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Sample records for affect stability binding

  1. Stability of the Octameric Structure Affects Plasminogen-Binding Capacity of Streptococcal Enolase

    PubMed Central

    Law, Ruby H. P.; Casey, Lachlan W.; Valkov, Eugene; Bertozzi, Carlo; Stamp, Anna; Jovcevski, Blagojce; Aquilina, J. Andrew; Whisstock, James C.; Walker, Mark J.; Kobe, Bostjan

    2015-01-01

    Group A Streptococcus (GAS) is a human pathogen that has the potential to cause invasive disease by binding and activating human plasmin(ogen). Streptococcal surface enolase (SEN) is an octameric α-enolase that is localized at the GAS cell surface. In addition to its glycolytic role inside the cell, SEN functions as a receptor for plasmin(ogen) on the bacterial surface, but the understanding of the molecular basis of plasmin(ogen) binding is limited. In this study, we determined the crystal and solution structures of GAS SEN and characterized the increased plasminogen binding by two SEN mutants. The plasminogen binding ability of SENK312A and SENK362A is ~2- and ~3.4-fold greater than for the wild-type protein. A combination of thermal stability assays, native mass spectrometry and X-ray crystallography approaches shows that increased plasminogen binding ability correlates with decreased stability of the octamer. We propose that decreased stability of the octameric structure facilitates the access of plasmin(ogen) to its binding sites, leading to more efficient plasmin(ogen) binding and activation. PMID:25807546

  2. “DNA Binding Region” of BRCA1 Affects Genetic Stability through modulating the Intra-S-Phase Checkpoint

    PubMed Central

    Masuda, Takaaki; Xu, Xiaoling; Dimitriadis, Emilios K.; Lahusen, Tyler; Deng, Chu-Xia

    2016-01-01

    The breast cancer associated gene 1 (BRCA1) contains 3 domains: an N-terminal RING domain with ubiquitin E3 ligase activity, C-terminal BRCT protein interaction domain and a central region. RING and BRCT domains are well characterized, yet the function of the central region remains unclear. In this study, we identified an essential DNA binding region (DBR: 421-701 amino acids) within the central region of human BRCA1, and found that BRCA1 brings DNA together and preferably binds to splayed-arm DNA in a sequence-independent manner. To investigate the biological role of the DBR, we generated mouse ES cells, which lack the DBR (ΔDBR) by using the TALEN method. The ΔDBR cells exhibited decreased survival as compared to the wild type (WT) cells treated with a PARP inhibitor, however they have an intact ability to conduct DNA repair mediated by homologous recombination (HR). The ΔDBR cells continued to incorporate more EdU in the presence of hydroxyurea (HU), which causes replication stress and exhibited reduced viability than the WT cells. Moreover, phosphorylation of CHK1, which regulates the intra-S phase checkpoint, was moderately decreased in ΔDBR cells. These data suggest that DNA binding by BRCA1 affects the stability of DNA replication folks, resulting in weakened intra-S-phase checkpoint control in the ΔDBR cells. The ΔDBR cells also exhibited an increased number of abnormal chromosome structures as compared with WT cells, indicating that the ΔDBR cells have increased genetic instability. Thus, we demonstrated that the DBR of BRCA1 modulates genetic stability through the intra-S-phase checkpoint activated by replication stress. PMID:26884712

  3. Amino Acid Substitutions That Affect Receptor Binding and Stability of the Hemagglutinin of Influenza A/H7N9 Virus.

    PubMed

    Schrauwen, Eefje J A; Richard, Mathilde; Burke, David F; Rimmelzwaan, Guus F; Herfst, Sander; Fouchier, Ron A M

    2016-01-01

    Receptor-binding preference and stability of hemagglutinin have been implicated as crucial determinants of airborne transmission of influenza viruses. Here, amino acid substitutions previously identified to affect these traits were tested in the context of an A/H7N9 virus. Some combinations of substitutions, most notably G219S and K58I, resulted in relatively high affinity for α2,6-linked sialic acid receptor and acid and temperature stability. Thus, the hemagglutinin of the A/H7N9 virus may adopt traits associated with airborne transmission. PMID:26792744

  4. COX7A2L Is a Mitochondrial Complex III Binding Protein that Stabilizes the III2+IV Supercomplex without Affecting Respirasome Formation.

