-coumaryl-coenzyme A and malonyl-coenzyme A in a two-step reaction sequence that is catalyzed by benzalacetone synthase and benzalacetone reductase (W. Borejsza-Wysocki and G. Hrazdina [1994] Phytochemistry 35: 623-628). Benzalacetone synthase condenses one malonate with p-coumarate to form the pathway intermediate p-hydroxyphenylbut-3-ene-2-one (p-hydroxybenzalacetone) in a reaction that is similar to those catalyzed by chalcone and stilbene synthases. We have obtained an enzyme preparation from ripe raspberries that was preferentially enriched in benzalacetone synthase (approximately 170-fold) over chalcone synthase (approximately 14-fold) activity. This preparation was used to characterize benzalacetone synthase and to develop polyclonal antibodies in rabbits. Benzalacetone synthase showed similarity in its molecular properties to chalcone synthase but differed distinctly in its substrate specificity, response to 2-mercaptoethanol and ethylene glycol, and induction in cell-suspension cultures. The product of the enzyme, p-hydroxybenzalacetone, inhibited mycelial growth of the raspberry pathogen Phytophthora fragariae var rubi at 250 [mu]M. We do not know whether the dual activity in the benzalacetone synthase preparation is the result of a bifunctional enzyme or is caused by contamination with chalcone synthase that was also present. The rapid induction of the enzyme in cell-suspension cultures upon addition of yeast extract and the toxicity of its product, p-hydroxybenzalacetone, to phytopathogenic fungi also suggest that the pathway may be part of a plant defense response. PMID:12226219

Mitochondrial F1Fo-ATP synthase translocates to cell surface in hepatocytes and has high activity in tumor-like acidic and hypoxic environment.

PubMed

Ma, Zhan; Cao, Manlin; Liu, Yiwen; He, Yiqing; Wang, Yingzhi; Yang, Cuixia; Wang, Wenjuan; Du, Yan; Zhou, Muqing; Gao, Feng

2010-08-01

F1Fo-ATP synthase was originally thought to exclusively locate in the inner membrane of the mitochondria. However, recent studies prove the existence of ectopic F1Fo-ATP synthase on the outside of the cell membrane. Ectopic ATP synthase was proposed as a marker for tumor target therapy. Nevertheless, the protein transport mechanism of the ectopic ATP synthase is still unclear. The specificity of the ectopic ATP synthase, with regard to tumors, is questioned because of its widespread expression. In the current study, we constructed green fluorescent protein-ATP5B fusion protein and introduced it into HepG2 cells to study the localization of the ATP synthase. The expression of ATP5B was analyzed in six cell lines with different 'malignancies'. These cells were cultured in both normal and tumor-like acidic and hypoxic conditions. The results suggested that the ectopic expression of ATP synthase is a consequence of translocation from the mitochondria. The expression and catalytic activity of ectopic ATP synthase were similar on the surface of malignant cells as on the surface of less malignant cells. Interestingly, the expression of ectopic ATP synthase was not up-regulated in tumor-like acidic and hypoxic microenvironments. However, the catalytic activity of ectopic ATP synthase was up-regulated in tumor-like microenvironments. Therefore, the specificity of ectopic ATP synthase for tumor target therapy relies on the high level of catalytic activity that is observed in acidic and hypoxic microenvironments in tumor tissues.

A competent catalytic active site is necessary for substrate induced dimer assembly in triosephosphate isomerase.

PubMed

Jimenez-Sandoval, Pedro; Vique-Sanchez, Jose Luis; Hidalgo, Marisol López; Velazquez-Juarez, Gilberto; Diaz-Quezada, Corina; Arroyo-Navarro, Luis Fernando; Moran, Gabriela Montero; Fattori, Juliana; Jessica Diaz-Salazar, A; Rudiño-Pinera, Enrique; Sotelo-Mundo, Rogerio; Figueira, Ana Carolina Migliorini; Lara-Gonzalez, Samuel; Benítez-Cardoza, Claudia G; Brieba, Luis G

2017-11-01

The protozoan parasite Trichomonas vaginalis contains two nearly identical triosephosphate isomerases (TvTIMs) that dissociate into stable monomers and dimerize upon substrate binding. Herein, we compare the role of the "ball and socket" and loop 3 interactions in substrate assisted dimer assembly in both TvTIMs. We found that point mutants at the "ball" are only 39 and 29-fold less catalytically active than their corresponding wild-type counterparts, whereas Δloop 3 deletions are 1502 and 9400-fold less active. Point and deletion mutants dissociate into stable monomers. However, point mutants assemble as catalytic competent dimers upon binding of the transition state substrate analog PGH, whereas loop 3 deletions remain monomeric. A comparison between crystal structures of point and loop 3 deletion monomeric mutants illustrates that the catalytic residues in point mutants and wild-type TvTIMs are maintained in the same orientation, whereas the catalytic residues in deletion mutants show an increase in thermal mobility and present structural disorder that may hamper their catalytic role. The high enzymatic activity present in monomeric point mutants correlates with the formation of dimeric TvTIMs upon substrate binding. In contrast, the low activity and lack of dimer assembly in deletion mutants suggests a role of loop 3 in promoting the formation of the active site as well as dimer assembly. Our results suggest that in TvTIMs the active site is assembled during dimerization and that the integrity of loop 3 and ball and socket residues is crucial to stabilize the dimer. Copyright © 2017 Elsevier B.V. All rights reserved.

Chronic inflammatory demyelinating polyneuropathy (CIDP): change of serum IgG dimer levels during treatment with intravenous immunoglobulins.

PubMed

Ritter, Christian; Bobylev, Ilja; Lehmann, Helmar C

2015-08-14

Intravenous immunoglobulin (IVIg) is an effective treatment in chronic inflammatory demyelinating polyneuropathy (CIDP). In most patients, the optimal IVIg dose and regime is unknown. Polyvalent immunoglobulin (Ig) G form idiotypic/anti-idiotypic antibody pairs in serum and IVIg preparations. We determined IgG dimer levels before and after IVIg treatment in CIDP patients with the aim to explore their utility to serve as a surrogate marker for treatment response. IgG was purified from serum of five controls without treatment, as well as from serum of 16 CIDP patients, two patients with Miller Fisher syndrome (MFS), and one patient with myasthenia gravis before and after treatment with IVIg. IgG dimer levels were determined by size exclusion chromatography. IgG dimer formation was correlated with clinical response to IVIg treatment in CIDP. Re-monomerized IgG dimer fractions were analyzed for immunoreactivity against peripheral nerve tissue. IgG dimer levels were significantly higher in post- compared to pre-IVIg infusion samples. Low post-treatment IgG dimer levels in CIDP patients were associated with clinical worsening during IVIg treatment. Re-monomerized IgG dimer fractions from CIDP patients showed immunoreactivity against peripheral nerve tissue, whereas similarly treated samples from MFS patients showed immunoreactivity against GQ1b. Assessment of IgG dimer levels could be a novel approach to monitor CIDP patients during IVIg treatment, but further studies in larger cohorts are warranted to explore their utility to serve as a potential therapeutic biomarker for IVIg treatment response in CIDP.

Mapping energetics of atom probe evaporation events through first principles calculations.

PubMed

Peralta, Joaquín; Broderick, Scott R; Rajan, Krishna

2013-09-01

The purpose of this work is to use atomistic modeling to determine accurate inputs into the atom probe tomography (APT) reconstruction process. One of these inputs is evaporation field; however, a challenge occurs because single ions and dimers have different evaporation fields. We have calculated the evaporation field of Al and Sc ions and Al-Al and Al-Sc dimers from an L1₂-Al₃Sc surface using ab initio calculations and with a high electric field applied to the surface. The evaporation field is defined as the electric field at which the energy barrier size is calculated as zero, corresponding to the minimum field that atoms from the surface can break their bonds and evaporate from the surface. The evaporation field of the surface atoms are ranked from least to greatest as: Al-Al dimer, Al ion, Sc ion, and Al-Sc dimer. The first principles results were compared with experimental data in the form of an ion evaporation map, which maps multi-ion evaporations. From the ion evaporation map of L1₂-Al₃Sc, we extract relative evaporation fields and identify that an Al-Al dimer has a lower evaporation field than an Al-Sc dimer. Additionally, comparatively an Al-Al surface dimer is more likely to evaporate as a dimer, while an Al-Sc surface dimer is more likely to evaporate as single ions. These conclusions from the experiment agree with the ab initio calculations, validating the use of this approach for modeling APT energetics. Copyright © 2013 Elsevier B.V. All rights reserved.

Dimer interface of bovine cytochrome c oxidase is influenced by local posttranslational modifications and lipid binding

PubMed Central

Liko, Idlir; Degiacomi, Matteo T.; Mohammed, Shabaz; Yoshikawa, Shinya; Schmidt, Carla; Robinson, Carol V.

2016-01-01

Bovine cytochrome c oxidase is an integral membrane protein complex comprising 13 protein subunits and associated lipids. Dimerization of the complex has been proposed; however, definitive evidence for the dimer is lacking. We used advanced mass spectrometry methods to investigate the oligomeric state of cytochrome c oxidase and the potential role of lipids and posttranslational modifications in its subunit interfaces. Mass spectrometry of the intact protein complex revealed that both the monomer and the dimer are stabilized by large lipid entities. We identified these lipid species from the purified protein complex, thus implying that they interact specifically with the enzyme. We further identified phosphorylation and acetylation sites of cytochrome c oxidase, located in the peripheral subunits and in the dimer interface, respectively. Comparing our phosphorylation and acetylation sites with those found in previous studies of bovine, mouse, rat, and human cytochrome c oxidase, we found that whereas some acetylation sites within the dimer interface are conserved, suggesting a role for regulation and stabilization of the dimer, phosphorylation sites were less conserved and more transient. Our results therefore provide insights into the locations and interactions of lipids with acetylated residues within the dimer interface of this enzyme, and thereby contribute to a better understanding of its structure in the natural membrane. Moreover dimeric cytochrome c oxidase, comprising 20 transmembrane, six extramembrane subunits, and associated lipids, represents the largest integral membrane protein complex that has been transferred via electrospray intact into the gas phase of a mass spectrometer, representing a significant technological advance. PMID:27364008

Theoretical design of the cyclic lipopeptide nanotube as a molecular channel in the lipid bilayer, molecular dynamics and quantum mechanics approach.

PubMed

Khavani, Mohammad; Izadyar, Mohammad; Housaindokht, Mohammad Reza

2015-10-14

In this article, cyclic peptides (CP) with lipid substituents were theoretically designed. The dynamical behavior of the CP dimers and the cyclic peptide nanotube (CPNT) without lipid substituents in the solution (water and chloroform) during the 50 ns molecular dynamic (MD) simulations has been investigated. As a result, the CP dimers and CPNT in a non-polar solvent are more stable than in a polar solvent and CPNT is a good container for non-polar small molecules such as chloroform. The effect of the lipid substituents on the CP dimers and CPNT has been investigated in the next stage of our studies. Accordingly, these substituents increase the stability of the CP dimers and CPNT, significantly, in polar solvents. MM-PBSA and MM-GBSA calculations confirm that substitution has an important effect on the stability of the CP dimers and CPNT. Finally, the dynamical behavior of CPNT with lipid substituents in a fully hydrated DMPC bilayer shows the high ability of this structure for molecule transmission across the lipid membrane. This structure is stable enough to be used as a molecular channel. DFT calculations on the CP dimers in the gas phase, water and chloroform, indicate that H-bond formation is the driving force for dimerization. CP dimers are more stable in the gas phase in comparison to in solution. HOMO-LUMO orbital analysis indicates that the interaction of the CP units in the dimer structures is due to the molecular orbital interactions between the NH and CO groups.

Different Epidermal Growth Factor (EGF) Receptor Ligands Show Distinct Kinetics and Biased or Partial Agonism for Homodimer and Heterodimer Formation*

PubMed Central

Macdonald-Obermann, Jennifer L.; Pike, Linda J.

2014-01-01

The EGF receptor has seven different cognate ligands. Previous work has shown that these different ligands are capable of inducing different biological effects, even in the same cell. To begin to understand the molecular basis for this variation, we used luciferase fragment complementation to measure ligand-induced dimer formation and radioligand binding to study the effect of the ligands on subunit-subunit interactions in EGF receptor (EGFR) homodimers and EGFR/ErbB2 heterodimers. In luciferase fragment complementation imaging studies, amphiregulin (AREG) functioned as a partial agonist, inducing only about half as much total dimerization as the other three ligands. However, unlike the other ligands, AREG showed biphasic kinetics for dimer formation, suggesting that its path for EGF receptor activation involves binding to both monomers and preformed dimers. EGF, TGFα, and betacellulin (BTC) appear to mainly stimulate receptor activation through binding to and dimerization of receptor monomers. In radioligand binding assays, EGF and TGFα exhibited increased affinity for EGFR/ErbB2 heterodimers compared with EGFR homodimers. By contrast, BTC and AREG showed a similar affinity for both dimers. Thus, EGF and TGFα are biased agonists, whereas BTC and AREG are balanced agonists with respect to selectivity of dimer formation. These data suggest that the differences in biological response to different EGF receptor ligands may result from partial agonism for dimer formation, differences in the kinetic pathway utilized to generate activated receptor dimers, and biases in the formation of heterodimers versus homodimers. PMID:25086039

Structural Determinants Underlying Constitutive Dimerization of Unoccupied Human Follitropin Receptors

PubMed Central

Guan, Rongbin; Wu, Xueqing; Feng, Xiuyan; Zhang, Meilin; Hébert, Terence E.; Segaloff, Deborah L.

2009-01-01

The human follitropin receptor (hFSHR) is a G protein-coupled receptor (GPCR) central to reproductive physiology that is composed of an extracellular domain (ECD) fused to a serpentine region. Using bioluminescence resonance energy transfer (BRET) in living cells, we show that hFSHR dimers form constitutively during their biosynthesis. Mutations in TM1 and TM4 had no effect on hFSHR dimerization, alone or when combined with mutation of Tyr110 in the ECD, a residue predicted to mediate dimerization of the soluble hormone-binding portion of the ECD complexed with FSH (Q. Fan and W. Hendrickson, Nature 433:269–277, 2005). Expressed individually, the serpentine region and a membrane-anchored form of the hFSHR ECD each exhibited homodimerization, suggesting that both domains contribute to dimerization of the full-length receptor. However, even in the context of only the membrane-anchored ECD, mutation of Tyr110 to alanine did not inhibit dimerization. The full-length hFSHR and the membrane-anchored ECD were then each engineered to introduce a consensus site for N-linked glycosylation at residue 110. Despite experimental validation of the presence of carbohydrate on residue 110, we failed to observe disruption of dimerization of either the full-length hFSHR or membrane-anchored ECD containing the inserted glycan wedge. Taken altogether, our data suggest that both the serpentine region and the ECD contribute to hFSHR dimerization and that the dimerization interface of the unoccupied hFSHR does not involve Tyr110 of the ECD. PMID:19800402

Characterization of a monoterpene synthase from Paeonia lactiflora producing α-pinene as its single product.

PubMed

Ma, Xiaohui; Guo, Juan; Ma, Ying; Jin, Baolong; Zhan, Zhilai; Yuan, Yuan; Huang, Luqi

2016-07-01

To identify a terpene synthase that catalyzes the conversion of geranyl pyrophosphate (GPP) to α-pinene and is involved in the biosynthesis of paeoniflorin. Two new terpene synthase genes were isolated from the transcriptome data of Peaonia lactiflora. Phylogenetic analysis and sequence characterization revealed that one gene, named PlPIN, encoded a monoterpene synthase that might be involved in the biosynthesis of paeoniflorin. In vitro enzyme assay showed that, in contrast to most monoterpene synthases, PlPIN encoded an α-pinene synthase which converted GPP into α-pinene as a single product. This newly identified α-pinene synthase could be used for improving paeoniflorin accumulation by metabolic engineering or for producing α-pinene via synthetic biology.

Dimerization of a flocculent protein from Moringa oleifera: experimental evidence and in silico interpretation.

PubMed

Pavankumar, Asalapuram R; Kayathri, Rajarathinam; Murugan, Natarajan A; Zhang, Qiong; Srivastava, Vaibhav; Okoli, Chuka; Bulone, Vincent; Rajarao, Gunaratna K; Ågren, Hans

2014-01-01

Many proteins exist in dimeric and other oligomeric forms to gain stability and functional advantages. In this study, the dimerization property of a coagulant protein (MO2.1) from Moringa oleifera seeds was addressed through laboratory experiments, protein-protein docking studies and binding free energy calculations. The structure of MO2.1 was predicted by homology modelling, while binding free energy and residues-distance profile analyses provided insight into the energetics and structural factors for dimer formation. Since the coagulation activities of the monomeric and dimeric forms of MO2.1 were comparable, it was concluded that oligomerization does not affect the biological activity of the protein.

Radiation-induced tetramer-to-dimer transition of Escherichia coli lactose repressor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goffinont, S.; Davidkova, M.; Spotheim-Maurizot, M., E-mail: spotheim@cnrs-orleans.fr

2009-08-21

The wild type lactose repressor of Escherichia coli is a tetrameric protein formed by two identical dimers. They are associated via a C-terminal 4-helix bundle (called tetramerization domain) whose stability is ensured by the interaction of leucine zipper motifs. Upon in vitro {gamma}-irradiation the repressor losses its ability to bind the operator DNA sequence due to damage of its DNA-binding domains. Using an engineered dimeric repressor for comparison, we show here that irradiation induces also the change of repressor oligomerisation state from tetramer to dimer. The splitting of the tetramer into dimers can result from the oxidation of the leucinemore » residues of the tetramerization domain.« less

Detection of protonated non-Watson-Crick base pairs using electrospray ionization mass spectrometry.

PubMed

Ishida, Riyoko; Iwahashi, Hideo

2018-03-01

Many studies have shown that protonated nucleic acid base pairs are involved in a wide variety of nucleic acid structures. However, little information is available on relative stability of hemiprotonated self- and non-self-dimers at monomer level. We used electrospray ionization mass spectrometry (ESI-MS) to evaluate the relative stability under various concentrations of hydrogen ion. These enable conjecture of the formation of protonated non-Watson-Crick base pairs based on DNA and RNA base sequence. In the present study, we observed that ESI-MS peaks corresponded to respective self-dimers for all examined nucleosides except for adenosine. Peak heights depended on the concentration of hydrogen ion. The ESI-MS peak heights of the hemiprotonated cytidine dimers and the hemiprotonated thymidine dimer sharply increased with increased concentration of hydrogen ion, suggesting direct participation of hydrogen ion in dimer formations. In ESI-MS measurements of the solutions containing adenosine, cytidine, thymidine and guanosine, we observed protonated cytidine-guanosine dimer (CH+-G) and protonated cytidine-thymidine dimer (CH+-T) in addition to hemiprotonated cytidine-cytidine dimer (CH+-C) with following relative peak height, (CH+-C) > (CH+-G) ≈ (CH+-T) > (CH+-A). Additionally, in the ESI-MS measurements of solutions containing adenosine, thymidine and guanosine, we observed a considerable amount of protonated adenosine-guanosine (AH+-G) and protonated adenosine-thymidine (AH+-T).

NUCLEAR-MAGNETIC-RESONANCE STUDIES OF HYDROGEN BONDING (thesis)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Davis, J.C. Jr.

1959-10-26

The nuclear-magnetic-resonance spectra of hydrogen bonding systems in noninteracting solvents were studied at several concentrations at 20 to 100 deg C. Chemical shifts mic, acetic, and benzoic acids in benzene. The shifts characteristic of the monomer and dimer species were calculated. Shieldings of the monomer species were calculated to be of the same order as those of alcohol monomers. The dimer shieldings were found to be in the range of 300 to 400 cps below the benzene reference. The dimer shieldings increase with the strength of the hydrogen bond. Chemical shifts were also measured for methanol, etanol, isopropanol, and tertiarymore » butanol in carbon tetrschloride and for ethanol in benzene. The enthalpies of dimerization were estimated from the change in the limiting slopes of shift vs. concentration curves with temperature and found to be --9.3 plus or minus 2.5, --7.4 plus or minus 2.0, --6.5 plus or minus 1.5, --5.4 plus or minus 1.8, and--5.6 plu11.6 kcal per mole of dimer, respectively. At 22 deg C, the dimerization constant for ethanol in carbon tetrachloride is 11.0 for a cyclic dimer and twice this for a linear dimer. Probable higher polymers were estimated for the ethanol system, and the experimental data were fitted by adjusting polymer shieldings and equilibrium constants. (auth)« less

Crystal structure and function of an unusual dimeric Hsp20.1 provide insight into the thermal protection mechanism of small heat shock proteins.

PubMed

Liu, Liang; Chen, Jiyun; Yang, Bo; Wang, Yonghua

2015-03-06

Small heat shock proteins (sHSPs) are ubiquitous chaperones that play a vital role in protein homeostasis. sHSPs are characterized by oligomeric architectures and dynamic exchange of subunits. The flexible oligomeric assembling associating with function remains poorly understood. Based on the structural data, it is certainly agreed that two dimerization models depend on the presence or absence of a β6 strand to differentiate nonmetazoan sHSPs from metazoan sHSPs. Here, we report the Sulfolobus solfataricus Hsp20.1 ACD dimer structure, which shows a distinct dimeric interface. We observed that, in the absence of β6, Hsp20.1 dimer does not depend on β7 strand for forming dimer interface as metazoan sHSPs, nor dissociates to monomers. This is in contrast to other published sHSPs. Our structure reveals a variable, highly polar dimer interface that has advantages for rapid subunits exchange and substrate binding. Remarkably, we find that the C-terminal truncation variant has chaperone activity comparable to that of wild-type despite lack of the oligomer structure. Our further study indicates that the N-terminal region is essential for the oligomer and dimer binding to the target protein. Together, the structure and function of Hsp20.1 give more insight into the thermal protection mechanism of sHSPs. Copyright © 2015 Elsevier Inc. All rights reserved.

Intra- and intermolecular H-bond mediated tautomerization and dimerization of 3-methyl-1,2-cyclopentanedione: Infrared spectroscopy in argon matrix and CCl 4 solution

NASA Astrophysics Data System (ADS)

Samanta, Amit K.; Pandey, Prasenjit; Bandyopadhyay, Biman; Mukhopadhyay, Anamika; Chakraborty, Tapas

2011-05-01

Mid-infrared spectra of 3-methyl-1,2-cyclopentanedione (3-MeCPD) have been recorded by isolating the molecule in a cold argon matrix (8 K) and also in CCl 4 solution at room temperature. The spectral features reveal that in both media, the molecule exists exclusively in an enol tautomeric form, which is stabilized by an intramolecular O sbnd H⋯O hydrogen bond. NBO analysis shows that the preferred conformer is further stabilized because of hyperconjugation interaction between the methyl and vinyl group of the enol tautomer. In CCl 4 solution, the molecule undergoes extensive self association and generates a doubly hydrogen bonded centrosymmetric dimer. The dimerization constant ( K d) is estimated to have a value of ˜9 L mol -1 at room temperature (25 °C) and the thermodynamic parameters, Δ H°, Δ S° and Δ G°, of dimerization are estimated by measuring K d at several temperatures within the range 22-60 °C. The same dimer is also produced when the matrix is annealed at a higher temperature. In addition, a non-centrosymmetric singly hydrogen bonded dimer is also identified in the argon matrix. A comparison between the spectral features of the two dimers indicates that the dimerization effect on doubly H-bonded case is influenced by cooperative interaction between the two H-bonds.

The Role of Water in the Stability of Wild Type and Mutant Insulin Dimers.

PubMed

Raghunathan, Shampa; El Hage, Krystel; Desmond, Jasmine; Zhang, Lixian; Meuwly, Markus

2018-06-19

Insulin dimerization and aggregation play important roles in the endogenous delivery of the hormone. One of the important residues at the insulin dimer interface is Phe B24 which is an invariant aromatic anchor that packs towards its own monomer inside a hydrophobic cavity formed by Val B12 , Leu B15 , Tyr B16 , Cys B19 and Tyr B26 . Using molecular dynamics and free energy simulations in explicit solvent, the structural and dynamical consequences of mutations of Phe at position B24 to Gly, Ala, and d-Ala and the des-PheB25 variant are quantified. Consistent with experiments it is found that the Gly and Ala modifications lead to insulin dimers with reduced stability by 4 and 5 kcal/mol from thermodynamic integration and 4 and 8 kcal/mol from results using MM-GBSA, respectively. Given the experimental difficulties to quantify the thermodynamic stability of modified insulin dimers, such computations provide a valuable complement. Interestingly, the Gly-mutant exists as a strongly and a weakly interacting dimer. Analysis of the molecular dynamics simulations shows that this can be explained by water molecules that replace direct monomer-monomer H-bonding contacts at the dimerization interface involving residues B24 to B26. It is concluded that such solvent molecules play an essential role and must be included in future insulin dimerization studies.

Improved anticancer effects of albumin-bound paclitaxel nanoparticle via augmentation of EPR effect and albumin-protein interactions using S-nitrosated human serum albumin dimer.

PubMed

Kinoshita, Ryo; Ishima, Yu; Chuang, Victor T G; Nakamura, Hideaki; Fang, Jun; Watanabe, Hiroshi; Shimizu, Taro; Okuhira, Keiichiro; Ishida, Tatsuhiro; Maeda, Hiroshi; Otagiri, Masaki; Maruyama, Toru

2017-09-01

In the latest trend of anticancer chemotherapy research, there were many macromolecular anticancer drugs developed based on enhanced permeability and retention (EPR) effect, such as albumin bound paclitaxel nanoparticle (nab- PTX, also called Abraxane ® ). However, cancers with low vascular permeability posed a challenge for these EPR based therapeutic systems. Augmenting the intrinsic EPR effect with an intrinsic vascular modulator such as nitric oxide (NO) could be a promising strategy. S-nitrosated human serum albumin dimer (SNO-HSA Dimer) shown promising activity previously was evaluated for the synergistic effect when used as a pretreatment agent in nab-PTX therapy against various tumor models. In the high vascular permeability C26 murine colon cancer subcutaneous inoculation model, SNO-HSA Dimer enhanced tumor selectivity of nab-PTX, and attenuated myelosuppression. SNO-HSA Dimer also augmented the tumor growth inhibition of nab-PTX in low vascular permeability B16 murine melanoma subcutaneous inoculation model. Furthermore, nab-PTX therapy combined with SNO-HSA Dimer showed higher antitumor activity and improved survival rate of SUIT2 human pancreatic cancer orthotopic model. In conclusion, SNO-HSA Dimer could enhance the therapeutic effect of nab-PTX even in low vascular permeability or intractable pancreatic cancers. The possible underlying mechanisms of action of SNO-HSA Dimer were discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

The E2 Domains of APP and APLP1 Share a Conserved Mode of Dimerization

DOE Office of Scientific and Technical Information (OSTI.GOV)

S Lee; Y Xue; J Hulbert

2011-12-31

Amyloid precursor protein (APP) is genetically linked to Alzheimer's disease. APP is a type I membrane protein, and its oligomeric structure is potentially important because this property may play a role in its function or affect the processing of the precursor by the secretases to generate amyloid {beta}-peptide. Several independent studies have shown that APP can form dimers in the cell, but how it dimerizes remains controversial. At least three regions of the precursor, including a centrally located and conserved domain called E2, have been proposed to contribute to dimerization. Here we report two new crystal structures of E2, onemore » from APP and the other from APLP1, a mammalian APP homologue. Comparison with an earlier APP structure, which was determined in a different space group, shows that the E2 domains share a conserved and antiparallel mode of dimerization. Biophysical measurements in solution show that heparin binding induces E2 dimerization. The 2.1 {angstrom} resolution electron density map also reveals phosphate ions that are bound to the protein surface. Mutational analysis shows that protein residues interacting with the phosphate ions are also involved in heparin binding. The locations of two of these residues, Arg-369 and His-433, at the dimeric interface suggest a mechanism for heparin-induced protein dimerization.« less

Members of the DAN family are BMP antagonists that form highly stable noncovalent dimers.

PubMed

Kattamuri, Chandramohan; Luedeke, David M; Nolan, Kristof; Rankin, Scott A; Greis, Kenneth D; Zorn, Aaron M; Thompson, Thomas B

2012-12-14

Signaling of bone morphogenetic protein (BMP) ligands is antagonized by a number of extracellular proteins, including noggin, follistatin and members of the DAN (differential screening selected gene abberative in neuroblastoma) family. Structural studies on the DAN family member sclerostin (a weak BMP antagonist) have previously revealed that the protein is monomeric and consists of an eight-membered cystine knot motif with a fold similar to transforming growth factor-β ligands. In contrast to sclerostin, certain DAN family antagonists, including protein related to DAN and cerberus (PRDC), have an unpaired cysteine that is thought to function in covalent dimer assembly (analogous to transforming growth factor-β ligands). Through a combination of biophysical and biochemical studies, we determined that PRDC forms biologically active dimers that potently inhibit BMP ligands. Furthermore, we showed that PRDC dimers, surprisingly, are not covalently linked, as mutation of the unpaired cysteine does not inhibit dimer formation or biological activity. We further demonstrated that the noncovalent PRDC dimers are highly stable under both denaturing and reducing conditions. This study was extended to the founding family member DAN, which also forms noncovalent dimers that are highly stable. These results demonstrate that certain DAN family members can form both monomers and noncovalent dimers, implying that biological activity of DAN family members might be linked to their oligomeric state. Published by Elsevier Ltd.

Rapid Reversion from Monomer to Dimer Regenerates the Ultraviolet-B Photoreceptor UV RESISTANCE LOCUS8 in Intact Arabidopsis Plants1[W][OA

PubMed Central

Heilmann, Monika; Jenkins, Gareth I.

2013-01-01

Arabidopsis (Arabidopsis thaliana) UV RESISTANCE LOCUS8 (UVR8) is a photoreceptor that specifically mediates photomorphogenic responses to ultraviolet (UV)-B in plants. UV-B photoreception induces the conversion of the UVR8 dimer into a monomer that interacts with the CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) protein to regulate gene expression. However, it is not known how the dimeric photoreceptor is regenerated in plants. Here, we show, by using inhibitors of protein synthesis and degradation via the proteasome, that the UVR8 dimer is not regenerated by rapid de novo synthesis following destruction of the monomer. Rather, regeneration occurs by reversion from the monomer to the dimer. However, regeneration of dimeric UVR8 in darkness following UV-B exposure occurs much more rapidly in vivo than in vitro with illuminated plant extracts or purified UVR8, indicating that rapid regeneration requires intact cells. Rapid dimer regeneration in vivo requires protein synthesis, the presence of a carboxyl-terminal 27-amino acid region of UVR8, and the presence of COP1, which is known to interact with the carboxyl-terminal region. However, none of these factors can account fully for the difference in regeneration kinetics in vivo and in vitro, indicating that additional proteins or processes are involved in UVR8 dimer regeneration in vivo. PMID:23129206

A novel dimeric thymosin beta 4 with enhanced activities accelerates the rate of wound healing

PubMed Central

Xu, Tian-Jiao; Wang, Qi; Ma, Xiao-Wen; Zhang, Zhen; Zhang, Wei; Xue, Xiao-Chang; Zhang, Cun; Hao, Qiang; Li, Wei-Na; Zhang, Ying-Qi; Li, Meng

2013-01-01

Objective Thymosin beta 4 (Tβ4) is a peptide with 43 amino acids that is critical for repair and remodeling tissues on the skin, eye, heart, and neural system following injury. To fully realize its utility as a treatment for disease caused by injury, the authors constructed a cost-effective novel Tβ4 dimer and demonstrated that it was better able to accelerate tissue repair than native Tβ4. Methods A prokaryotic vector harboring two complete Tβ4 genes with a short linker was constructed and expressed in Escherichia coli. A pilot-scale fermentation (10 L) was performed to produce engineered bacteria and the Tβ4 dimer was purified by one-step hydrophobic interaction chromatography. The activities of the Tβ4 dimer to promote endothelial cell proliferation, migration, and sprouting were assessed by tetramethylbenzidine (methylthiazol tetrazolium), trans-well, scratch, and tube formation assays. The ability to accelerate dermal healing was assessed on rats. Results After fermentation, the Tβ4 dimer accounted for about 30% of all the bacteria proteins. The purity of the Tβ4 dimer reached 98% after hydrophobic interaction chromatography purification. An average of 562.4 mg/L Tβ4 dimer was acquired using a 10 L fermenter. In each assay, the dimeric Tβ4 exhibited enhanced activities compared with native Tβ4. Notably, the ability of the dimeric Tβ4 to promote cell migration was almost two times higher than that of Tβ4. The rate of dermal healing in the dimeric Tβ4-treated rats was approximately 1 day faster than with native Tβ4-treated rats. Conclusion The dimeric Tβ4 exhibited enhanced activity on wound healing than native Tβ4, and the purification process was simple and cost-effective. This data could be of significant benefit for the high pain and morbidity associated with chronic wounds disease. A better strategy to develop Tβ4 as a treatment for other diseases caused by injuries such as heart attack, neurotrophic keratitis, and multiple sclerosis was also described. PMID:24109178

D-dimer and risk of thromboembolic and bleeding events in patients with atrial fibrillation--observations from the ARISTOTLE trial.

PubMed

Christersson, C; Wallentin, L; Andersson, U; Alexander, J H; Ansell, J; De Caterina, R; Gersh, B J; Granger, C B; Hanna, M; Horowitz, J D; Huber, K; Husted, S; Hylek, E M; Lopes, R D; Siegbahn, A

2014-09-01

D-dimer is related to adverse outcomes in arterial and venous thromboembolic diseases. To evaluate the predictive value of D-dimer level for stroke, other cardiovascular events, and bleeds, in patients with atrial fibrillation (AF) treated with oral anticoagulation with apixaban or warfarin; and to evaluate the relationship between the D-dimer levels at baseline and the treatment effect of apixaban vs. warfarin. In the ARISTOTLE trial, 18 201 patients with AF were randomized to apixaban or warfarin. D-dimer was analyzed in 14 878 patients at randomization. The cohort was separated into two groups; not receiving vitamin K antagonist (VKA) treatment and receiving VKA treatment at randomization. Higher D-dimer levels were associated with increased frequencies of stroke or systemic embolism (hazard ratio [HR] [Q4 vs. Q1] 1.72, 95% confidence interval [CI] 1.14-2.59, P = 0.003), death (HR [Q4 vs. Q1] 4.04, 95% CI 3.06-5.33) and major bleeding (HR [Q4 vs. Q1] 2.47, 95% CI 1.77-3.45, P < 0.0001) in the no-VKA group. Similar results were obtained in the on-VKA group. Adding D-dimer level to the CHADS2 score improved the C-index from 0.646 to 0.655 for stroke or systemic embolism, and from 0.598 to 0.662 for death, in the no-VKA group. D-dimer level improved the HAS-BLED score for prediction of major bleeds, with an increase in the C-index from 0.610 to 0.641. There were no significant interactions between efficacy and safety of study treatment and D-dimer level. In anticoagulated patients with AF, the level of D-dimer is related to the risk of stroke, death, and bleeding, and adds to the predictive value of clinical risk scores. The benefits of apixaban were consistent, regardless of the baseline D-dimer level. © 2014 International Society on Thrombosis and Haemostasis.

Long-Lived Triplet Excited States of Bent-Shaped Pentacene Dimers by Intramolecular Singlet Fission.

PubMed

Sakuma, Takao; Sakai, Hayato; Araki, Yasuyuki; Mori, Tadashi; Wada, Takehiko; Tkachenko, Nikolai V; Hasobe, Taku

2016-03-24

Intramolecular singlet fission (ISF) is a promising photophysical process to construct more efficient light energy conversion systems as one excited singlet state converts into two excited triplet states. Herein we synthesized and evaluated bent-shaped pentacene dimers as a prototype of ISF to reveal intrinsic characters of triplet states (e.g., lifetimes of triplet excited states). In this study, meta-phenylene-bridged TIPS-pentacene dimer (PcD-3Ph) and 2,2'-bipheynyl bridged TIPS-pentacene dimer (PcD-Biph) were newly synthesized as bent-shaped dimers. In the steady-state spectroscopy, absorption and emission bands of these dimers were fully characterized, suggesting the appropriate degree of electronic coupling between pentacene moieties in these dimers. In addition, the electrochemical measurements were also performed to check the electronic interaction between two pentacene moieties. Whereas the successive two oxidation peaks owing to the delocalization were observed in a directly linked-pentacene dimer (PcD) by a single bond, the cyclic voltammograms in PcD-Biph and PcD-3Ph implied the weaker interaction compared to that of p-phenylene-bridged TIPS-pentacene dimer (PcD-4Ph) and PcD. The femtosecond and nanosecond transient absorption spectra clearly revealed the slower ISF process in bent-shaped pentacene dimers (PcD-Biph and PcD-3Ph), more notably, the slower relaxation of the excited triplet states in PcD-Biph and PcD-3Ph. Namely, the quantum yields of triplet states (ΦT) by ISF approximately remain constant (ca. 180-200%) in all dimer systems, whereas the lifetimes of the triplet excited states became much longer (up to 360 ns) in PcD-Biph as compared to PcD-4Ph (15 ns). Additionally, the lifetimes of the corresponding triplet states in PcD-Biph and PcD-3Ph were sufficiently affected by solvent viscosity. In particular, the lifetimes of PcD-Biph triplet state in THF/paraffin (1.0 μs) increased up to approximately three times as compared to that in THF (360 ns), whereas those of PcD-4Ph were quite similar in both solvent.

Molecular Diversity of Terpene Synthases in the Liverwort Marchantia polymorpha[OPEN

PubMed Central

Zhuang, Xun; Jiang, Zuodong; Jia, Qidong; Babbitt, Patricia C.

2016-01-01

Marchantia polymorpha is a basal terrestrial land plant, which like most liverworts accumulates structurally diverse terpenes believed to serve in deterring disease and herbivory. Previous studies have suggested that the mevalonate and methylerythritol phosphate pathways, present in evolutionarily diverged plants, are also operative in liverworts. However, the genes and enzymes responsible for the chemical diversity of terpenes have yet to be described. In this study, we resorted to a HMMER search tool to identify 17 putative terpene synthase genes from M. polymorpha transcriptomes. Functional characterization identified four diterpene synthase genes phylogenetically related to those found in diverged plants and nine rather unusual monoterpene and sesquiterpene synthase-like genes. The presence of separate monofunctional diterpene synthases for ent-copalyl diphosphate and ent-kaurene biosynthesis is similar to orthologs found in vascular plants, pushing the date of the underlying gene duplication and neofunctionalization of the ancestral diterpene synthase gene family to >400 million years ago. By contrast, the mono- and sesquiterpene synthases represent a distinct class of enzymes, not related to previously described plant terpene synthases and only distantly so to microbial-type terpene synthases. The absence of a Mg2+ binding, aspartate-rich, DDXXD motif places these enzymes in a noncanonical family of terpene synthases. PMID:27650333

Interactions of citrate synthases from osmoconforming and osmoregulating animals with salt: possible signs of molecular eco-adaptation?

PubMed

Sarkissian, I V

1977-01-01

This study considers differential sensitivity of citrate synthase (citrate oxaloacetatelyase [CoA acetylating]) EC 4.1.3.7. from an osmoconforming animal (sea anemone) and an osmoregulating animal (the pig) to salt. Attention is drawn to the fact that the osmoconforming sea anemone is in essence a sessile creature while the pig is readily mobile and able to change its ionic environment at will. It had been shown earlier that citrate synthase from another osmoconformer (oyster) is also not sensitive to ionic strength while citrate synthase from osmoregulating white shrimp is sensitive to increasing levels of salt. However, these enzymes are characteristically regulated by ATP and alpha-ketoglutarate. Both forms of citrate synthase are denatured by 6 M guanidine hydrochloride and are aided by salt levels in their refolding but the rate and extent of refolding of the osmoconformer citrate synthase are greater than those of the osmoregulator citrate synthase. Catalytic activity of both forms of citrate synthase is inhibited by incubation in distilled water; osmoconformer citrate synthase was inhibited completely in 7 h while osmoregulator citrate synthase was inhibited only 60% in this time and 80% after 22 h in distilled water. The eco-adaptive and evolutionary implications of these findings are discussed.

Receptor signaling: when dimerization is not enough.

PubMed

Jiang, G; Hunter, T

Activation of receptors that signal via tyrosine kinase domains has been thought to involve receptor dimerization and transphosphorylation of juxtaposed catalytic domains. Recent results suggest things might be more complex - specific intersubunit conformational changes within a dimer can also be important.

Inhibition of Human Cytomegalovirus Replication by Artemisinins: Effects Mediated through Cell Cycle Modulation

PubMed Central

Roy, Sujayita; He, Ran; Kapoor, Arun; Forman, Michael; Mazzone, Jennifer R.; Posner, Gary H.

2015-01-01

Artemisinin-derived monomers and dimers inhibit human cytomegalovirus (CMV) replication in human foreskin fibroblasts (HFFs). The monomer artesunate (AS) inhibits CMV at micromolar concentrations, while dimers inhibit CMV replication at nanomolar concentrations, without increased toxicity in HFFs. We report on the variable anti-CMV activity of AS compared to the consistent and reproducible CMV inhibition by dimer 606 and ganciclovir (GCV). Investigation of this phenomenon revealed that the anti-CMV activity of AS correlated with HFFs synchronized to the G0/G1 stage of the cell cycle. In contact-inhibited serum-starved HFFs or cells arrested at early/late G1 with specific checkpoint regulators, AS and dimer 606 efficiently inhibited CMV replication. However, in cycling HFFs, in which CMV replication was productive, virus inhibition by AS was significantly reduced, but inhibition by dimer 606 and GCV was maintained. Cell cycle analysis in noninfected HFFs revealed that AS induced early G1 arrest, while dimer 606 partially blocked cell cycle progression. In infected HFFs, AS and dimer 606 prevented the progression of cell cycle toward the G1/S checkpoint. AS reduced the expression of cyclin-dependent kinases (CDK) 2, 4, and 6 in noninfected cycling HFFs, while the effect of dimer 606 on these CDKs was moderate. Neither compound affected CDK expression in noninfected contact-inhibited HFFs. In CMV-infected cells, AS activity correlated with reduced CDK2 levels. CMV inhibition by AS and dimer 606 also correlated with hypophosphorylation (activity) of the retinoblastoma protein (pRb). AS activity was strongly associated with pRb hypophosphorylation, while its reduced anti-CMV activity was marked by pRb phosphorylation. Roscovitine, a CDK2 inhibitor, antagonized the anti-CMV activities of AS and dimer 606. These data suggest that cell cycle modulation through CDKs and pRb might play a role in the anti-CMV activities of artemisinins. Proteins involved in this modulation may be identified and targeted for CMV inhibition. PMID:25870074

Impact of D-Dimer for Prediction of Incident Occult Cancer in Patients with Unprovoked Venous Thromboembolism

PubMed Central

Han, Donghee; ó Hartaigh, Bríain; Lee, Ji Hyun; Cho, In-Jeong; Shim, Chi Young; Chang, Hyuk-Jae; Hong, Geu-Ru; Ha, Jong-Won; Chung, Namsik

2016-01-01

Background Unprovoked venous thromboembolism (VTE) is related to a higher incidence of occult cancer. D-dimer is clinically used for screening VTE, and has often been shown to be present in patients with malignancy. We explored the predictive value of D-dimer for detecting occult cancer in patients with unprovoked VTE. Methods We retrospectively examined data from 824 patients diagnosed with deep vein thrombosis or pulmonary thromboembolism. Of these, 169 (20.5%) patients diagnosed with unprovoked VTE were selected to participate in this study. D-dimer was categorized into three groups as: <2,000, 2,000–4,000, and >4,000 ng/ml. Cox regression analysis was employed to estimate the odds of occult cancer and metastatic state of cancer according to D-dimer categories. Results During a median 5.3 (interquartile range: 3.4–6.7) years of follow-up, 24 (14%) patients with unprovoked VTE were diagnosed with cancer. Of these patients, 16 (67%) were identified as having been diagnosed with metastatic cancer. Log transformed D-dimer levels were significantly higher in those with occult cancer as compared with patients without diagnosis of occult cancer (3.5±0.5 vs. 3.2±0.5, P-value = 0.009, respectively). D-dimer levels >4,000 ng/ml was independently associated with occult cancer (HR: 4.12, 95% CI: 1.54–11.04, P-value = 0.005) when compared with D-dimer levels <2,000 ng/ml, even after adjusting for age, gender, and type of VTE (e.g., deep vein thrombosis or pulmonary thromboembolism). D-dimer levels >4000 ng/ml were also associated with a higher likelihood of metastatic cancer (HR: 9.55, 95% CI: 2.46–37.17, P-value <0.001). Conclusion Elevated D-dimer concentrations >4000 ng/ml are independently associated with the likelihood of occult cancer among patients with unprovoked VTE. PMID:27073982

Impact of D-Dimer for Prediction of Incident Occult Cancer in Patients with Unprovoked Venous Thromboembolism.

PubMed

Han, Donghee; ó Hartaigh, Bríain; Lee, Ji Hyun; Cho, In-Jeong; Shim, Chi Young; Chang, Hyuk-Jae; Hong, Geu-Ru; Ha, Jong-Won; Chung, Namsik

2016-01-01

Unprovoked venous thromboembolism (VTE) is related to a higher incidence of occult cancer. D-dimer is clinically used for screening VTE, and has often been shown to be present in patients with malignancy. We explored the predictive value of D-dimer for detecting occult cancer in patients with unprovoked VTE. We retrospectively examined data from 824 patients diagnosed with deep vein thrombosis or pulmonary thromboembolism. Of these, 169 (20.5%) patients diagnosed with unprovoked VTE were selected to participate in this study. D-dimer was categorized into three groups as: <2,000, 2,000-4,000, and >4,000 ng/ml. Cox regression analysis was employed to estimate the odds of occult cancer and metastatic state of cancer according to D-dimer categories. During a median 5.3 (interquartile range: 3.4-6.7) years of follow-up, 24 (14%) patients with unprovoked VTE were diagnosed with cancer. Of these patients, 16 (67%) were identified as having been diagnosed with metastatic cancer. Log transformed D-dimer levels were significantly higher in those with occult cancer as compared with patients without diagnosis of occult cancer (3.5±0.5 vs. 3.2±0.5, P-value = 0.009, respectively). D-dimer levels >4,000 ng/ml was independently associated with occult cancer (HR: 4.12, 95% CI: 1.54-11.04, P-value = 0.005) when compared with D-dimer levels <2,000 ng/ml, even after adjusting for age, gender, and type of VTE (e.g., deep vein thrombosis or pulmonary thromboembolism). D-dimer levels >4000 ng/ml were also associated with a higher likelihood of metastatic cancer (HR: 9.55, 95% CI: 2.46-37.17, P-value <0.001). Elevated D-dimer concentrations >4000 ng/ml are independently associated with the likelihood of occult cancer among patients with unprovoked VTE.

99mTc-labeling of HYNIC-conjugated cyclic RGDfK dimer and tetramer using EDDA as coligand.

PubMed

Wang, Jianjun; Kim, Young-Seung; Liu, Shuang

2008-03-01

In this study, EDDA (ethylenediamine- N, N'-diacetic acid) was used as the coligand for 99mTc-labeling of cyclic RGDfK conjugates: HYNIC-dimer (HYNIC = 6-hydrazinonicotinamide; dimer = E[c(RGDfK)]2) and HYNIC-tetramer (tetramer = E{E[c(RGDfK)]2}2). First, HYNIC-dimer was allowed to react with 99mTcO4 (-) in the presence of excess tricine and stannous chloride to form the intermediate complex [99mTc(HYNIC-dimer)(tricine)2], which was then allowed to react with EDDA to afford [99mTc(HYNIC-dimer)(EDDA)] with high yield (>90%) and high specific activity ( approximately 8.0 Ci/micromol). Under the same radiolabeling conditions, the yield for [99mTc(HYNIC-tetramer)(EDDA)] was always <65%. The results from a mixed-ligand experiment show that there is only one EDDA bonding to the 99mTc-HYNIC core in [99mTc(HYNIC-dimer)(EDDA)]. The athymic nude mice bearing subcutaneous U87MG human glioma xenografts were used to evaluate the impact of EDDA coligand on the biodistribution characteristics and excretion kinetics of the 99mTc-labeled HYNIC-dimer and HYNIC-tetramer. Surprisingly, [99mTc(HYNIC-dimer)(EDDA)] and [99mTc(HYNIC-tetramer)(EDDA)] had almost identical tumor uptake over the 2 h period. The use of EDDA as coligand to replace tricine/TPPTS (TPPTS = trisodium triphenylphosphine-3,3',3''-trisulfonate) did not significantly change the uptake of the 99mTc-labeled HYNIC-dimer in noncancerous organs, such as the liver, kidneys, and lungs; but it did result in a significantly lower kidney uptake for the 99mTc-labeled HYNIC-tetramer due to faster renal excretion. It was also found that the radiotracer tumor uptake decreases in a linear fashion as the tumor size increases. The smaller the tumors are, the higher the tumor uptake is regardless of the identity of radiotracer.

Effects of Cd{sup 2+} on cis-dimer structure of E-cadherin in living cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takeda, Hiroshi, E-mail: hirotake@sapmed.ac.jp

2014-02-21

Highlights: • The effects of Cd on the dimer of cadherin in living cells was analyzed. • Cd induced cadherin dimer formation was not detected in living cell with low Ca. • Ca mediated structural cooperativity and allostery in the native cadherin. • Ca concentration-dependent competitive displacement of Cd from cadherin is proposed. - Abstract: E-cadherin, a calcium (Ca{sup 2+})-dependent cell–cell adhesion molecule, plays a key role in the maintenance of tissue integrity. We have previously demonstrated that E-cadherin functions in vivo as a cis-dimer through chemical cross-linking reagents. Ca{sup 2+} plays an important role in the cis-dimer formation ofmore » cadherin. However, the molecular mechanisms by which Ca{sup 2+} interacts with the binding sites that regulate cis-dimer structures have not been completely elucidated. As expected for a Ca{sup 2+} antagonist, cadmium (Cd{sup 2+}) disrupts cadherin function by displacing Ca{sup 2+} from its binding sites on the cadherin molecules. We used Cd{sup 2+} as a probe for investigating the role of Ca{sup 2+} in the dynamics of the E-cadherin extracellular region that involve cis-dimer formation and adhesion. While cell–cell adhesion assembly was completely disrupted in the presence of Cd{sup 2+}, the amount of cis-dimers of E-cadherin that formed at the cell surface was not affected. In our “Cd{sup 2+}-switch” experiments, we did not find that Cd{sup 2+}-induced E-cadherin cis-dimer formation in EL cells when they were incubated in low-Ca{sup 2+} medium. In the present study, we demonstrated for the first time the effects of Cd{sup 2+} on the cis-dimer structure of E-cadherin in living cells using a chemical cross-link analysis.« less

Formation of an active dimer during storage of interleukin-1 receptor antagonist in aqueous solution.

PubMed Central

Chang, B S; Beauvais, R M; Arakawa, T; Narhi, L O; Dong, A; Aparisio, D I; Carpenter, J F

1996-01-01

The degradation products of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) formed during storage at 30 degrees C in aqueous solution were characterized. Cationic exchange chromatography of the stored sample showed two major, new peaks eluting before (P1) and after (L2) the native protein, which were interconvertible. Size-exclusion chromatography and electrophoresis documented that both the P1 and L2 fractions were irreversible dimers, formed by noncovalent interactions. A competition assay with interleukin-1 indicated that on a per monomer basis the P1 and L2 dimers retained about two-thirds of the activity of the native monomer. Infrared and far-UV circular dichroism spectroscopies showed that only minor alterations in secondary structure arose upon the formation of the P1 dimer. However, alteration in the near-UV circular dichroism spectrum suggested the presence of disulfide bonds in the P1 dimer, which are absent in the native protein. Mass spectroscopy and tryptic mapping, before and after carboxymethylation, demonstrated that the P1 dimer contained an intramolecular disulfide bond between Cys-66 and Cys-69. Although conversion of native protein to the P1 dimer was irreversible in buffer alone, the native monomer could be regained by denaturing the P1 dimer with guanidine hydrochloride and renaturing it by dialysis, suggesting that the intramolecular disulfide bond does not interfere with refolding. Analysis of the time course of P1 formation during storage at 30 degrees C indicated that the process followed first-order, and not second-order, kinetics, suggesting that the rate-limiting step was not dimerization. It is proposed that a conformational change in the monomer is the rate-limiting step in the formation of the P1 dimer degradation product. Sucrose stabilized the native monomer against this process. This result can be explained by the general stabilization mechanism for this additive, which is due to its preferential exclusion from the protein surface. PMID:8968609

The ratio of D-dimer to brain natriuretic peptide may help to differentiate between cerebral infarction with and without acute aortic dissection.