    PubMed

    Pérez-Pérez, Rafael; Lobo-Jarne, Teresa; Milenkovic, Dusanka; Mourier, Arnaud; Bratic, Ana; García-Bartolomé, Alberto; Fernández-Vizarra, Erika; Cadenas, Susana; Delmiro, Aitor; García-Consuegra, Inés; Arenas, Joaquín; Martín, Miguel A; Larsson, Nils-Göran; Ugalde, Cristina

    2016-08-30

    Mitochondrial respiratory chain (MRC) complexes I, III, and IV associate into a variety of supramolecular structures known as supercomplexes and respirasomes. While COX7A2L was originally described as a supercomplex-specific factor responsible for the dynamic association of complex IV into these structures to adapt MRC function to metabolic variations, this role has been disputed. Here, we further examine the functional significance of COX7A2L in the structural organization of the mammalian respiratory chain. As in the mouse, human COX7A2L binds primarily to free mitochondrial complex III and, to a minor extent, to complex IV to specifically promote the stabilization of the III2+IV supercomplex without affecting respirasome formation. Furthermore, COX7A2L does not affect the biogenesis, stabilization, and function of the individual oxidative phosphorylation complexes. These data show that independent regulatory mechanisms for the biogenesis and turnover of different MRC supercomplex structures co-exist. PMID:27545886

  5. Changes in thermodynamic stability of von Willebrand factor differentially affect the force-dependent binding to platelet GPIbalpha.

    PubMed

    Auton, Matthew; Sedlák, Erik; Marek, Jozef; Wu, Tao; Zhu, Cheng; Cruz, Miguel A

    2009-07-22

    In circulation, plasma glycoprotein von Willebrand Factor plays an important role in hemostasis and in pathological thrombosis under hydrodynamic forces. Mutations in the A1 domain of von Willebrand factor cause the hereditary types 2B and 2M von Willebrand disease that either enhance (2B) or inhibit (2M) the interaction of von Willebrand factor with the platelet receptor glycoprotein Ibalpha. To understand how type 2B and 2M mutations cause clinically opposite phenotypes, we use a combination of protein unfolding thermodynamics and atomic force microscopy to assess the effects of two type 2B mutations (R1306Q and I1309V) and a type 2M mutation (G1324S) on the conformational stability of the A1 domain and the single bond dissociation kinetics of the A1-GPIbalpha interaction. At physiological temperature, the type 2B mutations destabilize the structure of the A1 domain and shift the A1-GPIbalpha catch to slip bonding to lower forces. Conversely, the type 2M mutation stabilizes the structure of the A1 domain and shifts the A1-GPIbalpha catch to slip bonding to higher forces. As a function of increasing A1 domain stability, the bond lifetime at low force decreases and the critical force required for maximal bond lifetime increases. Our results are able to distinguish the clinical phenotypes of these naturally occurring mutations from a thermodynamic and biophysical perspective that provides a quantitative description of the allosteric coupling of A1 conformational stability with the force dependent catch to slip bonding between A1 and GPIbalpha. PMID:19619477

  6. Supersonic Wave Interference Affecting Stability

    NASA Technical Reports Server (NTRS)

    Love, Eugene S.

    1958-01-01

    Some of the significant interference fields that may affect stability of aircraft at supersonic speeds are briefly summarized. Illustrations and calculations are presented to indicate the importance of interference fields created by wings, bodies, wing-body combinations, jets, and nacelles.

  7. Stabilized Interleukin-6 receptor binding RNA aptamers

    PubMed Central

    Meyer, Cindy; Berg, Katharina; Eydeler-Haeder, Katja; Lorenzen, Inken; Grötzinger, Joachim; Rose-John, Stefan; Hahn, Ulrich

    2014-01-01

    Interleukin-6 (IL-6) is a multifunctional cytokine that is involved in the progression of various inflammatory diseases, such as rheumatoid arthritis and certain cancers; for example, multiple myeloma or hepatocellular carcinoma. To interfere with IL-6-dependent diseases, targeting IL-6 receptor (IL-6R)-presenting tumor cells using aptamers might be a valuable strategy to broaden established IL-6- or IL-6R-directed treatment regimens. Recently, we reported on the in vitro selection of RNA aptamers binding to the human IL-6 receptor (IL-6R) with nanomolar affinity. One aptamer, namely AIR-3A, was 19 nt in size and able to deliver bulky cargos into IL-6R-presenting cells. As AIR-3A is a natural RNA molecule, its use for in vivo applications might be limited due to its susceptibility to ubiquitous ribonucleases. Aiming at more robust RNA aptamers targeting IL-6R, we now report on the generation of stabilized RNA aptamers for potential in vivo applications. The new 2'-F-modified RNA aptamers bind to IL-6R via its extracellular portion with low nanomolar affinity comparable to the previously identified unmodified counterpart. Aptamers do not interfere with the IL-6 receptor complex formation. The work described here represents one further step to potentially apply stabilized IL-6R-binding RNA aptamers in IL-6R-connected diseases, like multiple myeloma and hepatocellular carcinoma. PMID:24440854