PubMed

Okazaki, Toshiyuki; Yamamoto, Yoko; Yoda, Keishi; Nagahiro, Shinji

2014-05-15

Previous studies reported that the plasma d-dimer level reflects the activity of thrombus formation in the left atrium of patients with acute cerebral infarction and acute aortic dissection (AAD). Brain natriuretic peptide (BNP) is considered to be a marker of chronic heart failure. The differential diagnosis in the emergency room between stroke due to cardioembolism and AAD is difficult but important for early treatment especially in patients requiring intravenous thrombolysis with a recombinant tissue-type plasminogen activator. We aimed to investigate the association between the plasma d-dimer and BNP levels in patients with cerebral infarction and AAD. We identified 115 consecutive patients with ischemic stroke who were admitted within 72 h of symptom onset and 15 consecutive patients with AAD and measured the level of plasma d-dimer and BNP and the d-dimer:BNP ratio. In patients with AAD the d-dimer level was significantly higher than that in patients with any other stroke subtypes and their BNP level was significantly lower than that in patients with cardioembolic stroke. The d-dimer:BNP ratio was significantly higher in patients with AAD than in those with any other stroke subtype. Compared to patients with a cardioembolic stroke subtype they manifested significantly higher d-dimer levels and d-dimer:BNP ratios suggesting that this ratio may help to diagnose cerebral infarction due to AAD (sensitivity 80%, specificity 93.5%, cut-off 0.074). When the population was limited to patients within 6h of onset, the ratio had higher sensitivity and specificity at the same cut-off value (sensitivity 81.8%, specificity 96.4%). We found that the d-dimer:BNP ratio may be helpful in distinguishing between cerebral infarction with and without AAD. Copyright © 2014 Elsevier B.V. All rights reserved.

Photochemical pathways of the dimeric, mixed dimer, and monomeric sulfophthalocyanines of cobalt(III) and iron(II)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ferraudi, G.

1979-04-01

The photochemical reactivity of the dimeric, mixed dimer, and monomeric sulfophthalocyanines of cobalt (III) and iron (II) was investigated by steady-state and flash irradiations. The dimeric species photodissociated into sulfophthalocyanine radicals which were coordinated to either Co(III) or Fe(II) metal centers. Reactions of such intermediates were investigated by interception with alcohols and O/sub 2/. Also, photoredox reactions were detected with monomeric acidocobalt(III) sulfophtahlocyanines. These processes produce the oxidation of the acido ligands (Cl/sup -/, Br/sup -/, N/sub 3//sup -/, I/sup -/) and the reduction of the metal center. The photoredox dissociation was also investigated by using mixed dimers of themore » cobalt sulfophthalocyanines with Cr(bpy)/sub 3//sup 3 +/ and Ru(bpy)/sub 3//sup 2 +/. The photogeneration of sulfophthalocyanine radicals was observed as a general reaction which was produced by excitation of either the Cr(bby)/sub 3//sup 3 +/ or Ru(bpy)/sub 3//sup 2 +/ units in the mixed dimer. The nature of the reactive excited states involved in the various photochemical reactions of the sulfophthalocyanines of Co(II), Co(III), Cu(II), and Fe(II) is discussed.« less

Light activation of the LOV protein vivid generates a rapidly exchanging dimer.

PubMed

Zoltowski, Brian D; Crane, Brian R

2008-07-08

The fungal photoreceptor Vivid (VVD) plays an important role in the adaptation of blue-light responses in Neurospora crassa. VVD, an FAD-binding LOV (light, oxygen, voltage) protein, couples light-induced cysteinyl adduct formation at the flavin ring to conformational changes in the N-terminal cap (Ncap) of the VVD PAS domain. Size-exclusion chromatography (SEC), equilibrium ultracentrifugation, and static and dynamic light scattering show that these conformational changes generate a rapidly exchanging VVD dimer, with an expanded hydrodynamic radius. A three-residue N-terminal beta-turn that assumes two different conformations in a crystal structure of a VVD C71V variant is essential for light-state dimerization. Residue substitutions at a critical hinge between the Ncap and PAS core can inhibit or enhance dimerization, whereas a Tyr to Trp substitution at the Ncap-PAS interface stabilizes the light-state dimer. Cross-linking through engineered disulfides indicates that the light-state dimer differs considerably from the dark-state dimer found in VVD crystal structures. These results verify the role of Ncap conformational changes in gating the photic response of N. crassa and indicate that LOV-LOV homo- or heterodimerization may be a mechanism for regulating light-activated gene expression.

Hydrodynamic Torques and Rotations of Superparamagnetic Bead Dimers

NASA Astrophysics Data System (ADS)

Pease, Christopher; Etheridge, J.; Wijesinghe, H. S.; Pierce, C. J.; Prikockis, M. V.; Sooryakumar, R.

Chains of micro-magnetic particles are often rotated with external magnetic fields for many lab-on-a-chip technologies such as transporting beads or mixing fluids. These applications benefit from faster responses of the actuated particles. In a rotating magnetic field, the magnetization of superparamagnetic beads, created from embedded magnetic nano-particles within a polymer matrix, is largely characterized by induced dipoles mip along the direction of the field. In addition there is often a weak dipole mop that orients out-of-phase with the external rotating field. On a two-bead dimer, the simplest chain of beads, mop contributes a torque Γm in addition to the torque from mip. For dimers with beads unbound to each other, mop rotates individual beads which generate an additional hydrodynamic torque on the dimer. Whereas, mop directly torques bound dimers. Our results show that Γm significantly alters the average frequency-dependent dimer rotation rate for both bound and unbound monomers and, when mop exceeds a critical value, increases the maximum dimer rotation frequency. Models that include magnetic and hydrodynamics torques provide good agreement with the experimental findings over a range of field frequencies.

Lifetime of Feshbach dimers in a Fermi-Fermi mixture of 6Li and 40K

NASA Astrophysics Data System (ADS)

Jag, M.; Cetina, M.; Lous, R. S.; Grimm, R.; Levinsen, J.; Petrov, D. S.

2016-12-01

We present a joint experimental and theoretical investigation of the lifetime of weakly bound dimers formed near narrow interspecies Feshbach resonances in mass-imbalanced Fermi-Fermi systems, considering the specific example of a mixture of 6Li and 40K atoms. Our work addresses the central question of the increase in the stability of the dimers resulting from Pauli suppression of collisional losses, which is a well-known effect in mass-balanced fermionic systems near broad resonances. We present measurements of the spontaneous dissociation of dimers in dilute samples, and of the collisional losses in dense samples arising from both dimer-dimer processes and from atom-dimer processes. We find that all loss processes are suppressed close to the Feshbach resonance. Our general theoretical approach for fermionic mixtures near narrow Feshbach resonances provides predictions for the suppression of collisional decay as a function of the detuning from resonance, and we find excellent agreement with the experimental benchmarks provided by our 40K-6Li system. We finally present model calculations for other Feshbach-resonant Fermi-Fermi systems, which are of interest for experiments in the near future.

Comparison of FDTD numerical computations and analytical multipole expansion method for plasmonics-active nanosphere dimers.

PubMed

Dhawan, Anuj; Norton, Stephen J; Gerhold, Michael D; Vo-Dinh, Tuan

2009-06-08

This paper describes a comparative study of finite-difference time-domain (FDTD) and analytical evaluations of electromagnetic fields in the vicinity of dimers of metallic nanospheres of plasmonics-active metals. The results of these two computational methods, to determine electromagnetic field enhancement in the region often referred to as "hot spots" between the two nanospheres forming the dimer, were compared and a strong correlation observed for gold dimers. The analytical evaluation involved the use of the spherical-harmonic addition theorem to relate the multipole expansion coefficients between the two nanospheres. In these evaluations, the spacing between two nanospheres forming the dimer was varied to obtain the effect of nanoparticle spacing on the electromagnetic fields in the regions between the nanostructures. Gold and silver were the metals investigated in our work as they exhibit substantial plasmon resonance properties in the ultraviolet, visible, and near-infrared spectral regimes. The results indicate excellent correlation between the two computational methods, especially for gold nanosphere dimers with only a 5-10% difference between the two methods. The effect of varying the diameters of the nanospheres forming the dimer, on the electromagnetic field enhancement, was also studied.

Lifecourse social position and D-dimer; findings from the 1958 British birth cohort.

PubMed

Tabassum, Faiza; Kumari, Meena; Rumley, Ann; Power, Chris; Strachan, David P; Lowe, Gordon

2014-01-01

The aim is to examine the association of lifecourse socioeconomic position (SEP) on circulating levels of D-dimer. Data from the 1958 British birth cohort were used, social class was determined at three stages of respondents' life: at birth, at 23 and at 42 years. A cumulative indicator score of SEP (CIS) was calculated ranging from 0 (always in the highest social class) to 9 (always in the lowest social class). In men and women, associations were observed between CIS and D-dimer (P<0.05). Thus, the respondents in more disadvantaged social classes had elevated levels of D-dimer compared to respondents in less disadvantaged social class. In multivariate analyses, the association of disadvantaged social position with D-dimer was largely explained by fibrinogen, C-reactive protein and von Willebrand Factor in women, and additionally by smoking, alcohol consumption and physical activity in men. Socioeconomic circumstances across the lifecourse at various stages also contribute independently to raised levels of D-dimer in middle age in women only. Risk exposure related to SEP accumulates across life and contributes to raised levels of D-dimer. The association of haemostatic markers and social differences in health may be mediated by inflammatory and other markers.

Adenylyl cyclase G is activated by an intramolecular osmosensor.

PubMed

Saran, Shweta; Schaap, Pauline

2004-03-01

Adenylyl cyclase G (ACG) is activated by high osmolality and mediates inhibition of spore germination by this stress factor. The catalytic domains of all eukaryote cyclases are active as dimers and dimerization often mediates activation. To investigate the role of dimerization in ACG activation, we coexpressed ACG with an ACG construct that lacked the catalytic domain (ACGDeltacat) and was driven by a UV-inducible promoter. After UV induction of ACGDeltacat, cAMP production by ACG was strongly inhibited, but osmostimulation was not reduced. Size fractionation of native ACG showed that dimers were formed between ACG molecules and between ACG and ACGDeltacat. However, high osmolality did not alter the dimer/monomer ratio. This indicates that ACG activity requires dimerization via a region outside the catalytic domain but that dimer formation does not mediate activation by high osmolality. To establish whether ACG required auxiliary sensors for osmostimulation, we expressed ACG cDNA in a yeast adenylyl cyclase null mutant. In yeast, cAMP production by ACG was similarly activated by high osmolality as in Dictyostelium. This strongly suggests that the ACG osmosensor is intramolecular, which would define ACG as the first characterized primary osmosensor in eukaryotes.

The role of prostacyclin synthase and thromboxane synthase signaling in the development and progression of cancer.

PubMed

Cathcart, Mary-Clare; Reynolds, John V; O'Byrne, Kenneth J; Pidgeon, Graham P

2010-04-01

Prostacyclin synthase and thromboxane synthase signaling via arachidonic acid metabolism affects a number of tumor cell survival pathways such as cell proliferation, apoptosis, tumor cell invasion and metastasis, and angiogenesis. However, the effects of these respective synthases differ considerably with respect to the pathways described. While prostacyclin synthase is generally believed to be anti-tumor, a pro-carcinogenic role for thromboxane synthase has been demonstrated in a variety of cancers. The balance of oppositely-acting COX-derived prostanoids influences many processes throughout the body, such as blood pressure regulation, clotting, and inflammation. The PGI(2)/TXA(2) ratio is of particular interest in-vivo, with the corresponding synthases shown to be differentially regulated in a variety of disease states. Pharmacological inhibition of thromboxane synthase has been shown to significantly inhibit tumor cell growth, invasion, metastasis and angiogenesis in a range of experimental models. In direct contrast, prostacyclin synthase overexpression has been shown to be chemopreventive in a murine model of the disease, suggesting that the expression and activity of this enzyme may protect against tumor development. In this review, we discuss the aberrant expression and known functions of both prostacyclin synthase and thromboxane synthase in cancer. We discuss the effects of these enzymes on a range of tumor cell survival pathways, such as tumor cell proliferation, induction of apoptosis, invasion and metastasis, and tumor cell angiogenesis. As downstream signaling pathways of these enzymes have also been implicated in cancer states, we examine the role of downstream effectors of PGIS and TXS activity in tumor growth and progression. Finally, we discuss current therapeutic strategies aimed at targeting these enzymes for the prevention/treatment of cancer.

Characterization of membrane protein interactions in plasma membrane derived vesicles with quantitative imaging Förster resonance energy transfer.

PubMed

Sarabipour, Sarvenaz; Del Piccolo, Nuala; Hristova, Kalina

2015-08-18

Here we describe an experimental tool, termed quantitative imaging Förster resonance energy transfer (QI-FRET), that enables the quantitative characterization of membrane protein interactions. The QI-FRET methodology allows us to acquire binding curves and calculate association constants for complex membrane proteins in the native plasma membrane environment. The method utilizes FRET detection, and thus requires that the proteins of interest are labeled with florescent proteins, either FRET donors or FRET acceptors. Since plasma membranes of cells have complex topologies precluding the acquisition of two-dimensional binding curves, the FRET measurements are performed in plasma membrane derived vesicles that bud off cells as a result of chemical or osmotic stress. The results overviewed here are acquired in vesicles produced with an osmotic vesiculation buffer developed in our laboratory, which does not utilize harsh chemicals. The concentrations of the donor-labeled and the acceptor-labeled proteins are determined, along with the FRET efficiencies, in each vesicle. The experiments utilize transient transfection, such that a wide variety of concentrations is sampled. Then, data from hundreds of vesicles are combined to yield dimerization curves. Here we discuss recent findings about the dimerization of receptor tyrosine kinases (RTKs), membrane proteins that control cell growth and differentiation via lateral dimerization in the plasma membrane. We focus on the dimerization of fibroblast growth factor receptor 3 (FGFR3), a RTK that plays a critically important role in skeletal development. We study the role of different FGFR3 domains in FGFR3 dimerization in the absence of ligand, and we show that FGFR3 extracellular domains inhibit unliganded dimerization, while contacts between the juxtamembrane domains, which connect the transmembrane domains to the kinase domains, stabilize the unliganded FGFR3 dimers. Since FGFR3 has been documented to harbor many pathogenic single amino acid mutations that cause skeletal and cranial dysplasias, as well as cancer, we also study the effects of these mutations on dimerization. First, we show that the A391E mutation, linked to Crouzon syndrome with acanthosis nigricans and to bladder cancer, significantly enhances FGFR3 dimerization in the absence of ligand and thus induces aberrant receptor interactions. Second, we present results about the effect of three cysteine mutations that cause thanatophoric dysplasia, a lethal phenotype. Such cysteine mutations have been hypothesized previously to cause constitutive dimerization, but we find instead that they have a surprisingly modest effect on dimerization. Most of the studied pathogenic mutations also altered FGFR3 dimer structure, suggesting that both increases in dimerization propensities and changes in dimer structure contribute to the pathological phenotypes. The results acquired with the QI-FRET method further our understanding of the interactions between FGFR3 molecules and RTK molecules in general. Since RTK dimerization regulates RTK signaling, our findings advance our knowledge of RTK activity in health and disease. The utility of the QI-FRET method is not restricted to RTKs, and we thus hope that in the future the QI-FRET method will be applied to other classes of membrane proteins, such as channels and G protein-coupled receptors.

Novel Isochroman Dimers from Stachybotrys sp. PH30583: Fermentation, Isolation, Structural Elucidation and Biological Activities.

PubMed

Li, Wei; Yang, Ya-Bin; Yang, Xue-Qiong; Xie, Hui-Ding; Shao, Zhi-Hui; Zhou, Hao; Miao, Cui-Ping; Zhao, Li-Xing; Ding, Zhong-Tao

2017-05-01

The rare anishidiol and five new isochromans, including three novel dimers with unprecedented skeletons, were isolated from Stachybotrys sp. PH30583. Their structures were determined by spectral analyses. The bioactivities of these compounds were also investigated. The dimers ( 6 - 10 ) inhibited acetylcholinesterase at 50 µM, but the monomers did not. To investigate the biogenesis of the novel dimers, a time-course investigation of metabolite production was undertaken. Georg Thieme Verlag KG Stuttgart · New York.

Photochemical dimerization and functionalization of alkanes, ethers, primary alcohols and silanes

DOEpatents

Crabtree, Robert H.; Brown, Stephen H.

1988-01-01

The space-time yield and/or the selectivity of the photochemical dimerization of alkanes, ethers, primary alcohols and tertiary silanes with Hg and U.V. light is enhanced by refluxing the substrate in the irradiated reaction zone at a temperature at which the dimer product condenses and remains condensed promptly upon its formation. Cross-dimerization of the alkanes, ethers and silanes with primary alcohols is disclosed, as is the functionalization to aldehydes of the alkanes with carbon monoxide.

Photochemical dimerization and functionalization of alkanes, ethers, primary alcohols and silanes

DOEpatents

Crabtree, R.H.; Brown, S.H.

1988-02-16

The space-time yield and/or the selectivity of the photochemical dimerization of alkanes, ethers, primary alcohols and tertiary silanes with Hg and U.V. light is enhanced by refluxing the substrate in the irradiated reaction zone at a temperature at which the dimer product condenses and remains condensed promptly upon its formation. Cross-dimerization of the alkanes, ethers and silanes with primary alcohols is disclosed, as is the functionalization to aldehydes of the alkanes with carbon monoxide.

Supramolecular organization of cytochrome c oxidase- and alternative oxidase-dependent respiratory chains in the filamentous fungus Podospora anserina.

PubMed

Krause, Frank; Scheckhuber, Christian Q; Werner, Alexandra; Rexroth, Sascha; Reifschneider, Nicole H; Dencher, Norbert A; Osiewacz, Heinz D

2004-06-18

To elucidate the molecular basis of the link between respiration and longevity, we have studied the organization of the respiratory chain of a wild-type strain and of two long-lived mutants of the filamentous fungus Podospora anserina. This established aging model is able to respire by either the standard or the alternative pathway. In the latter pathway, electrons are directly transferred from ubiquinol to the alternative oxidase and thus bypass complexes III and IV. We show that the cytochrome c oxidase pathway is organized according to the mammalian "respirasome" model (Schägger, H., and Pfeiffer, K. (2000) EMBO J. 19, 1777-1783). In contrast, the alternative pathway is composed of distinct supercomplexes of complexes I and III (i.e. I(2) and I(2)III(2)), which have not been described so far. Enzymatic analysis reveals distinct functional properties of complexes I and III belonging to either cytochrome c oxidase- or alternative oxidase-dependent pathways. By a gentle colorless-native PAGE, almost all of the ATP synthases from mitochondria respiring by either pathway were preserved in the dimeric state. Our data are of significance for the understanding of both respiratory pathways as well as lifespan control and aging.

Hemolysis in sickle cell mice causes pulmonary hypertension due to global impairment in nitric oxide bioavailability

PubMed Central

Champion, Hunter C.; Campbell-Lee, Sally A.; Bivalacqua, Trinity J.; Manci, Elizabeth A.; Diwan, Bhalchandra A.; Schimel, Daniel M.; Cochard, Audrey E.; Wang, Xunde; Schechter, Alan N.; Noguchi, Constance T.; Gladwin, Mark T.

2007-01-01

Pulmonary hypertension is a highly prevalent complication of sickle cell disease and is a strong risk factor for early mortality. However, the pathophysiologic mechanisms leading to pulmonary vasculopathy remain unclear. Transgenic mice provide opportunities for mechanistic studies of vascular pathophysiology in an animal model. By microcardiac catheterization, all mice expressing exclusively human sickle hemoglobin had pulmonary hypertension, profound pulmonary and systemic endothelial dysfunction, and vascular instability characterized by diminished responses to authentic nitric oxide (NO), NO donors, and endothelium-dependent vasodilators and enhanced responses to vasoconstrictors. However, endothelium-independent vasodilation in sickle mice was normal. Mechanisms of vasculopathy in sickle mice involve global dysregulation of the NO axis: impaired constitutive nitric oxide synthase activity (NOS) with loss of endothelial NOS (eNOS) dimerization, increased NO scavenging by plasma hemoglobin and superoxide, increased arginase activity, and depleted intravascular nitrite reserves. Light microscopy and computed tomography revealed no plexogenic arterial remodeling or thrombi/emboli. Transplanting sickle marrow into wild-type mice conferred the same phenotype, and similar pathobiology was observed in a nonsickle mouse model of acute alloimmune hemolysis. Although the time course is shorter than typical pulmonary hypertension in human sickle cell disease, these results demonstrate that hemolytic anemia is sufficient to produce endothelial dysfunction and global dysregulation of NO. PMID:17158223

Flavonoids of cocoa inhibit recombinant human 5-lipoxygenase.

PubMed

Schewe, Tankred; Kühn, Hartmut; Sies, Helmut

2002-07-01

(-)-Epicatechin and its related oligomers, the procyanidins, are present in sizable amounts in some cocoas and chocolates. Intake of flavonoid-rich chocolate in humans has been reported to increase the plasma level of (-)-epicatechin and concomitantly to significantly decrease the plasma level of proinflammatory cysteinyl leukotrienes. Because leukotrienes are formed via the 5-lipoxygenase pathway of arachidonic acid metabolism, we examined whether 5-lipoxygenase is a possible target for the flavonoids of cocoa. Recombinant human 5-lipoxygenase was reacted with arachidonic acid and yielded a mixture of mainly 5-hydroperoxy-6E,8Z, 11Z,14Z-eicosatetraenoic acid (5-HpETE) and hydrolysis products of 5,6-leukotriene A(4) (LTA(4)). The formation of these products was significantly inhibited by (-)-epicatechin in a dose-dependent manner with 50% inhibitory concentrations (IC(50)) of 22 and 50 micromol/L, respectively. Among the procyanidin fractions isolated from the seeds of Theobroma cacao, only the dimer fraction and, to a lesser extent, the trimer through pentamer fractions exhibited comparable effects, whereas the larger procyanidins (hexamer through nonamer) were almost inactive. We conclude that (-)-epicatechin and its low-molecular procyanidins inhibit both dioxygenase and LTA(4) synthase activities of human 5-lipoxygenase and that this action may contribute to a putative anti-inflammatory effect of cocoa products.

Chemical Excitation of Electrons: A Dark Path to Melanoma

PubMed Central

Premi, Sanjay; Brash, Douglas E.

2016-01-01

Sunlight’s ultraviolet wavelengths induce cyclobutane pyrimidine dimers (CPDs), which then cause mutations that lead to melanoma or to cancers of skin keratinocytes. In pigmented melanocytes, we found that CPDs arise both instantaneously and for hours after UV exposure ends. Remarkably, the CPDs arising in the dark originate by a novel pathway that resembles bioluminescence but does not end in light: First, UV activates the enzymes nitric oxide synthase (NOS) and NADPH oxidase (NOX), which generate the radicals nitric oxide (NO•) and superoxide (O2•−); these combine to form the powerful oxidant peroxynitrite (ONOO−). A fragment of the skin pigment melanin is then oxidized, exciting an electron to an energy level so high that it is rarely seen in biology. This process of chemically exciting electrons, termed “chemiexcitation”, is used by fireflies to generate light but it had never been seen in mammalian cells. In melanocytes, the energy transfers radiationlessly to DNA, inducing CPDs. Chemiexcitation is a new source of genome instability, and it calls attention to endogenous mechanisms of genome maintenance that prevent electronic excitation or dissipate the energy of excited states. Chemiexcitation may also trigger pathogenesis in internal tissues because the same chemistry should arise wherever superoxide and nitric oxide arise near cells that contain melanin. PMID:27262612

Chemical excitation of electrons: A dark path to melanoma.

PubMed

Premi, Sanjay; Brash, Douglas E

2016-08-01

Sunlight's ultraviolet wavelengths induce cyclobutane pyrimidine dimers (CPDs), which then cause mutations that lead to melanoma or to cancers of skin keratinocytes. In pigmented melanocytes, we found that CPDs arise both instantaneously and for hours after UV exposure ends. Remarkably, the CPDs arising in the dark originate by a novel pathway that resembles bioluminescence but does not end in light: First, UV activates the enzymes nitric oxide synthase (NOS) and NADPH oxidase (NOX), which generate the radicals nitric oxide (NO) and superoxide (O2(-)); these combine to form the powerful oxidant peroxynitrite (ONOO(-)). A fragment of the skin pigment melanin is then oxidized, exciting an electron to an energy level so high that it is rarely seen in biology. This process of chemically exciting electrons, termed "chemiexcitation", is used by fireflies to generate light but it had never been seen in mammalian cells. In melanocytes, the energy transfers radiationlessly to DNA, inducing CPDs. Chemiexcitation is a new source of genome instability, and it calls attention to endogenous mechanisms of genome maintenance that prevent electronic excitation or dissipate the energy of excited states. Chemiexcitation may also trigger pathogenesis in internal tissues because the same chemistry should arise wherever superoxide and nitric oxide arise near cells that contain melanin. Copyright © 2016 Elsevier B.V. All rights reserved.

Replica Exchange Molecular Dynamics Study of Dimerization in Prion Protein: Multiple Modes of Interaction and Stabilization.

PubMed

Chamachi, Neharika G; Chakrabarty, Suman

2016-08-04

The pathological forms of prions are known to be a result of misfolding, oligomerization, and aggregation of the cellular prion. While the mechanism of misfolding and aggregation in prions has been widely studied using both experimental and computational tools, the structural and energetic characterization of the dimer form have not garnered as much attention. On one hand dimerization can be the first step toward a nucleation-like pathway to aggregation, whereas on the other hand it may also increase the conformational stability preventing self-aggregation. In this work, we have used extensive all-atom replica exchange molecular dynamics simulations of both monomer and dimer forms of a mouse prion protein to understand the structural, dynamic, and thermodynamic stability of dimeric prion as compared to the monomeric form. We show that prion proteins can dimerize spontaneously being stabilized by hydrophobic interactions as well as intermolecular hydrogen bonding and salt bridge formation. We have computed the conformational free energy landscapes for both monomer and dimer forms to compare the thermodynamic stability and misfolding pathways. We observe large conformational heterogeneity among the various modes of interactions between the monomers and the strong intermolecular interactions may lead to as high as 20% β-content. The hydrophobic regions in helix-2, surrounding coil regions, terminal regions along with the natively present β-sheet region appear to actively participate in prion-prion intermolecular interactions. Dimerization seems to considerably suppress the inherent dynamic instability observed in monomeric prions, particularly because the regions of structural frustration constitute the dimer interface. Further, we demonstrate an interesting reversible coupling between the Q160-G131 interaction (which leads to inhibition of β-sheet extension) and the G131-V161 H-bond formation.

Repair of cyclobutyl pyrimidine dimers in human skin: variability among normal humans in nucleotide excision and in photorepair.

PubMed

Sutherland, Betsy M; Hacham, Haim; Bennett, Paula; Sutherland, John C; Moran, Michael; Gange, R W

2002-06-01

Photoreactivation (PR) of cyclobutyl pyrimidine dimers (CPD) in human skin remains controversial. Recently Whitmore et al. (1) reported negative results of experiments using two photorepair light (PRL) sources on UV-irradiated skin of volunteers. However, their PRL sources induced substantial levels of dimers in skin, suggesting that the additional dimers formed could have obscured PR. We met a similar problem of dimer induction by a PRL source. We designed and validated a PRL source of sufficient intensity to catalyse PR, but that did not induce CPD, and used it to measure photorepair in human skin. Using a solar simulator filtered with three types of UV-filters, we found significant dimer formation in skin, quantified by number average length analysis using electrophoretic gels of isolated skin DNA. To prevent scattered UV from reaching the skin, we interposed shields between the filters and skin, and showed that the UV-filtered/shielded solar simulator system did not induce damage in isolated DNA or in human skin. We exposed skin of seven healthy human volunteers to 302 nm radiation, then to the improved PRL source (control skin areas were kept in the dark for measurement of excision repair). Using a high intensity PRL source that did not induce dimers in skin, we found that three of seven subjects carried out rapid photorepair of dimers; two carried out moderate or slow dimer photorepair, and three did not show detectable photorepair. Excision repair was similarly variable in these volunteers. Subjects with slower excision repair showed rapid photorepair, whereas those with rapid excision generally showed little or no photoreactivation.

Heat Capacity Changes and Disorder-to-Order Transitions in Allosteric Activation.

PubMed

Cressman, William J; Beckett, Dorothy

2016-01-19

Allosteric coupling in proteins is ubiquitous but incompletely understood, particularly in systems characterized by coupling over large distances. Binding of the allosteric effector, bio-5'-AMP, to the Escherichia coli biotin protein ligase, BirA, enhances the protein's dimerization free energy by -4 kcal/mol. Previous studies revealed that disorder-to-order transitions at the effector binding and dimerization sites, which are separated by 33 Å, are integral to functional coupling. Perturbations to the transition at the ligand binding site alter both ligand binding and coupled dimerization. Alanine substitutions in four loops on the dimerization surface yield a range of energetic effects on dimerization. A glycine to alanine substitution at position 142 in one of these loops results in a complete loss of allosteric coupling, disruption of the disorder-to-order transitions at both functional sites, and a decreased affinity for the effector. In this work, allosteric communication between the effector binding and dimerization surfaces in BirA was further investigated by performing isothermal titration calorimetry measurements on nine proteins with alanine substitutions in three dimerization surface loops. In contrast to BirAG142A, at 20 °C all variants bind to bio-5'-AMP with free energies indistinguishable from that measured for wild-type BirA. However, the majority of the variants exhibit altered heat capacity changes for effector binding. Moreover, the ΔCp values correlate with the dimerization free energies of the effector-bound proteins. These thermodynamic results, combined with structural information, indicate that allosteric activation of the BirA monomer involves formation of a network of intramolecular interactions on the dimerization surface in response to bio-5'-AMP binding at the distant effector binding site.

Human glucose-6-phosphate dehydrogenase: the crystal structure reveals a structural NADP(+) molecule and provides insights into enzyme deficiency.

PubMed

Au, S W; Gover, S; Lam, V M; Adams, M J

2000-03-15

Glucose-6-phosphate dehydrogenase (G6PD) catalyses the first committed step in the pentose phosphate pathway; the generation of NADPH by this enzyme is essential for protection against oxidative stress. The human enzyme is in a dimer<-->tetramer equilibrium and its stability is dependent on NADP(+) concentration. G6PD deficiency results from many different point mutations in the X-linked gene encoding G6PD and is the most common human enzymopathy. Severe deficiency causes chronic non-spherocytic haemolytic anaemia; the usual symptoms are neonatal jaundice, favism and haemolytic anaemia. We have determined the first crystal structure of a human G6PD (the mutant Canton, Arg459-->Leu) at 3 A resolution. The tetramer is a dimer of dimers. Despite very similar dimer topology, there are two major differences from G6PD of Leuconostoc mesenteroides: a structural NADP(+) molecule, close to the dimer interface but integral to the subunit, is visible in all subunits of the human enzyme; and an intrasubunit disulphide bond tethers the otherwise disordered N-terminal segment. The few dimer-dimer contacts making the tetramer are charge-charge interactions. The importance of NADP(+) for stability is explained by the structural NADP(+) site, which is not conserved in prokaryotes. The structure shows that point mutations causing severe deficiency predominate close to the structural NADP(+) and the dimer interface, primarily affecting the stability of the molecule. They also indicate that a stable dimer is essential to retain activity in vivo. As there is an absolute requirement for some G6PD activity, residues essential for coenzyme or substrate binding are rarely modified.

N-glycosylation of the β2 adrenergic receptor regulates receptor function by modulating dimerization.

PubMed

Li, Xiaona; Zhou, Mang; Huang, Wei; Yang, Huaiyu

2017-07-01

N-glycosylation is a common post-translational modification of G-protein-coupled receptors (GPCRs). However, it remains unknown how N-glycosylation affects GPCR signaling. β 2 adrenergic receptor (β 2 AR) has three N-glycosylation sites: Asn6, Asn15 at the N-terminus, and Asn187 at the second extracellular loop (ECL2). Here, we show that deletion of the N-glycan did not affect receptor expression and ligand binding. Deletion of the N-glycan at the N-terminus rather than Asn187 showed decreased effects on isoproterenol-promoted G-protein-dependent signaling, β-arrestin2 recruitment, and receptor internalization. Both N6Q and N15Q showed decreased receptor dimerization, while N187Q did not influence receptor dimerization. As decreased β 2 AR homodimer accompanied with reduced efficiency for receptor function, we proposed that the N-glycosylation of β 2 AR regulated receptor function by influencing receptor dimerization. To verify this hypothesis, we further paid attention to the residues at the dimerization interface. Studies of Lys60 and Glu338, two residues at the receptor dimerization interface, exhibited that the K60A/E338A showed decreased β 2 AR dimerization and its effects on receptor signaling were similar to N6Q and N15Q, which further supported the importance of receptor dimerization for receptor function. This work provides new insights into the relationship among glycosylation, dimerization, and function of GPCRs. Peptide-N-glycosidase F (PNGase F, EC 3.2.2.11); endo-β-N-acetylglucosaminidase A (Endo-A, EC 3.2.1.96). © 2017 Federation of European Biochemical Societies.

In vitro resolution of the dimer bridge of the minute virus of mice (MVM) genome supports the modified rolling hairpin model for MVM replication.

PubMed

Liu, Q; Yong, C B; Astell, C R

1994-06-01

Previous characterization of the terminal sequences of the minute virus of mice (MVM) genome demonstrated that the right hand palindrome contains two sequences, each the inverted complement of the other. However, the left hand palindrome was shown to exist as a unique sequence [Astell et al., J. Virol. 54: 179-185 (1985)]. The modified rolling hairpin (MRH) model for MVM replication provided an explanation of how the right hand palindrome could undergo hairpin transfer to generate two sequences, while the left end palindrome within the dimer bridge could undergo asymmetric resolution and retain the unique left end sequence. This report describes in vitro resolution of the wild-type dimer bridge sequence of MVM using recombinant (baculovirus) expressed NS-1 and a replication extract from LA9 cells. The resolution products are consistent with those predicted by the MRH model, providing support for this replication mechanism. In addition, mutant dimer bridge clones were constructed and used in the resolution assay. The mutant structures included removal of the asymmetry in the hairpin stem, inversion of the sequence at the initiating nick site, and a 2-bp deletion within one stem of the dimer bridge. In all cases, the mutant dimer bridge structures are resolved; however, the resolution pattern observed with the mutant dimer bridge compared with the wild-type dimer bridge is shifted toward symmetrical resolution. These results suggest that sequences within the left hand hairpin (and hence dimer bridge sequence) are responsible for asymmetric resolution and conservation of the unique sequence within the left hand palindrome of the MVM genome.

Brief Report: Racial Comparison of D-Dimer Levels in US Male Military Personnel Before and After HIV Infection and Viral Suppression.

PubMed

OʼBryan, Thomas A; Agan, Brian K; Tracy, Russell P; Freiberg, Matthew S; Okulicz, Jason F; So-Armah, Kaku; Ganesan, Anuradha; Rimland, David; Lalani, Tahaniyat; Deiss, Robert G; Tramont, Edmund C

2018-04-15

D-dimer blood levels in persons with HIV infection are associated with risk of serious non-AIDS conditions and death. Black race has been correlated with higher D-dimer levels in several studies. We examined the effects of race and HIV on D-dimer over time and the impact of viral load suppression by longitudinally comparing changes in levels among healthy young adult male African Americans and whites before HIV seroconversion and before and after initiation of antiretroviral therapy (ART). We analyzed D-dimer levels and clinical and laboratory data of 192 participants enrolled in the US Military HIV Natural History Study, a 30-year cohort of military personnel infected with HIV. D-dimer levels were measured on stored sera from each participant at 3 time points: (1) before HIV seroconversion (Pre-SC), (2) ≥6 months after HIV seroconversion but before ART initiation (Post-SC), and (3) ≥6 months after ART with documented viral suppression (Post-ART). Levels were compared at each time point using nonparametric and logistic regression analysis. Compared with whites (n = 106), African Americans (n = 86) had higher D-dimer levels post-SC (P = 0.007), but in the same individuals, pre-SC baseline and post-ART levels were similar (P = 0.40 and P = 0.99, respectively). There were no racial differences in CD4 cell counts, HIV RNA viral load, time from estimated seroconversion to ART initiation, and duration on ART. Observed longitudinally, racial differences in D-dimer levels were seen only during HIV viremia. Higher levels of D-dimer commonly observed in African Americans are likely due to factors in addition to race.

Age-adjusted versus clinical probability-adjusted D-dimer to exclude pulmonary embolism.

PubMed

Takach Lapner, Sarah; Stevens, Scott M; Woller, Scott C; Snow, Gregory; Kearon, Clive

2018-05-05

A low D-dimer can exclude suspected pulmonary embolism (PE) in cases with low or intermediate clinical probability of disease. Yet D-dimer is nonspecific, so many cases without PE require imaging. D-dimer's specificity is improved by increasing the threshold for a positive test with age (age × 10 ng/mL; age-adjusted D-dimer; AADD) or clinical probability of PE (1000 ng/mL if low and 500 ng/mL if intermediate clinical probability; clinical probability-adjusted D-dimer; CPADD). It is unclear which approach is preferable. We report the sensitivity, specificity and negative predictive value (NPV) of AADD compared to CPADD in suspected PE. A retrospective cohort of 3500 consecutive cases imaged for suspected PE at two U.S. emergency departments was assembled. We analyzed cases with low or intermediate clinical probability of PE (Revised Geneva Score) who had a D-dimer. The outcome was acute PE on imaging at presentation. Of the 3500 cases, 1745 were eligible. 37% were low, and 63% were intermediate clinical probability of PE. PE was present in 145 (8.3%) cases. Sensitivity of CPADD was 87.5% vs. 96.6% for AADD (difference 9.1%; 95% CI 4.3% to 14.0%). NPV of CPADD was 97.1% vs. 99.0% for AADD (difference 1.9%; 95% CI, 0.7% to 3.1%). Specificity of CPADD was 37.5% vs. 30.2% for AADD (difference -7.3%; 95% CI -9.4% to -5.1%). D-dimer was negative in 35.4% of cases using CPADD vs. 28.0% using AADD. CPADD modestly improved the specificity of D-dimer, but had a lower NPV than AADD. AADD appears preferable in this analysis. Copyright © 2018 Elsevier Ltd. All rights reserved.

The nature of the [TTF]˙+···[TTF]˙+ interactions in the [TTF]2(2+) dimers embedded in charged [3]catenanes: room-temperature multicenter long bonds.

PubMed

Capdevila-Cortada, Marçal; Novoa, Juan J

2012-04-23

The properties of tetrathiafulvalene dimers ([TTF](2)(2+)) and the functionalized ring-shaped bispropargyl (BPP)-functionalized TTF dimers, [BPP-TTF](2)(2+), found at room temperature in charged [3]catenanes, were evaluated by M06L calculations. The results showed that their isolated [TTF](2)(2+) and [BPP-TTF](2)(2+) dimers are energetically unstable towards dissociation. When enclosed in the 4(+)-charged central cyclophane ring of charged [3]catenanes (CBPQT(4+)), [TTF](2)(2+) and [BPP-TTF](2)(2+) dimers are also energetically unstable with respect to leaving the CBPQT(4+) ring; since the barrier for the exiting process is only about 3 kcal mol(-1), that is, within the reach of thermal energies at room temperature (neutral [TTF](2)(0) dimers are stable within the CBPQT(4+) ring). However, the [BPP-TTF](2)(2+) dimers in charged [3]catenanes cannot exit, because this would imply breaking the covalent bonds of the BPP-TTF(+) macrocycle. Finally, it was shown that the [TTF](2)(2+), [BPP-TTF](2)(2+) dimers, and charged [3]catenanes are energetically stable in solution and in crystals of their salts, in the first case due to the interactions with the solvent, and in the second case mostly due to cation-anion interactions. In these environmental conditions at room temperature the TTF units of the [BPP-TTF](2)(2+) dimers make short contacts, thus allowing their SOMO orbitals to overlap: a room-temperature multicenter long bond is formed, similar to those previously found in other [TTF](2)(2+) salts and their solutions. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tea Catechin Auto-oxidation Dimers are Accumulated and Retained by Caco-2 Human Intestinal Cells

PubMed Central

Neilson, Andrew P.; Song, Brian J.; Sapper, Teryn N.; Bomser, Joshua A.; Ferruzzi, Mario G.

2010-01-01

Despite the presence of bioactive catechin B-ring auto-oxidation dimers in tea, little is known regarding their absorption in humans. Our hypothesis for this research is that catechin auto-oxidation dimers are present in teas and are absorbable by human intestinal epithelial cells. Dimers [theasinensins (THSNs) and P-2 analogs) were quantified in commercial teas by HPLC-MS. (−)-Epigallocatechin (EGC) and (−)-epigallocatechin gallate (EGCG) homodimers were present at 10–43 and 0–62 µmol/g leaf, respectively. EGC-EGCG heterodimers were present at 0–79 µmol/g. The potential intestinal absorption of these dimers was assessed using Caco-2 intestinal cells. Catechin monomers and dimers were detected in cells exposed to media containing monomers and preformed dimers. Accumulation of dimers was significantly greater than monomers from test media. Three h accumulation of EGC and EGCG was 0.19– 0.55% and 1.24–1.35% respectively. Comparatively, 3h accumulation of the EGC P-2 analog, and THSNs C/E was 0.89 ± 0.28% and 1.53 ± 0.36%. Accumulation of P-2, and THSNs A/D was 6.93 ± 2.1%, and 10.1 ± 3.6%. EGCG-EGC heterodimer P-2 analog, and THSN B 3h accumulation was 4.87 ± 2.2%, and 4.65 ± 2.8% respectively. One h retention of P-2, and THSNs A/D was 171 ± 22%, and 29.6 ± 9.3% of accumulated amount suggesting intracellular oxidative conversion of THSNs to P-2. These data suggest that catechin dimers present in the gut lumen may be readily absorbed by intestinal epithelium. PMID:20579525

Dynamics and asymmetry in the dimer of the norovirus major capsid protein.

PubMed

Tubiana, Thibault; Boulard, Yves; Bressanelli, Stéphane

2017-01-01

Noroviruses are the major cause of non-bacterial acute gastroenteritis in humans and livestock worldwide, despite being physically among the simplest animal viruses. The icosahedral capsid encasing the norovirus RNA genome is made of 90 dimers of a single ca 60-kDa polypeptide chain, VP1, arranged with T = 3 icosahedral symmetry. Here we study the conformational dynamics of this main building block of the norovirus capsid. We use molecular modeling and all-atom molecular dynamics simulations of the VP1 dimer for two genogroups with 50% sequence identity. We focus on the two points of flexibility in VP1 known from the crystal structure of the genogroup I (GI, human) capsid and from subsequent cryo-electron microscopy work on the GII capsid (also human). First, with a homology model of the GIII (bovine) VP1 dimer subjected to simulated annealing then classical molecular dynamics simulations, we show that the N-terminal arm conformation seen in the GI crystal structure is also favored in GIII VP1 but depends on the protonation state of critical residues. Second, simulations of the GI dimer show that the VP1 spike domain will not keep the position found in the GII electron microscopy work. Our main finding is a consistent propensity of the VP1 dimer to assume prominently asymmetric conformations. In order to probe this result, we obtain new SAXS data on GI VP1 dimers. These data are not interpretable as a population of symmetric dimers, but readily modeled by a highly asymmetric dimer. We go on to discuss possible implications of spontaneously asymmetric conformations in the successive steps of norovirus capsid assembly. Our work brings new lights on the surprising conformational range encoded in the norovirus major capsid protein.

Molecular interactions investigated with DFT calculations of QTAIM and NBO analyses: An application to dimeric structures of rice α-amylase/subtilisin inhibitor

NASA Astrophysics Data System (ADS)

Astani, Elahe K.; Hadipour, Nasser L.; Chen, Chun-Jung

2017-03-01

Characterization of the dimer interactions at the dimeric interface of the crystal structure of rice α-amylase/subtilisin inhibitor (RASI) were performed using the quantum theory of atoms in molecules (QTAIM) and natural bonding orbital (NBO) analyses at the density-functional theory (DFT) level. The results revealed that Gly27 and Arg151 of chain A are the main residues involved in hydrogen bonds, dipole-dipole, and charge-dipole interactions with Gly64, Ala66, Ala67 and Arg81 of chain B at the dimeric interface. Calcium ion of chain A plays the significant role in the stability of the dimeric structure through a strong charge-charge interaction with Ala66.

Relative stabilities and the spectral signatures of stacked and hydrogen-bonded dimers of serotonin

NASA Astrophysics Data System (ADS)

Dev, S.; Giri, K.; Majumder, M.; Sathyamurthy, N.

2015-10-01

The O-HṡṡṡN hydrogen-bonded dimer of serotonin is shown to be more stable than the stacked dimer in its ground electronic state, by using the Møller-Plesset second-order perturbation theory (MP2) and the 6-31g** basis set. The vertical excitation energy for the lowest π → π* transition for the monomer as well as the dimer is predicted by time-dependent density functional theory. The experimentally observed red shift of excitation wavelength on oligomerisation is explained in terms of the change in the HOMO-LUMO energy gap due to complex formation. The impact of dimer formation on the proton magnetic resonance spectrum of serotonin monomer is also examined.

Infrared spectra of C2H4 dimer and trimer

NASA Astrophysics Data System (ADS)

Barclay, A. J.; Esteki, K.; McKellar, A. R. W.; Moazzen-Ahmadi, N.

2018-05-01

Spectra of ethylene dimers and trimers are studied in the ν11 and (for the dimer) ν9 fundamental band regions of C2H4 (≈2990 and 3100 cm-1) using a tunable optical parametric oscillator source to probe a pulsed supersonic slit jet expansion. The deuterated trimer has been observed previously, but this represents the first rotationally resolved spectrum of (C2H4)3. The results support the previously determined cross-shaped (D2d) dimer and barrel-shaped (C3h or C3) trimer structures. However, the dimer spectrum in the ν9 fundamental region of C2H4 is apparently very perturbed and a previous rotational analysis is not well verified.

Detecting specific cytotoxic T lymphocytes against SARS-coronavirus with DimerX HLA-A2:Ig fusion protein.

PubMed

Wang, Yue-Dan; Chen, Wei Feng

2004-11-01

To assess specific cytotoxic T lymphocytes (CTLs) against Severe acute respiratory syndrome (SARS)-coronavirus, a modified DimerX flow cytometry assay was performed with peripheral blood mononuclear cell (PBMC) from HLA-A2+ SARS-recovered donors at different time points post disease. CD8+DimerX-S1203+ CTLs were detected in the PBMC from these donors up to 3 months after recovery. The percentages of CD8+DimerX-S1203+ cells paralleled the numbers of interferon-gamma-positive spots in an ELISPOT assay using the same antigenic peptide. In conclusion, DimerX-based flow cytometry staining may prove to be a real-time method to screen for CTL directed at epitopes from a newly identified virus.

Caspase enzymology and activation mechanisms.

PubMed

Mace, Peter D; Riedl, Stefan J; Salvesen, Guy S

2014-01-01

Apical caspases 8, 9, and 10 are only active as dimers. These dimers are unstable, and to characterize their activity they need to be maintained in vitro in a dimeric state. We provide updated methods for those looking to characterize various aspects of caspase function. We describe full methods for those looking to activate caspases in vitro using kosmotropic reagents, an essential step in characterizing upstream (apical) caspases. We detail methods for fusion of caspase domains to engineered dimerization domains as an alternative method to trigger regulated dimerization of caspases. We also describe methods to determine caspase activity profiles in cells and provide methods for studying the ability of SMAC-mimetic reagents to release inhibition of caspases by IAPs. © 2014 Elsevier Inc. All rights reserved.

Prediction of the wetting-induced collapse behaviour using the soil-water characteristic curve

NASA Astrophysics Data System (ADS)

Xie, Wan-Li; Li, Ping; Vanapalli, Sai K.; Wang, Jia-Ding

2018-01-01

Collapsible soils go through three distinct phases in response to matric suction decrease during wetting: pre-collapse phase, collapse phase and post-collapse phase. It is reasonable and conservative to consider a strain path that includes a pre-collapse phase in which constant volume is maintained and a collapse phase that extends to the final matric suction to be experienced by collapsible soils during wetting. Upon this assumption, a method is proposed for predicting the collapse behaviour due to wetting. To use the proposed method, two parameters, critical suction and collapse rate, are required. The former is the suction value below which significant collapse deformations take place in response to matric suction decease, and the later is the rate at which void ratio reduces with matric suction in the collapse phase. The value of critical suction can be estimated from the water-entry value taking account of both the microstructure characteristics and collapse mechanism of fine-grained collapsible soils; the wetting soil-water characteristic curve thus can be used as a tool. Five sets of data of wetting tests on both compacted and natural collapsible soils reported in the literature were used to validate the proposed method. The critical suction values were estimated from the water-entry value with parameter a that is suggested to vary between 0.10 and 0.25 for compacted soils and to be lower for natural collapsible soils. The results of a field permeation test in collapsible loess soils were also used to validate the proposed method. The relatively good agreement between the measured and estimated collapse deformations suggests that the proposed method can provide reasonable prediction of the collapse behaviour due to wetting.

DOE Office of Scientific and Technical Information (OSTI.GOV)

R Vasquez-Del Carpio; T Silverstein; S Lone

Exposure of DNA to UV radiation causes covalent linkages between adjacent pyrimidines. The most common lesion found in DNA from these UV-induced linkages is the cis-syn cyclobutane pyrimidine dimer. Human DNA polymerase {Kappa} (Pol{Kappa}), a member of the Y-family of DNA polymerases, is unable to insert nucleotides opposite the 3'T of a cis-syn T-T dimer, but it can efficiently extend from a nucleotide inserted opposite the 3'T of the dimer by another DNA polymerase. We present here the structure of human Pol{Kappa} in the act of inserting a nucleotide opposite the 5'T of the cis-syn T-T dimer. The structure revealsmore » a constrained active-site cleft that is unable to accommodate the 3'T of a cis-syn T-T dimer but is remarkably well adapted to accommodate the 5'T via Watson-Crick base pairing, in accord with a proposed role for Pol{Kappa} in the extension reaction opposite from cyclobutane pyrimidine dimers in vivo.« less

Topological edge states and impurities: Manifestation in the local static and dynamical characteristics of dimerized quantum chains

NASA Astrophysics Data System (ADS)

Zvyagin, A. A.

2018-04-01

Based on the results of exact analytic calculations, we show that topological edge states and impurities in quantum dimerized chains manifest themselves in various local static and dynamical characteristics, which can be measured in experiments. In particular, topological edge states can be observed in the magnetic field behavior of the local magnetization or magnetic susceptibility of dimerized spin chains as jumps (for the magnetization) and features (for the static susceptibility) at zero field. In contrast, impurities reveal themselves in similar jumps and features, however, at nonzero values of the critical field. We also show that dynamical characteristics of dimerized quantum chains also manifest the features, related to the topological edge states and impurities. Those features, as a rule, can be seen more sharply than the manifestation of bulk extended states in, e.g., the dynamical local susceptibility. Such peculiarities can be observed in one-dimensional dimerized spin chains, e.g., in NMR experiments, or in various realizations of quantum dimerized chains in optical experiments.