  8. Structural stabilization of GTP-binding domains in circularly permuted GTPases: Implications for RNA binding

    PubMed Central

    Anand, Baskaran; Verma, Sunil Kumar; Prakash, Balaji

    2006-01-01

    GTP hydrolysis by GTPases requires crucial residues embedded in a conserved G-domain as sequence motifs G1–G5. However, in some of the recently identified GTPases, the motif order is circularly permuted. All possible circular permutations were identified after artificially permuting the classical GTPases and subjecting them to profile Hidden Markov Model searches. This revealed G4–G5–G1–G2–G3 as the only possible circular permutation that can exist in nature. It was also possible to recognize a structural rationale for the absence of other permutations, which either destabilize the invariant GTPase fold or disrupt regions that provide critical residues for GTP binding and hydrolysis, such as Switch-I and Switch-II. The circular permutation relocates Switch-II to the C-terminus and leaves it unfastened, thus affecting GTP binding and hydrolysis. Stabilizing this region would require the presence of an additional domain following Switch-II. Circularly permuted GTPases (cpGTPases) conform to such a requirement and always possess an ‘anchoring’ C-terminal domain. There are four sub-families of cpGTPases, of which three possess an additional domain N-terminal to the G-domain. The biochemical function of these domains, based on available experimental reports and domain recognition analysis carried out here, are suggestive of RNA binding. The features that dictate RNA binding are unique to each subfamily. It is possible that RNA-binding modulates GTP binding or vice versa. In addition, phylogenetic analysis indicates a closer evolutionary relationship between cpGTPases and a set of universally conserved bacterial GTPases that bind the ribosome. It appears that cpGTPases are RNA-binding proteins possessing a means to relate GTP binding to RNA binding. PMID:16648363

  9. Changes to gonadotropin-releasing hormone (GnRH) receptor extracellular loops differentially affect GnRH analog binding and activation: evidence for distinct ligand-stabilized receptor conformations.

    PubMed

    Pfleger, Kevin D G; Pawson, Adam J; Millar, Robert P

    2008-06-01

    GnRH and its structural variants bind to GnRH receptors from different species with different affinities and specificities. By investigating chimeric receptors that combine regions of mammalian and nonmammalian GnRH receptors, a greater understanding of how different domains influence ligand binding and receptor activation can be achieved. Using human-catfish and human-chicken chimeric receptors, we demonstrate the importance of extracellular loop conformation for ligand binding and agonist potency, providing further evidence for GnRH and GnRH II stabilization of distinct active receptor conformations. We demonstrate examples of GnRH receptor gain-of-function mutations that apparently improve agonist potency independently of affinity, implicating a role for extracellular loops in stabilizing the inactive receptor conformation. We also show that entire extracellular loop substitution can overcome the detrimental effects of localized mutations, thereby demonstrating the importance of considering the conformation of entire domains when drawing conclusions from point-mutation studies. Finally, we present evidence implicating the configuration of extracellular loops 2 and 3 in combination differentiating GnRH analog binding modes. Because there are two endogenous forms of GnRH ligand but only one functional form of full-length GnRH receptor in humans, understanding how GnRH and GnRH II can elicit distinct functional effects through the same receptor is likely to provide important insights into how these ligands can have differential effects in both physiological and pathological situations. PMID:18356273

  10. Pentoxifylline affects idarubicin binding to DNA.

    PubMed

    Gołuński, Grzegorz; Borowik, Agnieszka; Lipińska, Andrea; Romanik, Monika; Derewońko, Natalia; Woziwodzka, Anna; Piosik, Jacek

    2016-04-01

    Anticancer drug idarubicin - derivative of doxorubicin - is commonly used in treatment of numerous cancer types. However, in contrast to doxorubicin, its biophysical properties are not well established yet. Additionally, potential direct interactions of idarubicin with other biologically active aromatic compounds, such as pentoxifylline - representative of methylxanthines - were not studied at all. Potential formation of such hetero-aggregates may result in sequestration of the anticancer drug and, in consequence, reduction of its biological activity. This work provide description of the idarubicin biophysical properties as well as assess influence of pentoxifylline on idarubicin interactions with DNA. To achieve these goals we employed spectrophotometric methods coupled with analysis with the appropriate mathematical models as well as flow cytometry and Ames test. Obtained results show influence of pentoxifylline on idarubicin binding to DNA and are well in agreement with the data previously published for other aromatic ligands. Additionally it may be hypothesized that direct interactions between idarubicin and pentoxifylline may influence the anticancer drug biological activity. PMID:26921593

  11. Stabilized sulfur binding using activated fillers

    DOEpatents

    Kalb, Paul D.; Vagin, Vyacheslav P.; Vagin, Sergey P.