Improvement of Aptamer Affinity by Dimerization

PubMed Central

Hasegawa, Hijiri; Taira, Ken-ichi; Sode, Koji; Ikebukuro, Kazunori

2008-01-01

To increase the affinities of aptamers for their targets, we designed an aptamer dimer for thrombin and VEGF. This design is based on the avidity of the antibody, which enables the aptamer to connect easily since it is a single-strand nucleic acid. In this study, we connected a 15-mer thrombin-binding aptamer with a 29-mer thrombin-binding aptamer. Each aptamer recognizes a different part of the thrombin molecule, and the aptamer dimer has a Kd value which is 1/10 of that of the monomers from which it is composed. Also, the designed aptamer dimer has higher inhibitory activity than the reported (15-mer) thrombin-inhibiting aptamer. Additionally, we connected together two identical aptamers against vascular endothelial growth factor (VEGF165), which is a homodimeric protein. As in the case of the anti-thrombin aptamer, the dimeric anti-VEGF aptamer had a much lower Kd value than that of the monomer. This study demonstrated that the dimerization of aptamers effectively improves the affinities of those aptamers for their targets. PMID:27879754

Observation of Resonant Effects in Ultracold Collisions between Heteronuclear Feshbach Molecules

NASA Astrophysics Data System (ADS)

Ye, Xin; Wang, Fudong; Zhu, Bing; Guo, Mingyang; Lu, Bo; Wang, Dajun

2016-05-01

Magnetic field dependent dimer-dimer collisional losses are studied with ultracold 23 Na87 Rb Feshbach molecules. By ramping the magnetic field across the 347.8 G inter-species Feshbach resonance and removing residual atoms with a magnetic field gradient, ~ 8000 pure NaRb Feshbach molecules with a temperature below 1 μK are produced. By holding the pure molecule sample in a crossed optical dipole trap and measuring the time-dependent loss curves under different magnetic fields near the Feshbach resonance, the dimer-dimer loss rates with respect to the atomic scattering length a are mapped out. We observe a resonant feature at around a = 600a0 and a rising tail at above a = 1600a0 . This behavior resembles previous theoretical works on homonuclear Feshbach molecule, where resonant effects between dimer-dimer collisions tied to tetramer bound states were predicted. Our work shows the possibility of exploring four-body physics within a heteronuclear system. We are supported by Hong Kong RGC General Research Fund no. CUHK403813.

Competition between inter- and intra-molecular hydrogen bonding: An infrared spectroscopic study of jet-cooled amino-ethanol and its dimer

NASA Astrophysics Data System (ADS)

Asselin, Pierre; Madebène, Bruno; Soulard, Pascale; Georges, Robert; Goubet, Manuel; Huet, Thérèse R.; Pirali, Olivier; Zehnacker-Rentien, Anne

2016-12-01

The Fourier transform IR vibrational spectra of amino-ethanol (AE) and its dimer have been recorded at room temperature and under jet-cooled conditions over the far and mid infrared ranges (50-4000 cm-1) using the White-type cell and the supersonic jet of the Jet-AILES apparatus at the synchrotron facility SOLEIL. Assignment of the monomer experimental frequencies has been derived from anharmonic frequencies calculated at a hybrid CCSD(T)-F12/MP2 level. Various thermodynamical effects in the supersonic expansion conditions including molar dilution of AE and nature of carrier gas have been used to promote or not the formation of dimers. Four vibrational modes of the observed dimer have been unambiguously assigned using mode-specific scaling factors deduced from the ratio between experimental and computed frequencies for the monomer. The most stable g'Gg' monomer undergoes strong deformation upon dimerization, leading to a homochiral head to head dimer involving two strong hydrogen bonds.

Exact Solution of a Two-Species Quantum Dimer Model for Pseudogap Metals

NASA Astrophysics Data System (ADS)

Feldmeier, Johannes; Huber, Sebastian; Punk, Matthias

2018-05-01

We present an exact ground state solution of a quantum dimer model introduced by Punk, Allais, and Sachdev [Quantum dimer model for the pseudogap metal, Proc. Natl. Acad. Sci. U.S.A. 112, 9552 (2015)., 10.1073/pnas.1512206112], which features ordinary bosonic spin-singlet dimers as well as fermionic dimers that can be viewed as bound states of spinons and holons in a hole-doped resonating valence bond liquid. Interestingly, this model captures several essential properties of the metallic pseudogap phase in high-Tc cuprate superconductors. We identify a line in parameter space where the exact ground state wave functions can be constructed at an arbitrary density of fermionic dimers. At this exactly solvable line the ground state has a huge degeneracy, which can be interpreted as a flat band of fermionic excitations. Perturbing around the exactly solvable line, this degeneracy is lifted and the ground state is a fractionalized Fermi liquid with a small pocket Fermi surface in the low doping limit.

Structure of FGFR3 transmembrane domain dimer: implications for signaling and human pathologies.

PubMed

Bocharov, Eduard V; Lesovoy, Dmitry M; Goncharuk, Sergey A; Goncharuk, Marina V; Hristova, Kalina; Arseniev, Alexander S

2013-11-05

Fibroblast growth factor receptor 3 (FGFR3) transduces biochemical signals via lateral dimerization in the plasma membrane, and plays an important role in human development and disease. Eight different pathogenic mutations, implicated in cancers and growth disorders, have been identified in the FGFR3 transmembrane segment. Here, we describe the dimerization of the FGFR3 transmembrane domain in membrane-mimicking DPC/SDS (9/1) micelles. In the solved NMR structure, the two transmembrane helices pack into a symmetric left-handed dimer, with intermolecular stacking interactions occurring in the dimer central region. Some pathogenic mutations fall within the helix-helix interface, whereas others are located within a putative alternative interface. This implies that although the observed dimer structure is important for FGFR3 signaling, the mechanism of FGFR3-mediated transduction across the membrane is complex. We propose an FGFR3 signaling mechanism that is based on the solved structure, available structures of isolated soluble FGFR domains, and published biochemical and biophysical data. Copyright © 2013 Elsevier Ltd. All rights reserved.

Fibrin d-dimer concentration, deep vein thrombosis symptom duration, and venous thrombus volume.

PubMed

Kurklinsky, Andrew K; Kalsi, Henna; Wysokinski, Waldemar E; Mauck, Karen F; Bhagra, Anjali; Havyer, Rachel D; Thompson, Carrie A; Hayes, Sharonne N; McBane, Robert D

2011-04-01

To determine the relationship between fibrin D-dimer levels, symptom duration, and thrombus volume, consecutive patients with incident deep venous thrombosis (DVT) were evaluated. In a cross-sectional study design, patient symptom onset was determined by careful patient questioning. Venous thrombosis was confirmed by compression duplex ultrasonography. Thrombus volume was estimated based on patient's femur length using a forensic anthropology method. Fibrin D-dimer was measured by latex immunoassay. 72 consecutive patients with confirmed leg DVT agreed to participate. The median symptom duration at the time of diagnosis was 10 days. The median D-dimer concentration was 1050 ng/dL. The median thrombus volume was 12.92 cm(3). D-Dimer levels correlated with estimated thrombus volume (P < .0006 CI 0.12-0.41; R(2) (adjusted) = .15) but not symptom duration, patient's age, or gender. Despite varying symptom duration prior to diagnosis, fibrin D-dimer remains a sensitive measure of venous thrombosis and correlates with thrombus volume.

Adhesive Dimerization of Human P-Cadherin Catalyzed by a Chaperone-like Mechanism.

PubMed

Kudo, Shota; Caaveiro, Jose M M; Tsumoto, Kouhei

2016-09-06

Orderly assembly of classical cadherins governs cell adhesion and tissue maintenance. A key event is the strand-swap dimerization of the extracellular ectodomains of two cadherin molecules from apposing cells. Here we have determined crystal structures of P-cadherin in six different conformational states to elaborate a motion picture of its adhesive dimerization at the atomic level. The snapshots revealed that cell-adhesive dimerization is facilitated by several intermediate states collectively termed X-dimer in analogy to other classical cadherins. Based on previous studies and on the combined structural, kinetic, thermodynamic, biochemical, and cellular data reported herein, we propose that the adhesive dimerization of human P-cadherin is achieved by a stepwise mechanism analogous to that of assembly chaperones. This mechanism, applicable to type I classical cadherins, confers high specificity and fast association rates. We expect these findings to guide innovative therapeutic approaches targeting P-cadherin in cancer. Copyright © 2016 Elsevier Ltd. All rights reserved.

Experimental study of transport of a dimer on a vertically oscillating plate

PubMed Central

Wang, Jiao; Liu, Caishan; Ma, Daolin

2014-01-01

It has recently been shown that a dimer, composed of two identical spheres rigidly connected by a rod, under harmonic vertical vibration can exhibit a self-ordered transport behaviour. In this case, the mass centre of the dimer will perform a circular orbit in the horizontal plane, or a straight line if confined between parallel walls. In order to validate the numerical discoveries, we experimentally investigate the temporal evolution of the dimer's motion in both two- and three-dimensional situations. A stereoscopic vision method with a pair of high-speed cameras is adopted to perform omnidirectional measurements. All the cases studied in our experiments are also simulated using an existing numerical model. The combined investigations detail the dimer's dynamics and clearly show that its transport behaviours originate from a series of combinations of different contact states. This series is critical to our understanding of the transport properties in the dimer's motion and related self-ordered phenomena in granular systems. PMID:25383029

Naproxen Interferes with the Assembly of Aβ Oligomers Implicated in Alzheimer's Disease

PubMed Central

Kim, Seongwon; Chang, Wenling E.; Kumar, Rashmi; Klimov, Dmitri K.

2011-01-01

Experimental and epidemiological studies have shown that the nonsteroidal antiinflammatory drug naproxen may be useful in the treatment of Alzheimer's disease. To investigate the interactions of naproxen with Aβ dimers, which are the smallest cytotoxic aggregated Aβ peptide species, we use united atom implicit solvent model and exhaustive replica exchange molecular dynamics. We show that naproxen ligands bind to Aβ dimer and penetrate its volume interfering with the interpeptide interactions. As a result naproxen induces a destabilizing effect on Aβ dimer. By comparing the free-energy landscapes of naproxen interactions with Aβ dimers and fibrils, we conclude that this ligand has stronger antiaggregation potential against Aβ fibrils rather than against dimers. The analysis of naproxen binding energetics shows that the location of ligand binding sites in Aβ dimer is dictated by the Aβ amino acid sequence. Comparison of the in silico findings with experimental observations reveals potential limitations of naproxen as an effective therapeutic agent in the treatment of Alzheimer's disease. PMID:21504739

Chloroperoxidase-catalyzed oxidation of 4,6-dimethyldibenzothiophene as dimer complexes: evidence for kinetic cooperativity.

PubMed

Torres, Eduardo; Aburto, Jorge

2005-05-15

A sigmoidal kinetic behavior of chloroperoxidase for the oxidation of 4,6-dimethyldibenzothiophene (4,6-DMDBT) in water-miscible organic solvent is for the first time reported. Kinetics of 4,6-DMDBT oxidation showed a cooperative profile probably due to the capacity of chloroperoxidase to recognize a substrate dimer (pi-pi dimer) in its active site. Experimental evidence is given for dimer formation and its presence in the active site of chloroperoxidase. The kinetic data were adjusted for a binding site able to interact with either monomer or dimer substrates, producing a cooperative model describing a one-site binding of two related species. Determination of kinetics constants by iterative calculations of possible oxidation paths of 4,6-DMDBT suggests that kinetics oxidation of dimer substrate is preferred when compared to monomer oxidation. Steady-state fluorometry of substrate in the absence and presence of chloroperoxidase, described by the spectral center of mass, supports this last conclusion.

Measuring Membrane Protein Dimerization Equilibrium in Lipid Bilayers by Single-Molecule Fluorescence Microscopy.

PubMed

Chadda, R; Robertson, J L

2016-01-01

Dimerization of membrane protein interfaces occurs during membrane protein folding and cell receptor signaling. Here, we summarize a method that allows for measurement of equilibrium dimerization reactions of membrane proteins in lipid bilayers, by measuring the Poisson distribution of subunit capture into liposomes by single-molecule photobleaching analysis. This strategy is grounded in the fact that given a comparable labeling efficiency, monomeric or dimeric forms of a membrane protein will give rise to distinctly different photobleaching probability distributions. These methods have been used to verify the dimer stoichiometry of the Fluc F - ion channel and the dimerization equilibrium constant of the ClC-ec1 Cl - /H + antiporter in lipid bilayers. This approach can be applied to any membrane protein system provided it can be purified, fluorescently labeled in a quantitative manner, and verified to be correctly folded by functional assays, even if the structure is not yet known. © 2016 Elsevier Inc. All rights reserved.

Haem-dependent dimerization of PGRMC1/Sigma-2 receptor facilitates cancer proliferation and chemoresistance.

PubMed

Kabe, Yasuaki; Nakane, Takanori; Koike, Ikko; Yamamoto, Tatsuya; Sugiura, Yuki; Harada, Erisa; Sugase, Kenji; Shimamura, Tatsuro; Ohmura, Mitsuyo; Muraoka, Kazumi; Yamamoto, Ayumi; Uchida, Takeshi; Iwata, So; Yamaguchi, Yuki; Krayukhina, Elena; Noda, Masanori; Handa, Hiroshi; Ishimori, Koichiro; Uchiyama, Susumu; Kobayashi, Takuya; Suematsu, Makoto

2016-03-18

Progesterone-receptor membrane component 1 (PGRMC1/Sigma-2 receptor) is a haem-containing protein that interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 to regulate cancer proliferation and chemoresistance; its structural basis remains unknown. Here crystallographic analyses of the PGRMC1 cytosolic domain at 1.95 Å resolution reveal that it forms a stable dimer through stacking interactions of two protruding haem molecules. The haem iron is five-coordinated by Tyr113, and the open surface of the haem mediates dimerization. Carbon monoxide (CO) interferes with PGRMC1 dimerization by binding to the sixth coordination site of the haem. Haem-mediated PGRMC1 dimerization is required for interactions with EGFR and cytochromes P450, cancer proliferation and chemoresistance against anti-cancer drugs; these events are attenuated by either CO or haem deprivation in cancer cells. This study demonstrates protein dimerization via haem-haem stacking, which has not been seen in eukaryotes, and provides insights into its functional significance in cancer.

Haem-dependent dimerization of PGRMC1/Sigma-2 receptor facilitates cancer proliferation and chemoresistance

PubMed Central

Kabe, Yasuaki; Nakane, Takanori; Koike, Ikko; Yamamoto, Tatsuya; Sugiura, Yuki; Harada, Erisa; Sugase, Kenji; Shimamura, Tatsuro; Ohmura, Mitsuyo; Muraoka, Kazumi; Yamamoto, Ayumi; Uchida, Takeshi; Iwata, So; Yamaguchi, Yuki; Krayukhina, Elena; Noda, Masanori; Handa, Hiroshi; Ishimori, Koichiro; Uchiyama, Susumu; Kobayashi, Takuya; Suematsu, Makoto

2016-01-01

Progesterone-receptor membrane component 1 (PGRMC1/Sigma-2 receptor) is a haem-containing protein that interacts with epidermal growth factor receptor (EGFR) and cytochromes P450 to regulate cancer proliferation and chemoresistance; its structural basis remains unknown. Here crystallographic analyses of the PGRMC1 cytosolic domain at 1.95 Å resolution reveal that it forms a stable dimer through stacking interactions of two protruding haem molecules. The haem iron is five-coordinated by Tyr113, and the open surface of the haem mediates dimerization. Carbon monoxide (CO) interferes with PGRMC1 dimerization by binding to the sixth coordination site of the haem. Haem-mediated PGRMC1 dimerization is required for interactions with EGFR and cytochromes P450, cancer proliferation and chemoresistance against anti-cancer drugs; these events are attenuated by either CO or haem deprivation in cancer cells. This study demonstrates protein dimerization via haem–haem stacking, which has not been seen in eukaryotes, and provides insights into its functional significance in cancer. PMID:26988023

Water Dimer Concentrations in The Atmosphere

NASA Astrophysics Data System (ADS)

Saykally, R. J.

2000-03-01

The water dimer concentration present in water vapor under equilibrium conditions is rigorously determined as a function of temperature, pressure, and relative humidity via explicit calculations of partition functions on the VRT (ASP-W) potential surface using the SWPS method. Dimer vapor fractions as large as 4.6x10*3 are calculated under tropospheric conditions, and should have observable consequences on chemistry and physical properties of the atmosphere. There has been much recent interest and speculation regarding possible effects of water clusters on the chemistry and radiation balance of the atmosphere. For example, it has been proposed that vibrational overtones of the water dimer absorb solar radiation and account for a significant part of the *anomalous absorption* of the atmosphere, although recent measurements do not support this claim. Similarly, the presence of water dimers has been predicted to accelerate the formation of acid rain, and homogeneous nucleation of raindrops. In all of these contexts, the crucial unknown is the concentration of water dimers present under the specified conditions of temperature, pressure, and relative humidity.

Can Vitamin A be Improved to Prevent Blindness due to Age-Related Macular Degeneration, Stargardt Disease and Other Retinal Dystrophies?

PubMed

Saad, Leonide; Washington, Ilyas

2016-01-01

We discuss how an imperfect visual cycle results in the formation of vitamin A dimers, thought to be involved in the pathogenesis of various retinal diseases, and summarize how slowing vitamin A dimerization has been a therapeutic target of interest to prevent blindness. To elucidate the molecular mechanism of vitamin A dimerization, an alternative form of vitamin A, one that forms dimers more slowly yet maneuvers effortlessly through the visual cycle, was developed. Such a vitamin A, reinforced with deuterium (C20-D3-vitamin A), can be used as a non-disruptive tool to understand the contribution of vitamin A dimers to vision loss. Eventually, C20-D3-vitamin A could become a disease-modifying therapy to slow or stop vision loss associated with dry age-related macular degeneration (AMD), Stargardt disease and retinal diseases marked by such vitamin A dimers. Human clinical trials of C20-D3-vitamin A (ALK-001) are underway.

Comparative study of 64Cu/NOTA-[D-Tyr6,βAla11,Thi13,Nle14]BBN(6-14) monomer and dimers for prostate cancer PET imaging

PubMed Central

2012-01-01

Background Gastrin-releasing peptide receptors [GRPR] are highly over-expressed in multiple cancers and have been studied as a diagnostic target. Multimeric gastrin-releasing peptides are expected to have enhanced tumor uptake and affinity for GRPR. In this study, a 64Cu-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid [NOTA]-monomer and two NOTA-dimers of [D-Tyr6,βAla11, Thi13, Nle14]bombesin(6-14) ] [BBN(6-14)] were compared. Methods Monomeric and dimeric peptides were synthesized on solid phase support and radiolabeled with 64Cu. NOTA-dimer 1 consists of asymmetrically linked BBN(6-14), while NOTA-dimer 2 has similar spacer between the two BBN(6-14) ligands and the chelator. In vitro GRPR-binding affinities were determined with competitive binding assays on PC3 human prostate cancer cells. In vivo stability and biodistribution of radiolabeled compounds were assessed in Balb/c mice. Cellular uptake and efflux were measured with radiolabeled NOTA-monomer and NOTA-dimer 2 on PC3 cells for up to 4 h. In vivo biodistribution kinetics were measured in PC3 tumor-bearing Balb/c nude mice by μ-positron emission tomography [μPET] imaging and confirmed by dissection and counting. Results NOTA-monomer, NOTA-dimers 1 and 2 were prepared with purity of 99%. The inhibition constants of the three BBN peptides were comparable and in the low nanomolar range. All 64Cu-labeled peptides were stable up to 24 h in mouse plasma and 1 h in vivo. 64Cu/NOTA-dimer 2 featuring a longer spacer between the two BBN(6-14) ligands is a more potent GRPR-targeting probe than 64Cu/NOTA-dimer 1. PC3 tumor uptake profiles are slightly different for 64Cu/NOTA-monomer and 64Cu/NOTA-dimer 2; the monomeric BBN-peptide tracer exhibited higher tumor uptake during the first 0.5 h and a fast renal clearance resulting in higher tumor-to-muscle ratio when compared to 64Cu/NOTA-dimer 2. The latter exhibited higher tumor-to-blood ratio and was retained longer at the tumor site when compared to 64Cu/NOTA-monomer. Lower ratios of tumor-to-blood and tumor-to-muscle in blocking experiments showed GRPR-dependant tumor uptake for both tracers. Conclusion Both 64Cu/NOTA-monomer and 64Cu/NOTA-dimer 2 are suitable for detecting GRPR-positive prostate cancer in vivo by PET. Tumor retention was improved in vivo with 64Cu/NOTA-dimer 2 by applying polyvalency effect and/or statistical rebinding. PMID:22333272

Comparative study of 64Cu/NOTA-[D-Tyr6,βAla11,Thi13,Nle14]BBN(6-14) monomer and dimers for prostate cancer PET imaging.

PubMed

Fournier, Patrick; Dumulon-Perreault, Véronique; Ait-Mohand, Samia; Langlois, Réjean; Bénard, François; Lecomte, Roger; Guérin, Brigitte

2012-02-14

Gastrin-releasing peptide receptors [GRPR] are highly over-expressed in multiple cancers and have been studied as a diagnostic target. Multimeric gastrin-releasing peptides are expected to have enhanced tumor uptake and affinity for GRPR. In this study, a 64Cu-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid [NOTA]-monomer and two NOTA-dimers of [D-Tyr6,βAla11, Thi13, Nle14]bombesin(6-14) ] [BBN(6-14)] were compared. Monomeric and dimeric peptides were synthesized on solid phase support and radiolabeled with 64Cu. NOTA-dimer 1 consists of asymmetrically linked BBN(6-14), while NOTA-dimer 2 has similar spacer between the two BBN(6-14) ligands and the chelator. In vitro GRPR-binding affinities were determined with competitive binding assays on PC3 human prostate cancer cells. In vivo stability and biodistribution of radiolabeled compounds were assessed in Balb/c mice. Cellular uptake and efflux were measured with radiolabeled NOTA-monomer and NOTA-dimer 2 on PC3 cells for up to 4 h. In vivo biodistribution kinetics were measured in PC3 tumor-bearing Balb/c nude mice by μ-positron emission tomography [μPET] imaging and confirmed by dissection and counting. NOTA-monomer, NOTA-dimers 1 and 2 were prepared with purity of 99%. The inhibition constants of the three BBN peptides were comparable and in the low nanomolar range. All 64Cu-labeled peptides were stable up to 24 h in mouse plasma and 1 h in vivo. 64Cu/NOTA-dimer 2 featuring a longer spacer between the two BBN(6-14) ligands is a more potent GRPR-targeting probe than 64Cu/NOTA-dimer 1. PC3 tumor uptake profiles are slightly different for 64Cu/NOTA-monomer and 64Cu/NOTA-dimer 2; the monomeric BBN-peptide tracer exhibited higher tumor uptake during the first 0.5 h and a fast renal clearance resulting in higher tumor-to-muscle ratio when compared to 64Cu/NOTA-dimer 2. The latter exhibited higher tumor-to-blood ratio and was retained longer at the tumor site when compared to 64Cu/NOTA-monomer. Lower ratios of tumor-to-blood and tumor-to-muscle in blocking experiments showed GRPR-dependant tumor uptake for both tracers. Both 64Cu/NOTA-monomer and 64Cu/NOTA-dimer 2 are suitable for detecting GRPR-positive prostate cancer in vivo by PET. Tumor retention was improved in vivo with 64Cu/NOTA-dimer 2 by applying polyvalency effect and/or statistical rebinding.

Isolation and bacterial expression of a sesquiterpene synthase CDNA clone from peppermint(mentha .chi. piperita, L.) that produces the aphid alarm pheromone (E)-.beta.-farnesene

DOEpatents

Croteau, Rodney Bruce; Wildung, Mark Raymond; Crock, John E.

1999-01-01

A cDNA encoding (E)-.beta.-farnesene synthase from peppermint (Mentha piperita) has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID NO:1) is provided which codes for the expression of (E)-.beta.-farnesene synthase (SEQ ID NO:2), from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for (E)-.beta.-farnesene synthase, or for a base sequence sufficiently complementary to at least a portion of (E)-.beta.-farnesene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (E)-.beta.-farnesene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant (E)-.beta.-farnesene synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant (E)-.beta.-farnesene synthase may be used to obtain expression or enhanced expression of (E)-.beta.-farnesene synthase in plants in order to enhance the production of (E)-.beta.-farnesene, or may be otherwise employed for the regulation or expression of (E)-.beta.-farnesene synthase, or the production of its product.

Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint (Mentha x piperita, L.) that produces the aphid alarm pheromone (E)-.beta.-farnesene

DOEpatents

Croteau, Rodney Bruce; Crock, John E.

2005-01-25

A cDNA encoding (E)-.beta.-farnesene synthase from peppermint (Mentha piperita) has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID NO:1) is provided which codes for the expression of (E)-.beta.-farnesene synthase (SEQ ID NO:2), from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for (E)-.beta.-farnesene synthase, or for a base sequence sufficiently complementary to at least a portion of (E)-.beta.-farnesene synthase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding (E)-.beta.-farnesene synthase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant (E)-.beta.-famesene synthase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant (E)-.beta.-farnesene synthase may be used to obtain expression or enhanced expression of (E)-.beta.-famesene synthase in plants in order to enhance the production of (E)-.beta.-farnesene, or may be otherwise employed for the regulation or expression of (E)-.beta.-farnesene synthase, or the production of its product.

CTP synthase forms cytoophidia in the cytoplasm and nucleus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gou, Ke-Mian; State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193; Chang, Chia-Chun

2014-04-15

CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthasemore » 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus.« less

Evolution of glutamine amidotransferase genes. Nucleotide sequences of the pabA genes from Salmonella typhimurium, Klebsiella aerogenes and Serratia marcescens.

PubMed

Kaplan, J B; Merkel, W K; Nichols, B P

1985-06-05

The amide group of glutamine is a source of nitrogen in the biosynthesis of a variety of compounds. These reactions are catalyzed by a group of enzymes known as glutamine amidotransferases; two of these, the glutamine amidotransferase subunits of p-aminobenzoate synthase and anthranilate synthase have been studied in detail and have been shown to be structurally and functionally related. In some micro-organisms, p-aminobenzoate synthase and anthranilate synthase share a common glutamine amidotransferase subunit. We report here the primary DNA and deduced amino acid sequences of the p-aminobenzoate synthase glutamine amidotransferase subunits from Salmonella typhimurium, Klebsiella aerogenes and Serratia marcescens. A comparison of these glutamine amidotransferase sequences to the sequences of ten others, including some that function specifically in either the p-aminobenzoate synthase or anthranilate synthase complexes and some that are shared by both synthase complexes, has revealed several interesting features of the structure and organization of these genes, and has allowed us to speculate as to the evolutionary history of this family of enzymes. We propose a model for the evolution of the p-aminobenzoate synthase and anthranilate synthase glutamine amidotransferase subunits in which the duplication and subsequent divergence of the genetic information encoding a shared glutamine amidotransferase subunit led to the evolution of two new pathway-specific enzymes.

Dimerization in Highly Concentrated Solutions of Phosphoimidazolide Activated Mononucleotides

NASA Technical Reports Server (NTRS)

Kanavarioti, Anastassia

1997-01-01

Phosphoimidazolide activated ribomononucleotides (*pN) are useful substrates for the non-enzymatic synthesis of polynucleotides. However, dilute neutral aqueous solutions of *pN typically yield small amounts of dimers and traces of polymers; most of *pN hydrolyzes to yield nucleoside 5'-monophosphate. Here we report the self-condensation of nucleoside 5'-phosphate 2- methylimidazolide (2-MeImpN with N = cytidine, uridine or guanosine) in the presence of Mg2(+) in concentrated solutions, such as might have been found in an evaporating lagoon on prebiotic Earth. The product distribution indicates that oligomerization is favored at the expense of hydrolysis. At 1.0 M, 2-MelmpU and 2-MelmpC produce about 65% of oligomers including 4% of the 3',5'-Iinked dimer. Examination of the product distribution of the three isomeric dimers in a self-condensation allows identification of reaction pathways that lead to dimer formation. Condensations in a concentrated mixture of all three nucleotides (U,C,G mixtures) is made possible by the enhanced solubility of 2-MeImpG in such mixtures. Although percent yield of intemucleotide linked dimers is enhanced as a function of initial monomer concentration, pyrophosphate dimer yields remain practically unchanged at about 20% for 2-MelmpU, 16% for 2-MeImpC and 25% of the total pyrophosphate in the U,C,G mixtures. The efficiency by which oligomers are produced in these concentrated solutions makes the evaporating lagoon scenario a potentially interesting medium for the prebiotic synthesis of dimers and short RNAs.

An Alternative Mechanism for the Dimerization of Formic Acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brinkman, Nicole R.; Tschumper, Gregory; Yan, Ge

Gas-phase formic acid exists primarily as a cyclic dimer. The mechanism of dimerization has been traditionally considered to be a synchronous process; however, recent experimental findings suggest a possible alternative mechanism by which two formic acid monomers proceed through an acyclic dimer to the cyclic dimer in a stepwise process. To investigate this newly proposed process of dimerization in formic acid, density functional theory and second-order Moeller-Plesset perturbation theory (MP2) have been used to optimize cis and trans monomers of formic acid, the acyclic and cyclic dimers, and the acyclic and cyclic transition states between minima. Single-point energies of themore » trans monomer, dimer minima, and transition states at the MP2/TZ2P+diff optimized geometries were computed at the coupled-cluster level of theory including singles and doubles with perturbatively applied triple excitations [CCSD(T)] with an aug-cc-pVTZ basis set to obtain an accurate determination of energy barriers and dissociation energies. A counterpoise correction was performed to determine an estimate of the basis set superposition error in computing relative energies. The explicitly correlated MP2 method of Kutzelnigg and Klopper (MP2-R12) was used to provide an independent means for obtaining the MP2 one-particle limit. The cyclic minimum is predicted to be 6.3 kcal/mol more stable than the acyclic minimum, and the barrier to double proton transfer is 7.1 kcal/mol.« less

Hydrogen Dimers in Giant-planet Infrared Spectra

NASA Astrophysics Data System (ADS)

Fletcher, Leigh N.; Gustafsson, Magnus; Orton, Glenn S.

2018-03-01

Despite being one of the weakest dimers in nature, low-spectral-resolution Voyager/IRIS observations revealed the presence of (H2)2 dimers on Jupiter and Saturn in the 1980s. However, the collision-induced H2–H2 opacity databases widely used in planetary science have thus far only included free-to-free transitions and have neglected the contributions of dimers. Dimer spectra have both fine-scale structure near the S(0) and S(1) quadrupole lines (354 and 587 cm‑1, respectively), and broad continuum absorption contributions up to ±50 cm‑1 from the line centers. We develop a new ab initio model for the free-to-bound, bound-to-free, and bound-to-bound transitions of the hydrogen dimer for a range of temperatures (40–400 K) and para-hydrogen fractions (0.25–1.0). The model is validated against low-temperature laboratory experiments, and used to simulate the spectra of the giant planets. The new collision-induced opacity database permits high-resolution (0.5–1.0 cm‑1) spectral modeling of dimer spectra near S(0) and S(1) in both Cassini Composite Infrared Spectrometer observations of Jupiter and Saturn, and in Spitzer Infrared Spectrometer (IRS) observations of Uranus and Neptune for the first time. Furthermore, the model reproduces the dimer signatures observed in Voyager/IRIS data near S(0) on Jupiter and Saturn, and generally lowers the amount of para-H2 (and the extent of disequilibrium) required to reproduce IRIS observations.

Microtubules as mechanical force sensors.

PubMed

Karafyllidis, Ioannis G; Lagoudas, Dimitris C

2007-03-01

Microtubules are polymers of tubulin subunits (dimers) arranged on a hexagonal lattice. Each tubulin dimer comprises two monomers, the alpha-tubulin and beta-tubulin, and can be found in two states. In the first state a mobile negative charge is located into the alpha-tubulin monomer and in the second into the beta-tubulin monomer. Each tubulin dimer is modeled as an electrical dipole coupled to its neighbors by electrostatic forces. The location of the mobile charge in each dimer depends on the location of the charges in the dimer's neighborhood. Mechanical forces that act on the microtubule affect the distances between the dimers and alter the electrostatic potential. Changes in this potential affect the mobile negative charge location in each dimer and the charge distribution in the microtubule. The net effect is that mechanical forces affect the charge distribution in microtubules. We propose to exploit this effect and use microtubules as mechanical force sensors. We model each dimer as a two-state quantum system and, following the quantum computation paradigm, we use discrete quantum random walk on the hexagonal microtubule lattice to determine the charge distribution. Different forces applied on the microtubule are modeled as different coin biases leading to different probability distributions of the quantum walker location, which are directly connected to different charge distributions. Simulation results show that there is a strong indication that microtubules can be used as mechanical force sensors and that they can also detect the force directions and magnitudes.

Development and validation of a high-content bimolecular fluorescence complementation assay for small-molecule inhibitors of HIV-1 Nef dimerization.

PubMed

Poe, Jerrod A; Vollmer, Laura; Vogt, Andreas; Smithgall, Thomas E

2014-04-01

Nef is a human immunodeficiency virus 1 (HIV-1) accessory factor essential for viral pathogenesis and AIDS progression. Many Nef functions require dimerization, and small molecules that block Nef dimerization may represent antiretroviral drug leads. Here we describe a cell-based assay for Nef dimerization inhibitors based on bimolecular fluorescence complementation (BiFC). Nef was fused to nonfluorescent, complementary fragments of yellow fluorescent protein (YFP) and coexpressed in the same cell population. Dimerization of Nef resulted in juxtaposition of the YFP fragments and reconstitution of the fluorophore. For automation, the Nef-YFP fusion proteins plus a monomeric red fluorescent protein (mRFP) reporter were expressed from a single vector, separated by picornavirus "2A" linker peptides to permit equivalent translation of all three proteins. Validation studies revealed a critical role for gating on the mRFP-positive subpopulation of transfected cells, as well as use of the mRFP signal to normalize the Nef-BiFC signal. Nef-BiFC/mRFP ratios resulting from cells expressing wild-type versus dimerization-defective Nef were very clearly separated, with Z factors consistently in the 0.6 to 0.7 range. A fully automated pilot screen of the National Cancer Institute Diversity Set III identified several hit compounds that reproducibly blocked Nef dimerization in the low micromolar range. This BiFC-based assay has the potential to identify cell-active small molecules that directly interfere with Nef dimerization and function.

Development and Validation of a High-Content Bimolecular Fluorescence Complementation Assay for Small Molecule Inhibitors of HIV-1 Nef Dimerization

PubMed Central

Poe, Jerrod A.; Vollmer, Laura; Vogt, Andreas; Smithgall, Thomas E.

2014-01-01

Nef is an HIV-1 accessory factor essential for viral pathogenesis and AIDS progression. Many Nef functions require dimerization, and small molecules that block Nef dimerization may represent antiretroviral drug leads. Here we describe a cell-based assay for Nef dimerization inhibitors based on bimolecular fluorescence complementation (BiFC). Nef was fused to non-fluorescent, complementary fragments of YFP and co-expressed in the same cell population. Dimerization of Nef resulted in juxtaposition of the YFP fragments and reconstitution of the fluorophore. For automation, the Nef-YFP fusion proteins plus an mRFP reporter were expressed from a single vector, separated by picornavirus ‘2A’ linker peptides to permit equivalent translation of all three proteins. Validation studies revealed a critical role for gating on the mRFP-positive subpopulation of transfected cells, as well as use of the mRFP signal to normalize the Nef-BiFC signal. Nef-BiFC/mRFP ratios resulting from cells expressing wild-type vs. dimerization-defective Nef were very clearly separated, with Z-factors consistently in the 0.6–0.7 range. A fully automated pilot screen of the NIH Diversity Set III identified several hit compounds that reproducibly blocked Nef dimerization in the low micromolar range. This BiFC-based assay has the potential to identify cell-active small molecules that directly interfere with Nef dimerization and function. PMID:24282155

Molecular docking studies to map the binding site of squalene synthase inhibitors on dehydrosqualene synthase of Staphylococcus aureus.

PubMed

Kahlon, Amandeep Kaur; Roy, Sudeep; Sharma, Ashok

2010-10-01

Dehydrosqualene synthase of Staphylococcus aureus is involved in the synthesis of golden carotenoid pigment staphyloxanthin. This pigment of S. aureus provides the antioxidant property to this bacterium to survive inside the host cell. Dehydrosqualene synthase (CrtM) is having structural similarity with the human squalene synthase enzyme which is involved in the cholesterol synthesis pathway in humans (Liu et al., 2008). Cholesterol lowering drugs were found to have inhibitory effect on dehydrosqualene synthase enzyme of S. aureus. The present study attempts to focus on squalene synthase inhibitors, lapaquistat acetate and squalestatins reported as cholesterol lowering agents in vitro and in vivo but not studied in context to dehydrosqualene synthase of S. aureus. Mode of binding of lapaquistat acetate and squalestatin analogs on dehydrosqualene synthase (CrtM) enzyme of S. aureus was identified by performing docking analysis with Scigress Explorer Ultra 7.7 docking software. Based on the molecular docking analysis, it was found that the His18, Arg45, Asp48, Asp52, Tyr129, Gln165, Asn168 and Asp172 residues interacted with comparatively high frequency with the inhibitors studied. Comparative docking study with Discovery studio 2.0 also confirmed the involvement of these residues of dehydrosqualene synthase enzyme with the inhibitors studied. This further confirms the importance of these residues in the enzyme function. In silico ADMET analysis was done to predict the ADMET properties of the standard drugs and test compounds. This might provide insights to develop new drugs to target the virulence factor, dehydrosqualene synthase of S. aureus.

In Planta Recapitulation of Isoprene Synthase Evolution from Ocimene Synthases

PubMed Central

Li, Mingai; Xu, Jia; Algarra Alarcon, Alberto; Carlin, Silvia; Barbaro, Enrico; Cappellin, Luca; Velikova, Violeta; Vrhovsek, Urska; Loreto, Francesco; Varotto, Claudio

2017-01-01

Abstract Isoprene is the most abundant biogenic volatile hydrocarbon compound naturally emitted by plants and plays a major role in atmospheric chemistry. It has been proposed that isoprene synthases (IspS) may readily evolve from other terpene synthases, but this hypothesis has not been experimentally investigated. We isolated and functionally validated in Arabidopsis the first isoprene synthase gene, AdoIspS, from a monocotyledonous species (Arundo donax L., Poaceae). Phylogenetic reconstruction indicates that AdoIspS and dicots isoprene synthases most likely originated by parallel evolution from TPS-b monoterpene synthases. Site-directed mutagenesis demonstrated invivo the functional and evolutionary relevance of the residues considered diagnostic for IspS function. One of these positions was identified by saturating mutagenesis as a major determinant of substrate specificity in AdoIspS able to cause invivo a dramatic change in total volatile emission from hemi- to monoterpenes and supporting evolution of isoprene synthases from ocimene synthases. The mechanism responsible for IspS neofunctionalization by active site size modulation by a single amino acid mutation demonstrated in this study might be general, as the very same amino acidic position is implicated in the parallel evolution of different short-chain terpene synthases from both angiosperms and gymnosperms. Based on these results, we present a model reconciling in a unified conceptual framework the apparently contrasting patterns previously observed for isoprene synthase evolution in plants. These results indicate that parallel evolution may be driven by relatively simple biophysical constraints, and illustrate the intimate molecular evolutionary links between the structural and functional bases of traits with global relevance. PMID:28637270

Accumulation of prenyl alcohols by terpenoid biosynthesis inhibitors in various microorganisms.

PubMed

Muramatsu, Masayoshi; Ohto, Chikara; Obata, Shusei; Sakuradani, Eiji; Shimizu, Sakayu

2008-09-01

Squalene synthase inhibitors significantly accelerate the production of farnesol by various microorganisms. However, farnesol production by Saccharomyces cerevisiae ATCC 64031, in which the squalene synthase gene is deleted, was not affected by the inhibitors, indicating that farnesol accumulation is enhanced in the absence of squalene synthase activity. The combination of diphenylamine as an inhibitor of carotenoid biosynthesis and a squalene synthase inhibitor increases geranylgeraniol production by a yeast, Rhodotorula rubra NBRC 0870. An ent-kauren synthase inhibitor also enhances the production of farnesol and geranylgeraniol by a filamentous fungus, Gibberella fujikuroi NBRC 30336. These results indicate that the inhibition of downstream enzymes from prenyl diphosphate synthase leads to the production of farnesol and geranylgeraniol.

76 FR 8895 - Polymerized Fatty Acid Esters With Aminoalcohol Alkoxylates; Exemption From the Requirement of a...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-02-16

..., reaction products with fatty acid dimers (CAS Reg. No. 1173188-38-9); dimethylaminoethanol, ethoxylated, propoxylated, reaction products with fatty acid dimers (CAS Reg. No. 1173188-42-5 diethylaminoethanol, ethoxylated, reaction product with fatty acid dimers (CAS Reg. No. 1173188-72-1); diethylaminoethanol...

Excitation Localization/Delocalization Isomerism in a Strongly Coupled Covalent Dimer of 1,3-Diphenylisobenzofuran

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schrauben, Joel N.; Akdag, Akin; Wen, Jin

Two isomers of both the lowest excited singlet (S1) and triplet (T1) states of the directly para, para'-connected covalent dimer of the singlet-fission chromophore 1,3-diphenylisobenzofuran have been observed. In one isomer, excitation is delocalized over both halves of the dimer, and in the other, it is localized on one or the other half. For a covalent dimer in solution, such 'excitation isomerism' is extremely rare. The vibrationally relaxed isomers do not interconvert, and their photophysical properties, including singlet fission, differ significantly.

Identification of two conformationally trapped n-propanol-water dimers in a supersonic expansion

NASA Astrophysics Data System (ADS)

Mead, Griffin J.; Alonso, Elena R.; Finneran, Ian A.; Carroll, P. Brandon; Blake, Geoffrey A.

2017-05-01

Two conformers of the n-propanol-water dimer have been observed in a supersonic expansion using chirped-pulse Fourier-transform microwave (CPFTMW) spectroscopy. Structural assignments reveal the n-propanol sub-unit is conformationally trapped, with its methyl group in both Gauche and Trans orientations. Despite different carbon backbone conformations, both dimers display the same water-donor/alcohol-acceptor hydrogen bonding motif. This work builds upon other reported alcohol-water dimers and upon previous work detailing the trapping of small molecules into multiple structural minima in rare gas supersonic expansions.

Quantum dimer model for the pseudogap metal

PubMed Central

Punk, Matthias; Allais, Andrea; Sachdev, Subir

2015-01-01

We propose a quantum dimer model for the metallic state of the hole-doped cuprates at low hole density, p. The Hilbert space is spanned by spinless, neutral, bosonic dimers and spin S=1/2, charge +e fermionic dimers. The model realizes a “fractionalized Fermi liquid” with no symmetry breaking and small hole pocket Fermi surfaces enclosing a total area determined by p. Exact diagonalization, on lattices of sizes up to 8×8, shows anisotropic quasiparticle residue around the pocket Fermi surfaces. We discuss the relationship to experiments. PMID:26195771

Photochemical dimerization and functionalization of alkanes, ethers, primary and secondary alcohols, phosphine oxides and silanes

DOEpatents

Crabtree, Robert H.; Brown, Stephen H.

1989-01-01

The space-time yield and/or the selectivity of the photochemical dimerization of alkanes, ethers, primary and secondary alcohols, phosphine oxides and primary, secondary and tertiary silanes with Hg and U.V. light is enhanced by refluxing the substrate in the irradiated reaction zone at a temperature at which the dimer product condenses and remains condensed promptly upon its formation. Cross-dimerization of the alkanes, ethers and silanes with primary alcohols is disclosed, as is the functionalization to aldehydes of the alkanes with carbon monoxide.

Photochemical dimerization and functionalization of alkanes, ethers, primary and secondary alcohols, phosphine oxides and silanes

DOEpatents

Crabtree, R.H.; Brown, S.H.

1989-10-17

The space-time yield and/or the selectivity of the photochemical dimerization of alkanes, ethers, primary and secondary alcohols, phosphine oxides and primary, secondary and tertiary silanes with Hg and U.V. light is enhanced by refluxing the substrate in the irradiated reaction zone at a temperature at which the dimer product condenses and remains condensed promptly upon its formation. Cross-dimerization of the alkanes, ethers and silanes with primary alcohols is disclosed, as is the functionalization to aldehydes of the alkanes with carbon monoxide.

One-dimensional Kondo lattice model at quarter filling

NASA Astrophysics Data System (ADS)

Xavier, J. C.; Miranda, E.

2008-10-01

We revisit the problem of the quarter-filled one-dimensional Kondo lattice model, for which the existence of a dimerized phase and a nonzero charge gap had been reported by Xavier [Phys. Rev. Lett. 90, 247204 (2003)]. Recently, some objections were raised claiming that the system is neither dimerized nor has a charge gap. In the interest of clarifying this important issue, we show that these objections are based on results obtained under conditions in which the dimer order is artificially suppressed. We use the incontrovertible dimerized phase of the Majumdar-Ghosh point of the J1-J2 Heisenberg model as a paradigm with which to illustrate this artificial suppression. Finally, by means of extremely accurate density-matrix renormalization-group calculations, we show that the charge gap is indeed nonzero in the dimerized phase.

Estimation of quantum yields of weak fluorescence from eosin Y dimers formed in aqueous solutions.

PubMed

Enoki, Masami; Katoh, Ryuzi

2018-05-17

We studied the weak fluorescence from the dimer of eosin Y (EY) in aqueous solutions. We used a newly developed ultrathin optical cell with a thickness ranging from of the order of microns to several hundreds of microns to successfully measure the fluorescence spectra of highly concentrated aqueous solutions of EY without artifacts caused by the reabsorption of fluorescence. The spectra we obtained were similar to the fluorescence spectrum of the EY monomer; almost no fluorescence was observed from the EY dimer. By a careful comparison of the spectra of solutions at low and high concentrations of EY, we succeeded in extracting the fluorescence spectrum of the EY dimer. The fluorescence quantum yield of the EY dimer was estimated to be 0.005.

Dimer-based model for heptaspanning membrane receptors.

PubMed

Franco, Rafael; Casadó, Vicent; Mallol, Josefa; Ferré, Sergi; Fuxe, Kjell; Cortés, Antonio; Ciruela, Francisco; Lluis, Carmen; Canela, Enric I

2005-07-01

The existence of intramembrane receptor-receptor interactions for heptaspanning membrane receptors is now fully accepted, but a model considering dimers as the basic unit that binds to two ligand molecules is lacking. Here, we propose a two-state-dimer model in which the ligand-induced conformational changes from one component of the dimer are communicated to the other. Our model predicts cooperativity in binding, which is relevant because the other current models fail to address this phenomenon satisfactorily. Our two-state-dimer model also predicts the variety of responses elicited by full or partial agonists, neutral antagonists and inverse agonists. This model can aid our understanding of the operation of heptaspanning receptors and receptor channels, and, potentially, be important for improving the treatment of cardiovascular, neurological and neuropsychyatric diseases.

Exact exchange and Wilson-Levy correlation: a pragmatic device for studying complex weakly-bonded systems.

PubMed

Walsh, T R

2005-02-07

The Wilson-Levy (WL) correlation functional is used together with Hartree-Fock (HF) theory to evaluate interaction energies at intermediate separations (i.e. around equilibrium separation) for several weakly-bonded systems. The HF+WL approach reproduces binding trends for all complexes studied: selected rare-gas dimers, isomers of the methane dimer, benzene dimer and naphthalene dimer, and base-pair stacking structures for pyrimidine, cytosine, uracil and guanine dimers. These HF+WL data are contrasted against results obtained from some popular functionals (including B3LYP and PBE), as well as two newly-developed functionals, X3LYP and xPBE. The utility of HF+WL, with reference to exact-exchange (EXX) density-functional theory, is discussed in terms of a suggested EXXWL exchange-correlation functional.

Dimerization of the Bacterial Biotin Carboxylase Subunit Is Required for Acetyl Coenzyme A Carboxylase Activity In Vivo

PubMed Central

Smith, Alexander C.

2012-01-01

Acetyl coenzyme A (acteyl-CoA) carboxylase (ACC) is the first committed enzyme of the fatty acid synthesis pathway. Escherichia coli ACC is composed of four different proteins. The first enzymatic activity of the ACC complex, biotin carboxylase (BC), catalyzes the carboxylation of the protein-bound biotin moiety of another subunit with bicarbonate in an ATP-dependent reaction. Although BC is found as a dimer in cell extracts and the carboxylase activities of the two subunits of the dimer are interdependent, mutant BC proteins deficient in dimerization are reported to retain appreciable activity in vitro (Y. Shen, C. Y. Chou, G. G. Chang, and L. Tong, Mol. Cell 22:807–818, 2006). However, in vivo BC must interact with the other proteins of the complex, and thus studies of the isolated BC may not reflect the intracellular function of the enzyme. We have tested the abilities of three BC mutant proteins deficient in dimerization to support growth and report that the two BC proteins most deficient in dimerization fail to support growth unless expressed at high levels. In contrast, the wild-type protein supports growth at low expression levels. We conclude that BC must be dimeric to fulfill its physiological function. PMID:22037404

Convergent evolution involving dimeric and trimeric dUTPases in pathogenicity island mobilization.

PubMed

Donderis, Jorge; Bowring, Janine; Maiques, Elisa; Ciges-Tomas, J Rafael; Alite, Christian; Mehmedov, Iltyar; Tormo-Mas, María Angeles; Penadés, José R; Marina, Alberto

2017-09-01

The dUTPase (Dut) enzymes, encoded by almost all free-living organisms and some viruses, prevent the misincorporation of uracil into DNA. We previously proposed that trimeric Duts are regulatory proteins involved in different cellular processes; including the phage-mediated transfer of the Staphylococcus aureus pathogenicity island SaPIbov1. Recently, it has been shown that the structurally unrelated dimeric Dut encoded by phage ϕNM1 is similarly able to mobilize SaPIbov1, suggesting dimeric Duts could also be regulatory proteins. How this is accomplished remains unsolved. Here, using in vivo, biochemical and structural approaches, we provide insights into the signaling mechanism used by the dimeric Duts to induce the SaPIbov1 cycle. As reported for the trimeric Duts, dimeric Duts contain an extremely variable region, here named domain VI, which is involved in the regulatory capacity of these enzymes. Remarkably, our results also show that the dimeric Dut signaling mechanism is modulated by dUTP, as with the trimeric Duts. Overall, our results demonstrate that although unrelated both in sequence and structure, dimeric and trimeric Duts control SaPI transfer by analogous mechanisms, representing a fascinating example of convergent evolution. This conserved mode of action highlights the biological significance of Duts as regulatory molecules.

Convergent evolution involving dimeric and trimeric dUTPases in pathogenicity island mobilization

PubMed Central

Ciges-Tomas, J. Rafael; Mehmedov, Iltyar; Tormo-Mas, María Angeles; Penadés, José R.

2017-01-01

The dUTPase (Dut) enzymes, encoded by almost all free-living organisms and some viruses, prevent the misincorporation of uracil into DNA. We previously proposed that trimeric Duts are regulatory proteins involved in different cellular processes; including the phage-mediated transfer of the Staphylococcus aureus pathogenicity island SaPIbov1. Recently, it has been shown that the structurally unrelated dimeric Dut encoded by phage ϕNM1 is similarly able to mobilize SaPIbov1, suggesting dimeric Duts could also be regulatory proteins. How this is accomplished remains unsolved. Here, using in vivo, biochemical and structural approaches, we provide insights into the signaling mechanism used by the dimeric Duts to induce the SaPIbov1 cycle. As reported for the trimeric Duts, dimeric Duts contain an extremely variable region, here named domain VI, which is involved in the regulatory capacity of these enzymes. Remarkably, our results also show that the dimeric Dut signaling mechanism is modulated by dUTP, as with the trimeric Duts. Overall, our results demonstrate that although unrelated both in sequence and structure, dimeric and trimeric Duts control SaPI transfer by analogous mechanisms, representing a fascinating example of convergent evolution. This conserved mode of action highlights the biological significance of Duts as regulatory molecules. PMID:28892519

Free radicals impair the anti-oxidative stress activity of DJ-1 through the formation of SDS-resistant dimer.

PubMed

Yasuda, Tatsuki; Niki, Takeshi; Ariga, Hiroyoshi; Iguchi-Ariga, Sanae M M

2017-04-01

DJ-1 is a causative gene for familial Parkinson's disease (PD). Loss-of-function of DJ-1 protein is suggested to contribute to the onset of PD, but the causes of DJ-1 dysfunction remain insufficiently elucidated. In this study, we found that the SDS-resistant irreversible dimer of DJ-1 protein was formed in human dopaminergic neuroblastoma SH-SY5Y cells when the cells were exposed to massive superoxide inducers such as paraquat and diquat. The dimer was also formed in vitro by superoxide in PQ redox cycling system and hydroxyl radical produced in Fenton reaction. We, thus, found a novel phenomenon that free radicals directly affect DJ-1 to form SDS-resistant dimers. Moreover, the formation of the SDS-resistant dimer impaired anti-oxidative stress activity of DJ-1 both in cell viability assay and H 2 O 2 -elimination assay in vitro. Similar SDS-resistant dimers were steadily formed with several mutants of DJ-1 found in familial PD patients. These findings suggest that DJ-1 is impaired due to the formation of SDS-resistant dimer when the protein is directly attacked by free radicals yielded by external and internal stresses and that the DJ-1 impairment is one of the causes of sporadic PD.

Dimers of nineteen-electron sandwich compounds: crystal and electronic structures, and comparison of reducing strengths.

PubMed

Mohapatra, Swagat K; Fonari, Alexandr; Risko, Chad; Yesudas, Kada; Moudgil, Karttikay; Delcamp, Jared H; Timofeeva, Tatiana V; Brédas, Jean-Luc; Marder, Seth R; Barlow, Stephen

2014-11-17

The dimers of some Group 8 metal cyclopentadienyl/arene complexes and Group 9 metallocenes can be handled in air, yet are strongly reducing, making them useful n-dopants in organic electronics. In this work, the X-ray molecular structures are shown to resemble those of Group 8 metal cyclopentadienyl/pentadienyl or Group 9 metal cyclopentadienyl/diene model compounds. Compared to those of the model compounds, the DFT HOMOs of the dimers are significantly destabilized by interactions between the metal and the central CC σ-bonding orbital, accounting for the facile oxidation of the dimers. The lengths of these CC bonds (X-ray or DFT) do not correlate with DFT dissociation energies, the latter depending strongly on the monomer stabilities. Ru and Ir monomers are more reducing than their Fe and Rh analogues, but the corresponding dimers also exhibit much higher dissociation energies, so the estimated monomer cation/neutral dimer potentials are, with the exception of that of [RhCp2 ]2 , rather similar (-1.97 to -2.15 V vs. FeCp2 (+/0) in THF). The consequences of the variations in bond strength and redox potentials for the reactivity of the dimers are discussed. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Designed Angiopoietin-1 Variant, Dimeric CMP-Ang1 Activates Tie2 and Stimulates Angiogenesis and Vascular Stabilization in N-glycan Dependent Manner

PubMed Central

Oh, Nuri; Kim, Kangsan; Jin Kim, Soo; Park, Intae; Lee, Jung-Eun; Suk Seo, Young; Joo An, Hyun; Min Kim, Ho; Young Koh, Gou

2015-01-01

Angiopoietin-1 (Ang1), a potential growth factor for therapeutic angiogenesis and vascular stabilization, is known to specifically cluster and activate Tie2 in high oligomeric forms, which is a unique and essential process in this ligand-receptor interaction. However, highly oligomeric native Ang1 and Ang1 variants are difficult to produce, purify, and store in a stable and active form. To overcome these limitations, we developed a simple and active dimeric CMP-Ang1 by replacing the N-terminal of native Ang1 with the coiled-coil domain of cartilage matrix protein (CMP) bearing mutations in its cysteine residues. This dimeric CMP-Ang1 effectively increased the migration, survival, and tube formation of endothelial cells via Tie2 activation. Furthermore, dimeric CMP-Ang1 induced angiogenesis and suppressed vascular leakage in vivo. Despite its dimeric structure, the potencies of such Tie2-activation-induced effects were comparable to those of a previously engineered protein, COMP-Ang1. We also revealed that these effects of dimeric CMP-Ang1 were affected by specified N-glycosylation in its fibrinogen-like domain. Taken together, our results indicate that dimeric CMP-Ang1 is capable of activating Tie2 and stimulating angiogenesis in N-glycan dependent manner. PMID:26478188

Dimeric, trimeric and tetrameric complexes of immunoglobulin G fix complement.

PubMed Central

Wright, J K; Tschopp, J; Jaton, J C; Engel, J

1980-01-01

The binding of pure dimers, trimers and tetramers of randomly cross-linked non-immune rabbit immunoglobulin G to the first component and subcomponent of the complement system, C1 and C1q respectively, was studied. These oligomers possessed open linear structures. All three oligomers fixed complement with decreasing affinity in the order: tetramer, trimer, dimer. Complement fixation by dimeric immunoglobulin exhibited the strongest concentration-dependence. No clear distinction between a non-co-operative and a co-operative binding mechanism could be achieved, although the steepness of the complement-fixation curves for dimers and trimers was better reflected by the co-operative mechanism. Intrinsic binding constants were about 10(6)M-1 for dimers, 10(7)M-1 for trimers and 3 X 10(9)M-1 for tetramers, assuming non-co-operative binding. The data are consistent with a maximum valency of complement component C1 for immunoglobulin G protomers in the range 6-18. The binding of dimers to purified complement subcomponent C1q was demonstrated by sedimentation-velocity ultracentrifugation. Mild reduction of the complexes by dithioerythritol caused the immunoglobulin to revert to the monomeric state (S20,w = 6.2-6.5S) with concomitant loss of complement-fixing ability. Images Fig. 2. PMID:6985362

Enhancing action of positive allosteric modulators through the design of dimeric compounds.

PubMed

Drapier, Thomas; Geubelle, Pierre; Bouckaert, Charlotte; Nielsen, Lise; Laulumaa, Saara; Goffin, Eric; Dilly, Sébastien; Francotte, Pierre; Hanson, Julien; Pochet, Lionel; Kastrup, Jette Sandholm; Pirotte, Bernard

2018-05-18

The present study describes the identification of highly potent dimeric 1,2,4-benzothiadiazine 1,1-dioxide (BTD)-type positive allosteric modulators of the AMPA receptors (AMPApams) obtained by linking two monomeric BTD scaffolds through their respective 6-positions. Using previous X-ray data from monomeric BTDs co-crystallized with the GluA2o ligand-binding domain (LBD), a molecular modeling approach was performed to predict the preferred dimeric combinations. Two 6,6-ethylene-linked dimeric BTD compounds (16 and 22) were prepared and evaluated as AMPApams on HEK293 cells expressing GluA2o(Q) (calcium flux experiment). These compounds were found to be about 10,000 times more potent than their respective monomers, the most active dimeric compound being the bis-4-cyclopropyl-substituted compound 22 [6,6'-(ethane-1,2-diyl)bis(4-cyclopropyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide], with an EC50 value of 1.4 nM. As a proof of concept, the bis-4-methyl-substituted dimeric compound 16 (EC50 = 13 nM) was successfully co-crystallized with the GluA2o-LBD and was found to occupy the two BTD binding sites at the LBD dimer interface.

Sub-nanometer pore formation in single-molecule-thick polyurea molecular-sieving membrane: a computational study.

PubMed

Park, Seongjin; Lansac, Yves; Jang, Yun Hee

2018-06-07

A polymeric network of 1-(4-tritylphenyl)urea (TPU) built via layer-by-layer cross-linking polymerization has been proposed to be an excellent mesh equipped with single-molecule-thick pores (i.e., cyclic poly-TPU rings), which can sieve glucose (∼0.7 nm) out of its mixture with urea for hemodialysis applications. Monte Carlo search for the lowest-energy conformation of various sizes of poly-TPU rings unravels the origin of narrow pore size distribution, which is around the sizes of dimer and trimer rings (0.3-0.8 nm). Flexible rings larger than the dimer and trimer rings, in particular tetramer rings, prefer a twisted conformation in the shape of the infinity symbol (∞, which looks like two dimer rings joined together) locked by a hydrogen bond between diphenylurea linker groups facing each other. Translocation energy profiles across these TPU rings reveal their urea-versus-glucose sieving mechanism: glucose is either too large (to enter dimers and twisted tetramers) or too perfectly fit (to exit trimers), leaving only a dimer-sized free space in the ring, whereas smaller-sized urea and water pass through these effective dimer-sized rings (bare dimers, twisted tetramers, and glucose-filled trimers) without encountering a substantial energy barrier or trap.

Computational Equilibrium Thermodynamic and Kinetic Analysis of K-Ras Dimerization through an Effector Binding Surface Suggests Limited Functional Role.

PubMed

Sayyed-Ahmad, Abdallah; Cho, Kwang-Jin; Hancock, John F; Gorfe, Alemayehu A

2016-08-25

Dimer formation is believed to have a substantial impact on regulating K-Ras function. However, the evidence for dimerization and the molecular details of the process are scant. In this study, we characterize a K-Ras pseudo-C2-symmetric dimerization interface involving the effector interacting β2-strand. We used structure matching and all-atom molecular dynamics (MD) simulations to predict, refine, and investigate the stability of this interface. Our MD simulation suggested that the β2-dimer is potentially stable and remains relatively close to its initial conformation due to the presence of a number of hydrogen bonds, ionic salt bridges, and other favorable interactions. We carried out potential of mean force calculations to determine the relative binding strength of the interface. The results of these calculations indicated that the β2 dimerization interface provides a weak binding free energy in solution and a dissociation constant that is close to 1 mM. Analyses of Brownian dynamics simulations suggested an association rate kon ≈ 10(5)-10(6) M(-1) s(-1). Combining these observations with available literature data, we propose that formation of auto-inhibited β2 K-Ras dimers is possible but its fraction in cells is likely very small under normal physiologic conditions.

Observation of Solvent Penetration during Cold Denaturation of E. coli Phosphofructokinase-2

PubMed Central

Ramírez-Sarmiento, César A.; Baez, Mauricio; Wilson, Christian A.M.; Babul, Jorge; Komives, Elizabeth A.; Guixé, Victoria

2013-01-01

Phosphofructokinase-2 is a dimeric enzyme that undergoes cold denaturation following a highly cooperative N2 2I mechanism with dimer dissociation and formation of an expanded monomeric intermediate. Here, we use intrinsic fluorescence of a tryptophan located at the dimer interface to show that dimer dissociation occurs slowly, over several hours. We then use hydrogen-deuterium exchange mass spectrometry experiments, performed by taking time points over the cold denaturation process, to measure amide exchange throughout the protein during approach to the cold denatured state. As expected, a peptide corresponding to the dimer interface became more solvent exposed over time at 3°C; unexpectedly, amide exchange increased throughout the protein over time at 3°C. The rate of increase in amide exchange over time at 3°C was the same for each region and equaled the rate of dimer dissociation measured by tryptophan fluorescence, suggesting that dimer dissociation and formation of the cold denatured intermediate occur without appreciable buildup of folded monomer. The observation that throughout the protein amide exchange increases as phosphofructokinase-2 cold denatures provides experimental evidence for theoretical predictions that cold denaturation primarily occurs by solvent penetration into the hydrophobic core of proteins in a sequence-independent manner. PMID:23708365

Dehaloperoxidase-Hemoglobin from Amphitrite ornata Is Primarily a Monomer in Solution

DOE Office of Scientific and Technical Information (OSTI.GOV)

M Thompson; S Franzen; M Davis

2011-12-31

The crystal structures of the dehaloperoxidase-hemoglobin from A. ornata (DHP A) each report a crystallographic dimer in the unit cell. Yet, the largest dimer interface observed is 450 {angstrom}{sup 2}, an area significantly smaller than the typical value of 1200-2000 {angstrom}{sup 2} and in contrast to the extensive interface region of other known dimeric hemoglobins. To examine the oligomerization state of DHP A in solution, we used gel permeation by fast protein liquid chromatography and small-angle X-ray scattering (SAXS). Gel permeation experiments demonstrate that DHP A elutes as a monomer (15.5 kDa) and can be separated from green fluorescent protein,more » which has a molar mass of 27 kDa, near the 31 kDa expected for the DHP A dimer. By SAXS, we found that DHP A is primarily monomeric in solution, but with a detectable level of dimer (10%), under all conditions studied up to a protein concentration of 3.0 mM. These concentrations are likely 10-100-fold lower than the K{sub d} for dimer formation. Additionally, there was no significant effect either on the overall conformation of DHP A or its monomer-dimer equilibrium upon addition of the DHP A inhibitor, 4-iodophenol.« less

Light activation of the LOV protein Vivid generates a rapidly exchanging dimer†‡

PubMed Central

Zoltowski, Brian D.; Crane, Brian R.

2009-01-01

The fungal photoreceptor Vivid (VVD) plays an important role in the adaptation of blue-light responses in Neurospora crassa. VVD, an FAD-binding LOV (Light, Oxygen, Voltage) protein, couples light-induced cysteinyl-adduct formation at the flavin ring to conformational changes in the N-terminal cap (Ncap) of the VVD PAS domain. Size-exclusion chromatography (SEC), equilibrium ultracentrifugation, and static and dynamic light scattering show that these conformational changes generate a rapidly exchanging VVD dimer, with an expanded hydrodynamic radius. A three-residue N-terminal β-turn that assumes two different conformations in a crystal structure of a VVD C71V variant is essential for light-state dimerization. Residue substitutions at a critical hinge between the Ncap and PAS core can inhibit or enhance dimerization, whereas a Tyr to Trp substitution at the Ncap-to-PAS interface stabilizes the light-state dimer. Cross-linking through engineered disulfides indicates that the light-state dimer differs considerably from the dark-state dimer found in VVD crystal structures. These results verify the role of Ncap conformational changes in gating the photic response of Neurospora crassa, and indicate that LOV:LOV homo or hetero dimerization may be a mechanism for regulating light-activated gene expression. PMID:18553928

Observation of solvent penetration during cold denaturation of E. coli phosphofructokinase-2.

PubMed

Ramírez-Sarmiento, César A; Baez, Mauricio; Wilson, Christian A M; Babul, Jorge; Komives, Elizabeth A; Guixé, Victoria

2013-05-21

Phosphofructokinase-2 is a dimeric enzyme that undergoes cold denaturation following a highly cooperative N2 2I mechanism with dimer dissociation and formation of an expanded monomeric intermediate. Here, we use intrinsic fluorescence of a tryptophan located at the dimer interface to show that dimer dissociation occurs slowly, over several hours. We then use hydrogen-deuterium exchange mass spectrometry experiments, performed by taking time points over the cold denaturation process, to measure amide exchange throughout the protein during approach to the cold denatured state. As expected, a peptide corresponding to the dimer interface became more solvent exposed over time at 3°C; unexpectedly, amide exchange increased throughout the protein over time at 3°C. The rate of increase in amide exchange over time at 3°C was the same for each region and equaled the rate of dimer dissociation measured by tryptophan fluorescence, suggesting that dimer dissociation and formation of the cold denatured intermediate occur without appreciable buildup of folded monomer. The observation that throughout the protein amide exchange increases as phosphofructokinase-2 cold denatures provides experimental evidence for theoretical predictions that cold denaturation primarily occurs by solvent penetration into the hydrophobic core of proteins in a sequence-independent manner. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Primary and secondary dimer interfaces of the fibroblast growth factor receptor 3 transmembrane domain: characterization via multiscale molecular dynamics simulations.

PubMed

Reddy, Tyler; Manrique, Santiago; Buyan, Amanda; Hall, Benjamin A; Chetwynd, Alan; Sansom, Mark S P

2014-01-21

Receptor tyrosine kinases are single-pass membrane proteins that form dimers within the membrane. The interactions of their transmembrane domains (TMDs) play a key role in dimerization and signaling. Fibroblast growth factor receptor 3 (FGFR3) is of interest as a G380R mutation in its TMD is the underlying cause of ~99% of the cases of achondroplasia, the most common form of human dwarfism. The structural consequences of this mutation remain uncertain: the mutation shifts the position of the TMD relative to the lipid bilayer but does not alter the association free energy. We have combined coarse-grained and all-atom molecular dynamics simulations to study the dimerization of wild-type, heterodimer, and mutant FGFR3 TMDs. The simulations reveal that the helices pack together in the dimer to form a flexible interface. The primary packing mode is mediated by a Gx3G motif. There is also a secondary dimer interface that is more highly populated in heterodimer and mutant configurations that may feature in the molecular mechanism of pathology. Both coarse-grained and atomistic simulations reveal a significant shift of the G380R mutant dimer TMD relative to the bilayer to allow interactions of the arginine side chain with lipid headgroup phosphates.

Primary and Secondary Dimer Interfaces of the FGFR3 Transmembrane Domain: Characterization via Multiscale Molecular Dynamics Simulations

PubMed Central

Reddy, Tyler; Manrique, Santiago; Buyan, Amanda; Hall, Benjamin A.; Chetwynd, Alan; Sansom, Mark S.P.

2016-01-01

Receptor tyrosine kinases are single pass membrane proteins which form dimers within the membrane. The interactions of their transmembrane domains (TMDs) play a key role in dimerization and signaling. The fibroblast growth factor receptor 3 (FGFR3) is of interest as a G380R mutation in its TMD is the underlying cause of ~99% of cases of achondroplasia, the most common form of human dwarfism. The structural consequences of this mutation remain uncertain: the mutation shifts the position relative of the TMD relative to the lipid bilayer but does not alter the association free energy. We have combined coarse-grained and all-atom molecular dynamics simulations to study the dimerization of wild-type, heterodimer, and mutant FGFR3 TMDs. The simulations reveal that the helices pack together in the dimer to form a flexible interface. The primary packing mode is mediated by a Gx3G motif. There is also a secondary dimer interface which is more highly populated in heterodimer and mutant configurations which may feature in the molecular mechanism of pathology. Both coarse-grained and atomistic simulations reveal a significant shift of the G380R mutant dimer TMD relative to the bilayer so as to enable interactions of the arginine sidechain with lipid head group phosphates. PMID:24397339

Impaired thromboxane receptor dimerization reduces signaling efficiency: A potential mechanism for reduced platelet function in vivo.

PubMed

Capra, Valérie; Mauri, Mario; Guzzi, Francesca; Busnelli, Marta; Accomazzo, Maria Rosa; Gaussem, Pascale; Nisar, Shaista P; Mundell, Stuart J; Parenti, Marco; Rovati, G Enrico

2017-01-15

Thromboxane A 2 is a potent mediator of inflammation and platelet aggregation exerting its effects through the activation of a G protein-coupled receptor (GPCR), termed TP. Although the existence of dimers/oligomers in Class A GPCRs is widely accepted, their functional significance still remains controversial. Recently, we have shown that TPα and TPβ homo-/hetero-dimers interact through an interface of residues in transmembrane domain 1 (TM1) whose disruption impairs dimer formation. Here, biochemical and pharmacological characterization of this dimer deficient mutant (DDM) in living cells indicates a significant impairment in its response to agonists. Interestingly, two single loss-of-function TPα variants, namely W29C and N42S recently identified in two heterozygous patients affected by bleeding disorders, match some of the residues mutated in our DDM. These two naturally occurring variants display a reduced potency to TP agonists and are characterized by impaired dimer formation in transfected HEK-293T cells. These findings provide proofs that lack of homo-dimer formation is a crucial process for reduced TPα function in vivo, and might represent one molecular mechanism through which platelet TPα receptor dysfunction affects the patient(s) carrying these mutations. Copyright © 2016 Elsevier Inc. All rights reserved.

Overall conformation of covalently stabilized domain-swapped dimer of human cystatin C in solution

NASA Astrophysics Data System (ADS)

Murawska, Magdalena; Szymańska, Aneta; Grubb, Anders; Kozak, Maciej

2017-11-01

Human cystatin C (HCC), a small protein, plays a crucial role in inhibition of cysteine proteases. The most common structural form of human cystatin C in crystals is a dimer, which has been evidenced both for the native protein and its mutants. In these structures, HCC dimers were formed through the mechanism of domain swapping. The structure of the monomeric form of human cystatin C was determined for V57N mutant and the mutant with the engineered disulfide bond (L47C)-(G69C) (known as stab1-HCC). On the basis of stab1-HCC, a number of covalently stabilized oligomers, including also dimers have been obtained. The aim of this study was to analyze the structure of the covalently stabilized dimer HCC in solution by the small angle X-ray scattering (SAXS) technique and synchrotron radiation. Experimental data confirmed that in solution this protein forms a dimer, which is characterized by the radius of gyration RG = 3.1 nm and maximum intramolecular distance Dmax = 10.3 nm. Using the ab initio method and program DAMMIN, we propose a low resolution structure of stabilized covalently cystatin C in solution. Stab-HCC dimer adopts in solution an elongated conformation, which is well reconstructed by the ab initio model.

Surface characterization and orientation interaction between diamond- like carbon layer structure and dimeric liquid crystals

NASA Astrophysics Data System (ADS)

Naradikian, H.; Petrov, M.; Katranchev, B.; Milenov, T.; Tinchev, S.

2017-01-01

Diamond-like carbon (DLC) and amorphous carbon films are very promising type of semiconductor materials. Depending on the hybridization sp2/sp3 ratio, the material’s band gap varies between 0.8 and 3 eV. Moreover carbon films possess different interesting for practice properties: comparable to the Silicon, Diamond like structure has 22-time better thermal conductivity etc. Here we present one type of implementation of such type nanostructure. That is one attempt for orientation of dimeric LC by using of pre-deposited DLC layer with different ratio of sp2/sp3 hybridized carbon content. It could be expected a pronounced π1-π2interaction between s and p orbital levels on the surface and the dimeric ring of LC. We present comparison of surface anchoring strengths of both orientation inter-surfaces DLC/dimeric LC and single wall carbon nanotubes (SWCNT)/dimeric LC. The mechanism of interaction of dimeric LC and activated surfaces with DLC or SWCNT will be discussed. In both cases we have π-π interaction, which in combination with hydrogen bonding, typical for the dimeric LCs, influence the LC alignment. The Raman spectroscopy data evidenced the presence of charge transfer between contacting hexagonal rings of DLC and the C = O groups of the LC molecules.

I222 crystal form of despentapeptide (B26-B30) insulin provides new insights into the properties of monomeric insulin.

PubMed

Whittingham, Jean L; Youshang, Zhang; Záková, Lenka; Dodson, Eleanor J; Turkenburg, Johan P; Brange, Jens; Dodson, G Guy

2006-05-01

Despentapeptide (des-B26-B30) insulin (DPI), an active modified insulin, has been crystallized in the presence of 20% acetic acid pH 2. A crystal structure analysis to 1.8 A spacing (space group I222) revealed that the DPI molecule, which is unable to make beta-strand interactions for physiological dimer formation and is apparently monomeric in solution, formed an alternative lattice-generated dimer. The formation of this dimer involved interactions between surfaces which included the B9-B19 alpha-helices (usually buried by the dimer-dimer contacts within the native hexamer). The two crystallographically independent molecules within the dimer were essentially identical and were similar in conformation to T-state insulin as seen in the T(6) insulin hexamer. An unusual feature of each molecule in the dimer was the presence of two independent conformations at the B-chain C-terminus (residues B20-B25). Both conformations were different from that of native insulin, involving a 3.5 A displacement of the B20-B23 beta-turn and a repositioning of residue PheB25 such that it made close van der Waals contact with the main body of the molecule, appearing to stabilize the B-chain C-terminus.

Differential speciation of ferriprotoporphyrin IX in the presence of free base and diprotic 4-aminoquinoline antimalarial drugs

NASA Astrophysics Data System (ADS)

Gildenhuys, Johandie; Müller, Ronel; le Roex, Tanya; de Villiers, Katherine A.

2017-03-01

The crystal structures of the μ-propionato dimer and π-π dimer of ferriprotoporphyrin IX (Fe(III)PPIX) have been determined by single crystal X-ray diffraction (SCD). Both species were obtained in the presence of the synthetic 4-aminoquinoline antimalarial drug, amodiaquine (AQ). The solution that afforded the μ-propionato dimer contained AQ as a free base (i.e. with both quinoline and terminal amine nitrogen atoms neutral). On the other hand, when the diprotic salt of AQ was included in the crystallization medium, the Fe(III)PPIX π-π dimer was obtained. The structure of the μ-propionato dimer, which is the discrete structural unit that constitutes haemozoin (malaria pigment), is identical to that obtained previously in presence of chloroquine free base. We suspect that the drug, via its two available basic sites, facilitates dissociation of one of the two Fe(III)PPIX propionic acid groups to yield a propionate group that is required for reciprocal coordination of the metal centre to form the centrosymmetric dimer. On the other hand, this proton transfer is not possible when the drug is present as a diprotic salt. In this case, the π-π dimer of Fe(III)PPIX is obtained. In the current study, the π-π dimer of haemin (chloro-Fe(III)PPIX) was obtained as a DMF solvate from non-aqueous aprotic solution (dimethyl formamide and chloroform), however the π-π dimer is also known to exist in aqueous solution (as aqua- or hydroxo-Fe(III)PPIX), where it is purportedly involved in the nucleation of haemozoin. We have been able to unambiguously determine the positions of all non-hydrogen atoms, as well as locate or assign all hydrogen atoms in the structure of the π-π dimer, which was not possible in the SCD structure of haemin reported by Koenig in 1965 owing to disorder in the vinyl and methyl substituents. Interestingly, no disorder in the methyl and vinyl groups is observed in the current structure. Both the π-π and μ-propionato dimers of Fe(III)PPIX are important species in the haem detoxification pathway in the malaria parasite and other blood-feeding organisms, and the structural insight gained in this study may assist target-driven design of new chemotherapeutic agents.

Structural Basis for a Unique ATP Synthase Core Complex from Nanoarcheaum equitans*

PubMed Central

Mohanty, Soumya; Jobichen, Chacko; Chichili, Vishnu Priyanka Reddy; Velázquez-Campoy, Adrián; Low, Boon Chuan; Hogue, Christopher W. V.; Sivaraman, J.

2015-01-01

ATP synthesis is a critical and universal life process carried out by ATP synthases. Whereas eukaryotic and prokaryotic ATP synthases are well characterized, archaeal ATP synthases are relatively poorly understood. The hyperthermophilic archaeal parasite, Nanoarcheaum equitans, lacks several subunits of the ATP synthase and is suspected to be energetically dependent on its host, Ignicoccus hospitalis. This suggests that this ATP synthase might be a rudimentary machine. Here, we report the crystal structures and biophysical studies of the regulatory subunit, NeqB, the apo-NeqAB, and NeqAB in complex with nucleotides, ADP, and adenylyl-imidodiphosphate (non-hydrolysable analog of ATP). NeqB is ∼20 amino acids shorter at its C terminus than its homologs, but this does not impede its binding with NeqA to form the complex. The heterodimeric NeqAB complex assumes a closed, rigid conformation irrespective of nucleotide binding; this differs from its homologs, which require conformational changes for catalytic activity. Thus, although N. equitans possesses an ATP synthase core A3B3 hexameric complex, it might not function as a bona fide ATP synthase. PMID:26370083

Interaction of Constitutive Nitric Oxide Synthases with Cyclooxygenases in Regulation of Bicarbonate Secretion in the Gastric Mucosa.

PubMed

Zolotarev, V A; Andreeva, Yu V; Vershinina, E; Khropycheva, R P

2017-05-01

Neuronal NO synthase blocker 7-nitroindazole suppressed bicarbonate secretion in rat gastric mucosa induced by mild local irritation with 1 M NaCl (pH 2.0). Non-selective blocker of neuronal and endothelial synthases, Nω-nitro-L-arginine (L-NNA), did not affect HCO 3 - production, but inhibited secretion after pretreatment with omeprazole. Non-selective cyclooxygenase blocker indomethacin inhibited HCO 3 - production under conditions of normal synthase activity and in the presence of L-NNA, but was ineffective when co-administered with 7-nitroindazole. It was concluded that neuronal and endothelial synthases are involved in different mechanisms of regulation of HCO 3 - secretion in the gastric mucosa induced by mild irritation. Activation of neuronal synthase stimulated HCO 3 - production, which is mediated mainly through activation of cyclooxygenase. Theoretically, activation of endothelial synthase should suppress HCO 3 - production. The effect of endothelial synthase depends on acid secretion in the stomach and bicarbonate concentration in the submucosa, as it was demonstrated in experiments with intravenous NaHCO 3 infusion.

Nitric Oxide Synthase and Neuronal NADPH Diaphorase are Identical in Brain and Peripheral Tissues

NASA Astrophysics Data System (ADS)

Dawson, Ted M.; Bredt, David S.; Fotuhi, Majid; Hwang, Paul M.; Snyder, Solomon H.

1991-09-01

NADPH diaphorase staining neurons, uniquely resistant to toxic insults and neurodegenerative disorders, have been colocalized with neurons in the brain and peripheral tissue containing nitric oxide synthase (EC 1.14.23.-), which generates nitric oxide (NO), a recently identified neuronal messenger molecule. In the corpus striatum and cerebral cortex, NO synthase immunoreactivity and NADPH diaphorase staining are colocalized in medium to large aspiny neurons. These same neurons colocalize with somatostatin and neuropeptide Y immunoreactivity. NO synthase immunoreactivity and NADPH diaphorase staining are colocalized in the pedunculopontine nucleus with choline acetyltransferase-containing cells and are also colocalized in amacrine cells of the inner nuclear layer and ganglion cells of the retina, myenteric plexus neurons of the intestine, and ganglion cells of the adrenal medulla. Transfection of human kidney cells with NO synthase cDNA elicits NADPH diaphorase staining. The ratio of NO synthase to NADPH diaphorase staining in the transfected cells is the same as in neurons, indicating that NO synthase fully accounts for observed NADPH staining. The identity of neuronal NO synthase and NADPH diaphorase suggests a role for NO in modulating neurotoxicity.

Isolation and functional effects of monoclonal antibodies binding to thymidylate synthase.

PubMed

Jastreboff, M M; Todd, M B; Malech, H L; Bertino, J R

1985-01-29

Monoclonal antibodies against electrophoretically pure thymidylate synthase from HeLa cells have been produced. Antibodies (M-TS-4 and M-TS-9) from hybridoma clones were shown by enzyme-linked immunoassay to recognize thymidylate synthase from a variety of human cell lines, but they did not bind to thymidylate synthase from mouse cell lines. The strongest binding of antibodies was observed to enzyme from HeLa cells. These two monoclonal antibodies bind simultaneously to different antigenic sites on thymidylate synthase purified from HeLa cells, as reflected by a high additivity index and results of cross-linked radioimmunoassay. Both monoclonal antibodies inhibit the activity of thymidylate synthase from human cell lines. The strongest inhibition was observed with thymidylate synthase from HeLa cells. Monoclonal antibody M-TS-9 (IgM subclass) decreased the rate of binding of [3H]FdUMP to thymidylate synthase in the presence of 5,10-methylenetetrahydrofolate while M-TS-4 (IgG1) did not change the rate of ternary complex formation. These data indicate that the antibodies recognize different epitopes on the enzyme molecule.

Testing collapse models by a thermometer

NASA Astrophysics Data System (ADS)

Bahrami, M.

2018-05-01

Collapse models postulate that space is filled with a collapse noise field, inducing quantum Brownian motions, which are dominant during the measurement, thus causing collapse of the wave function. An important manifestation of the collapse noise field, if any, is thermal energy generation, thus disturbing the temperature profile of a system. The experimental investigation of a collapse-driven heating effect has provided, so far, the most promising test of collapse models against standard quantum theory. In this paper, we calculate the collapse-driven heat generation for a three-dimensional multi-atomic Bravais lattice by solving stochastic Heisenberg equations. We perform our calculation for the mass-proportional continuous spontaneous localization collapse model with nonwhite noise. We obtain the temperature distribution of a sphere under stationary-state and insulated surface conditions. However, the exact quantification of the collapse-driven heat-generation effect highly depends on the actual value of cutoff in the collapse noise spectrum.

Friedelin Synthase from Maytenus ilicifolia: Leucine 482 Plays an Essential Role in the Production of the Most Rearranged Pentacyclic Triterpene

PubMed Central

Souza-Moreira, Tatiana M.; Alves, Thaís B.; Pinheiro, Karina A.; Felippe, Lidiane G.; De Lima, Gustavo M. A.; Watanabe, Tatiana F.; Barbosa, Cristina C.; Santos, Vânia A. F. F. M.; Lopes, Norberto P.; Valentini, Sandro R.; Guido, Rafael V. C.; Furlan, Maysa; Zanelli, Cleslei F.

2016-01-01

Among the biologically active triterpenes, friedelin has the most-rearranged structure produced by the oxidosqualene cyclases and is the only one containing a cetonic group. In this study, we cloned and functionally characterized friedelin synthase and one cycloartenol synthase from Maytenus ilicifolia (Celastraceae). The complete coding sequences of these 2 genes were cloned from leaf mRNA, and their functions were characterized by heterologous expression in yeast. The cycloartenol synthase sequence is very similar to other known OSCs of this type (approximately 80% identity), although the M. ilicifolia friedelin synthase amino acid sequence is more related to β-amyrin synthases (65–74% identity), which is similar to the friedelin synthase cloned from Kalanchoe daigremontiana. Multiple sequence alignments demonstrated the presence of a leucine residue two positions upstream of the friedelin synthase Asp-Cys-Thr-Ala-Glu (DCTAE) active site motif, while the vast majority of OSCs identified so far have a valine or isoleucine residue at the same position. The substitution of the leucine residue with valine, threonine or isoleucine in M. ilicifolia friedelin synthase interfered with substrate recognition and lead to the production of different pentacyclic triterpenes. Hence, our data indicate a key role for the leucine residue in the structure and function of this oxidosqualene cyclase. PMID:27874020

Friedelin Synthase from Maytenus ilicifolia: Leucine 482 Plays an Essential Role in the Production of the Most Rearranged Pentacyclic Triterpene

NASA Astrophysics Data System (ADS)

Souza-Moreira, Tatiana M.; Alves, Thaís B.; Pinheiro, Karina A.; Felippe, Lidiane G.; de Lima, Gustavo M. A.; Watanabe, Tatiana F.; Barbosa, Cristina C.; Santos, Vânia A. F. F. M.; Lopes, Norberto P.; Valentini, Sandro R.; Guido, Rafael V. C.; Furlan, Maysa; Zanelli, Cleslei F.

2016-11-01

Among the biologically active triterpenes, friedelin has the most-rearranged structure produced by the oxidosqualene cyclases and is the only one containing a cetonic group. In this study, we cloned and functionally characterized friedelin synthase and one cycloartenol synthase from Maytenus ilicifolia (Celastraceae). The complete coding sequences of these 2 genes were cloned from leaf mRNA, and their functions were characterized by heterologous expression in yeast. The cycloartenol synthase sequence is very similar to other known OSCs of this type (approximately 80% identity), although the M. ilicifolia friedelin synthase amino acid sequence is more related to β-amyrin synthases (65-74% identity), which is similar to the friedelin synthase cloned from Kalanchoe daigremontiana. Multiple sequence alignments demonstrated the presence of a leucine residue two positions upstream of the friedelin synthase Asp-Cys-Thr-Ala-Glu (DCTAE) active site motif, while the vast majority of OSCs identified so far have a valine or isoleucine residue at the same position. The substitution of the leucine residue with valine, threonine or isoleucine in M. ilicifolia friedelin synthase interfered with substrate recognition and lead to the production of different pentacyclic triterpenes. Hence, our data indicate a key role for the leucine residue in the structure and function of this oxidosqualene cyclase.

Multiple defects in muscle glycogen synthase activity contribute to reduced glycogen synthesis in non-insulin dependent diabetes mellitus.

PubMed Central

Thorburn, A W; Gumbiner, B; Bulacan, F; Brechtel, G; Henry, R R

1991-01-01

To define the mechanisms of impaired muscle glycogen synthase and reduced glycogen formation in non-insulin dependent diabetes mellitus (NIDDM), glycogen synthase activity was kinetically analyzed during the basal state and three glucose clamp studies (insulin approximately equal to 300, 700, and 33,400 pmol/liter) in eight matched nonobese NIDDM and eight control subjects. Muscle glycogen content was measured in the basal state and following clamps at insulin levels of 33,400 pmol/liter. NIDDM subjects had glucose uptake matched to controls in each clamp by raising serum glucose to 15-20 mmol/liter. The insulin concentration required to half-maximally activate glycogen synthase (ED50) was approximately fourfold greater for NIDDM than control subjects (1,004 +/- 264 vs. 257 +/- 110 pmol/liter, P less than 0.02) but the maximal insulin effect was similar. Total glycogen synthase activity was reduced approximately 38% and glycogen content was approximately 30% lower in NIDDM. A positive correlation was present between glycogen content and glycogen synthase activity (r = 0.51, P less than 0.01). In summary, defects in muscle glycogen synthase activity and reduced glycogen content are present in NIDDM. NIDDM subjects also have less total glycogen synthase activity consistent with reduced functional mass of the enzyme. These findings and the correlation between glycogen synthase activity and glycogen content support the theory that multiple defects in glycogen synthase activity combine to cause reduced glycogen formation in NIDDM. PMID:1899428

Glycogen synthase activation by sugars in isolated hepatocytes.

PubMed

Ciudad, C J; Carabaza, A; Bosch, F; Gòmez I Foix, A M; Guinovart, J J

1988-07-01

We have investigated the activation by sugars of glycogen synthase in relation to (i) phosphorylase a activity and (ii) changes in the intracellular concentration of glucose 6-phosphate and adenine nucleotides. All the sugars tested in this work present the common denominator of activating glycogen synthase. On the other hand, phosphorylase a activity is decreased by mannose and glucose, unchanged by galactose and xylitol, and increased by tagatose, glyceraldehyde, and fructose. Dihydroxyacetone exerts a biphasic effect on phosphorylase. These findings provide additional evidence proving that glycogen synthase can be activated regardless of the levels of phosphorylase a, clearly establishing that a nonsequential mechanism for the activation of glycogen synthase occurs in liver cells. The glycogen synthase activation state is related to the concentrations of glucose 6-phosphate and adenine nucleotides. In this respect, tagatose, glyceraldehyde, and fructose deplete ATP and increase AMP contents, whereas glucose, mannose, galactose, xylitol, and dihydroxyacetone do not alter the concentration of these nucleotides. In addition, all these sugars, except glyceraldehyde, increase the intracellular content of glucose 6-phosphate. The activation of glycogen synthase by sugars is reflected in decreases on both kinetic constants of the enzyme, M0.5 (for glucose 6-phosphate) and S0.5 (for UDP-glucose). We propose that hepatocyte glycogen synthase is activated by monosaccharides by a mechanism triggered by changes in glucose 6-phosphate and adenine nucleotide concentrations which have been described to modify glycogen synthase phosphatase activity. This mechanism represents a metabolite control of the sugar-induced activation of hepatocyte glycogen synthase.

Cell wall composition contributes to the control of transpiration efficiency in Arabidopsis thaliana.

PubMed

Liang, Yun-Kuan; Xie, Xiaodong; Lindsay, Shona E; Wang, Yi Bing; Masle, Josette; Williamson, Lisa; Leyser, Ottoline; Hetherington, Alistair M

2010-11-01

To identify loci in Arabidopsis involved in the control of transpirational water loss and transpiration efficiency (TE) we carried out an infrared thermal imaging-based screen. We report the identification of a new allele of the Arabidopsis CesA7 cellulose synthase locus designated AtCesA7(irx3-5) involved in the control of TE. Leaves of the AtCesA7(irx3-5) mutant are warmer than the wild type (WT). This is due to reduced stomatal pore widths brought about by guard cells that are significantly smaller than the WT. The xylem of the AtCesA7(irx3-5) mutant is also partially collapsed, and we suggest that the small guard cells in the mutant result from decreased water supply to the developing leaf. We used carbon isotope discrimination to show that TE is increased in AtCesA7(irx3-5) when compared with the WT. Our work identifies a new class of genes that affects TE and raises the possibility that other genes involved in cell wall biosynthesis will have an impact on water use efficiency. © 2010 The Authors. The Plant Journal © 2010 Blackwell Publishing Ltd.

In Planta Recapitulation of Isoprene Synthase Evolution from Ocimene Synthases.

PubMed

Li, Mingai; Xu, Jia; Algarra Alarcon, Alberto; Carlin, Silvia; Barbaro, Enrico; Cappellin, Luca; Velikova, Violeta; Vrhovsek, Urska; Loreto, Francesco; Varotto, Claudio

2017-10-01

Isoprene is the most abundant biogenic volatile hydrocarbon compound naturally emitted by plants and plays a major role in atmospheric chemistry. It has been proposed that isoprene synthases (IspS) may readily evolve from other terpene synthases, but this hypothesis has not been experimentally investigated. We isolated and functionally validated in Arabidopsis the first isoprene synthase gene, AdoIspS, from a monocotyledonous species (Arundo donax L., Poaceae). Phylogenetic reconstruction indicates that AdoIspS and dicots isoprene synthases most likely originated by parallel evolution from TPS-b monoterpene synthases. Site-directed mutagenesis demonstrated invivo the functional and evolutionary relevance of the residues considered diagnostic for IspS function. One of these positions was identified by saturating mutagenesis as a major determinant of substrate specificity in AdoIspS able to cause invivo a dramatic change in total volatile emission from hemi- to monoterpenes and supporting evolution of isoprene synthases from ocimene synthases. The mechanism responsible for IspS neofunctionalization by active site size modulation by a single amino acid mutation demonstrated in this study might be general, as the very same amino acidic position is implicated in the parallel evolution of different short-chain terpene synthases from both angiosperms and gymnosperms. Based on these results, we present a model reconciling in a unified conceptual framework the apparently contrasting patterns previously observed for isoprene synthase evolution in plants. These results indicate that parallel evolution may be driven by relatively simple biophysical constraints, and illustrate the intimate molecular evolutionary links between the structural and functional bases of traits with global relevance. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Polyester synthases: natural catalysts for plastics.

PubMed Central

Rehm, Bernd H A

2003-01-01

Polyhydroxyalkanoates (PHAs) are biopolyesters composed of hydroxy fatty acids, which represent a complex class of storage polyesters. They are synthesized by a wide range of different Gram-positive and Gram-negative bacteria, as well as by some Archaea, and are deposited as insoluble cytoplasmic inclusions. Polyester synthases are the key enzymes of polyester biosynthesis and catalyse the conversion of (R)-hydroxyacyl-CoA thioesters to polyesters with the concomitant release of CoA. These soluble enzymes turn into amphipathic enzymes upon covalent catalysis of polyester-chain formation. A self-assembly process is initiated resulting in the formation of insoluble cytoplasmic inclusions with a phospholipid monolayer and covalently attached polyester synthases at the surface. Surface-attached polyester synthases show a marked increase in enzyme activity. These polyester synthases have only recently been biochemically characterized. An overview of these recent findings is provided. At present, 59 polyester synthase structural genes from 45 different bacteria have been cloned and the nucleotide sequences have been obtained. The multiple alignment of the primary structures of these polyester synthases show an overall identity of 8-96% with only eight strictly conserved amino acid residues. Polyester synthases can been assigned to four classes based on their substrate specificity and subunit composition. The current knowledge on the organization of the polyester synthase genes, and other genes encoding proteins related to PHA metabolism, is compiled. In addition, the primary structures of the 59 PHA synthases are aligned and analysed with respect to highly conserved amino acids, and biochemical features of polyester synthases are described. The proposed catalytic mechanism based on similarities to alpha/beta-hydrolases and mutational analysis is discussed. Different threading algorithms suggest that polyester synthases belong to the alpha/beta-hydrolase superfamily, with a conserved cysteine residue as catalytic nucleophile. This review provides a survey of the known biochemical features of these unique enzymes and their proposed catalytic mechanism. PMID:12954080

Deciphering Dimerization Modes of PAS Domains: Computational and Experimental Analyses of the AhR:ARNT Complex Reveal New Insights Into the Mechanisms of AhR Transformation

PubMed Central

Corrada, Dario; Soshilov, Anatoly A.; Denison, Michael S.

2016-01-01

The Aryl hydrocarbon Receptor (AhR) is a transcription factor that mediates the biochemical response to xenobiotics and the toxic effects of a number of environmental contaminants, including dioxins. Recently, endogenous regulatory roles for the AhR in normal physiology and development have also been reported, thus extending the interest in understanding its molecular mechanisms of activation. Since dimerization with the AhR Nuclear Translocator (ARNT) protein, occurring through the Helix-Loop-Helix (HLH) and PER-ARNT-SIM (PAS) domains, is needed to convert the AhR into its transcriptionally active form, deciphering the AhR:ARNT dimerization mode would provide insights into the mechanisms of AhR transformation. Here we present homology models of the murine AhR:ARNT PAS domain dimer developed using recently available X-ray structures of other bHLH-PAS protein dimers. Due to the different reciprocal orientation and interaction surfaces in the different template dimers, two alternative models were developed for both the PAS-A and PAS-B dimers and they were characterized by combining a number of computational evaluations. Both well-established hot spot prediction methods and new approaches to analyze individual residue and residue-pairwise contributions to the MM-GBSA binding free energies were adopted to predict residues critical for dimer stabilization. On this basis, a mutagenesis strategy for both the murine AhR and ARNT proteins was designed and ligand-dependent DNA binding ability of the AhR:ARNT heterodimer mutants was evaluated. While functional analysis disfavored the HIF2α:ARNT heterodimer-based PAS-B model, most mutants derived from the CLOCK:BMAL1-based AhR:ARNT dimer models of both the PAS-A and the PAS-B dramatically decreased the levels of DNA binding, suggesting this latter model as the most suitable for describing AhR:ARNT dimerization. These novel results open new research directions focused at elucidating basic molecular mechanisms underlying the functional activity of the AhR. PMID:27295348

Deciphering Dimerization Modes of PAS Domains: Computational and Experimental Analyses of the AhR:ARNT Complex Reveal New Insights Into the Mechanisms of AhR Transformation.

PubMed

Corrada, Dario; Soshilov, Anatoly A; Denison, Michael S; Bonati, Laura

2016-06-01

The Aryl hydrocarbon Receptor (AhR) is a transcription factor that mediates the biochemical response to xenobiotics and the toxic effects of a number of environmental contaminants, including dioxins. Recently, endogenous regulatory roles for the AhR in normal physiology and development have also been reported, thus extending the interest in understanding its molecular mechanisms of activation. Since dimerization with the AhR Nuclear Translocator (ARNT) protein, occurring through the Helix-Loop-Helix (HLH) and PER-ARNT-SIM (PAS) domains, is needed to convert the AhR into its transcriptionally active form, deciphering the AhR:ARNT dimerization mode would provide insights into the mechanisms of AhR transformation. Here we present homology models of the murine AhR:ARNT PAS domain dimer developed using recently available X-ray structures of other bHLH-PAS protein dimers. Due to the different reciprocal orientation and interaction surfaces in the different template dimers, two alternative models were developed for both the PAS-A and PAS-B dimers and they were characterized by combining a number of computational evaluations. Both well-established hot spot prediction methods and new approaches to analyze individual residue and residue-pairwise contributions to the MM-GBSA binding free energies were adopted to predict residues critical for dimer stabilization. On this basis, a mutagenesis strategy for both the murine AhR and ARNT proteins was designed and ligand-dependent DNA binding ability of the AhR:ARNT heterodimer mutants was evaluated. While functional analysis disfavored the HIF2α:ARNT heterodimer-based PAS-B model, most mutants derived from the CLOCK:BMAL1-based AhR:ARNT dimer models of both the PAS-A and the PAS-B dramatically decreased the levels of DNA binding, suggesting this latter model as the most suitable for describing AhR:ARNT dimerization. These novel results open new research directions focused at elucidating basic molecular mechanisms underlying the functional activity of the AhR.

Effect of dimer dissociation on activity and thermostability of the alpha-glucuronidase from Geobacillus stearothermophilus: dissecting the different oligomeric forms of family 67 glycoside hydrolases.

PubMed

Shallom, Dalia; Golan, Gali; Shoham, Gil; Shoham, Yuval

2004-10-01

The oligomeric organization of enzymes plays an important role in many biological processes, such as allosteric regulation, conformational stability and thermal stability. alpha-Glucuronidases are family 67 glycosidases that cleave the alpha-1,2-glycosidic bond between 4-O-methyl-D-glucuronic acid and xylose units as part of an array of hemicellulose-hydrolyzing enzymes. Currently, two crystal structures of alpha-glucuronidases are available, those from Geobacillus stearothermophilus (AguA) and from Cellvibrio japonicus (GlcA67A). Both enzymes are homodimeric, but surprisingly their dimeric organization is different, raising questions regarding the significance of dimerization for the enzymes' activity and stability. Structural comparison of the two enzymes suggests several elements that are responsible for the different dimerization organization. Phylogenetic analysis shows that the alpha-glucuronidases AguA and GlcA67A can be classified into two distinct subfamilies of bacterial alpha-glucuronidases, where the dimer-forming residues of each enzyme are conserved only within its own subfamily. It seems that the different dimeric forms of AguA and GlcA67A represent the two alternative dimeric organizations of these subfamilies. To study the biological significance of the dimerization in alpha-glucuronidases, we have constructed a monomeric form of AguA by mutating three of its interface residues (W328E, R329T, and R665N). The activity of the monomer was significantly lower than the activity of the wild-type dimeric AguA, and the optimal temperature for activity of the monomer was around 35 degrees C, compared to 65 degrees C of the wild-type enzyme. Nevertheless, the melting temperature of the monomeric protein, 72.9 degrees C, was almost identical to that of the wild-type, 73.4 degrees C. It appears that the dimerization of AguA is essential for efficient catalysis and that the dissociation into monomers results in subtle conformational changes in the structure which indirectly influence the active site region and reduce the activity. Structural and mechanistic explanations for these effects are discussed.

Effect of Dimer Dissociation on Activity and Thermostability of the α-Glucuronidase from Geobacillus stearothermophilus: Dissecting the Different Oligomeric Forms of Family 67 Glycoside Hydrolases

PubMed Central

Shallom, Dalia; Golan, Gali; Shoham, Gil; Shoham, Yuval

2004-01-01

The oligomeric organization of enzymes plays an important role in many biological processes, such as allosteric regulation, conformational stability and thermal stability. α-Glucuronidases are family 67 glycosidases that cleave the α-1,2-glycosidic bond between 4-O-methyl-d-glucuronic acid and xylose units as part of an array of hemicellulose-hydrolyzing enzymes. Currently, two crystal structures of α-glucuronidases are available, those from Geobacillus stearothermophilus (AguA) and from Cellvibrio japonicus (GlcA67A). Both enzymes are homodimeric, but surprisingly their dimeric organization is different, raising questions regarding the significance of dimerization for the enzymes' activity and stability. Structural comparison of the two enzymes suggests several elements that are responsible for the different dimerization organization. Phylogenetic analysis shows that the α-glucuronidases AguA and GlcA67A can be classified into two distinct subfamilies of bacterial α-glucuronidases, where the dimer-forming residues of each enzyme are conserved only within its own subfamily. It seems that the different dimeric forms of AguA and GlcA67A represent the two alternative dimeric organizations of these subfamilies. To study the biological significance of the dimerization in α-glucuronidases, we have constructed a monomeric form of AguA by mutating three of its interface residues (W328E, R329T, and R665N). The activity of the monomer was significantly lower than the activity of the wild-type dimeric AguA, and the optimal temperature for activity of the monomer was around 35°C, compared to 65°C of the wild-type enzyme. Nevertheless, the melting temperature of the monomeric protein, 72.9°C, was almost identical to that of the wild-type, 73.4°C. It appears that the dimerization of AguA is essential for efficient catalysis and that the dissociation into monomers results in subtle conformational changes in the structure which indirectly influence the active site region and reduce the activity. Structural and mechanistic explanations for these effects are discussed. PMID:15466046

Ligand-induced perturbation of the HIF-2α:ARNT dimer dynamics

PubMed Central

Motta, Stefano

2018-01-01

Hypoxia inducible factors (HIFs) are transcription factors belonging to the basic helix−loop−helix PER-ARNT-SIM (bHLH-PAS) protein family with a role in sensing oxygen levels in the cell. Under hypoxia, the HIF-α degradation pathway is blocked and dimerization with the aryl hydrocarbon receptor nuclear translocator (ARNT) makes HIF-α transcriptionally active. Due to the common hypoxic environment of tumors, inhibition of this mechanism by destabilization of HIF-α:ARNT dimerization has been proposed as a promising therapeutic strategy. Following the discovery of a druggable cavity within the PAS-B domain of HIF-2α, research efforts have been directed to identify artificial ligands that can impair heterodimerization. Although the crystallographic structures of the HIF-2α:ARNT complex have elucidated the dimer architecture and the 0X3-inhibitor placement within the HIF-2α PAS-B, unveiling the inhibition mechanism requires investigation of how ligand-induced perturbations could dynamically propagate through the structure and affect dimerization. To this end, we compared evolutionary features, intrinsic dynamics and energetic properties of the dimerization interfaces of HIF-2α:ARNT in both the apo and holo forms. Residue conservation analysis highlighted inter-domain connecting elements that have a role in dimerization. Analysis of domain contributions to the dimerization energy demonstrated the importance of bHLH and PAS-A of both partners and of HIF-2α PAS-B domain in dimer stabilization. Among quaternary structure oscillations revealed by Molecular Dynamics simulations, the hinge-bending motion of the ARNT PAS-B domain around the flexible PAS-A/PAS-B linker supports a general model for ARNT dimerization in different heterodimers. Comparison of the HIF-2α:ARNT dynamics in the apo and 0X3-bound forms indicated a model of inhibition where the HIF-2α-PAS-B interfaces are destabilised as a result of water-bridged ligand-protein interactions and these local effects allosterically propagate to perturb the correlated motions of the domains and inter-domain communication. These findings will guide the design of improved inhibitors to contrast cell survival in tumor masses. PMID:29489822