    2015-07-21

    A method of making a stable, sulfur binding composite comprising impregnating a solid aggregate with an organic modifier comprising unsaturated hydrocarbons with at least one double or triple covalent bond between adjacent carbon atoms to create a modifier-impregnated aggregate; heating and drying the modifier-impregnated aggregate to activate the surface of the modifier-impregnated aggregate for reaction with sulfur.

  12. Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins

    NASA Technical Reports Server (NTRS)

    Schoeffler, Allyn J.; Ruiz, Carmen R.; Joubert, Allison M.; Yang, Xuemei; LiCata, Vince J.

    2003-01-01

    Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.

  13. Stability and Sugar Recognition Ability of Ricin-Like Carbohydrate Binding Domains

    SciTech Connect

    Yao, Jianzhuang; Nellas, Ricky B; Glover, Mary M; Shen, Tongye

    2011-01-01

    Lectins are a class of proteins known for their novel binding to saccharides. Understanding this sugar recognition process can be crucial in creating structure-based designs of proteins with various biological roles. We focus on the sugar binding of a particular lectin, ricin, which has two -trefoil carbohydrate-binding domains (CRDs) found in several plant protein toxins. The binding ability of possible sites of ricin-like CRD has been puzzling. The apo and various (multiple) ligand-bound forms of the sugar-binding domains of ricin were studied by molecular dynamics simulations. By evaluating structural stability, hydrogen bond dynamics, flexibility, and binding energy, we obtained a detailed picture of the sugar recognition of the ricin-like CRD. Unlike what was previously believed, we found that the binding abilities of the two known sites are not independent of each other. The binding ability of one site is positively affected by the other site. While the mean positions of different binding scenarios are not altered significantly, the flexibility of the binding pockets visibly decreases upon multiple ligand binding. This change in flexibility seems to be the origin of the binding cooperativity. All the hydrogen bonds that are strong in the monoligand state are also strong in the double-ligand complex, although the stability is much higher in the latter form due to cooperativity. These strong hydrogen bonds in a monoligand state are deemed to be the essential hydrogen bonds. Furthermore, by examining the structural correlation matrix, the two domains are structurally one entity. Galactose hydroxyl groups, OH4 and OH3, are the most critical parts in both site 1 and site 2 recognition.

  14. Organic additives stabilize RNA aptamer binding of malachite green.

    PubMed

    Zhou, Yubin; Chi, Hong; Wu, Yuanyuan; Marks, Robert S; Steele, Terry W J

    2016-11-01

    Aptamer-ligand binding has been utilized for biological applications due to its specific binding and synthetic nature. However, the applications will be limited if the binding or the ligand is unstable. Malachite green aptamer (MGA) and its labile ligand malachite green (MG) were found to have increasing apparent dissociation constants (Kd) as determined through the first order rate loss of emission intensity of the MGA-MG fluorescent complex. The fluorescent intensity loss was hypothesized to be from the hydrolysis of MG into malachite green carbinol base (MGOH). Random screening organic additives were found to reduce or retain the fluorescence emission and the calculated apparent Kd of MGA-MG binding. The protective effect became more apparent as the percentage of organic additives increased up to 10% v/v. The mechanism behind the organic additive protective effects was primarily from a ~5X increase in first order rate kinetics of MGOH→MG (kMGOH→MG), which significantly changed the equilibrium constant (Keq), favoring the generation of MG, versus MGOH without organic additives. A simple way has been developed to stabilize the apparent Kd of MGA-MG binding over 24h, which may be beneficial in stabilizing other triphenylmethane or carbocation ligand-aptamer interactions that are susceptible to SN1 hydrolysis. PMID:27591602

  15. Stability and Change in Affect among Centenarians

    ERIC Educational Resources Information Center

    Martin, Peter; da Rosa, Grace; Margrett, Jennifer A.; Garasky, Steven; Franke, Warren

    2012-01-01

    Much information is available about physical and functional health among very old adults, but little knowledge exists about the mental health and mental health changes in very late life. This study reports findings concerning positive and negative affect changes among centenarians. Nineteen centenarians from a Midwestern state participated in four…

  16. Mouthrinses affect color stability of composite

    PubMed Central

    Baig, Arshia Rashid; Shori, Deepa Deepak; Shenoi, Pratima Ramakrishna; Ali, Syed Navid; Shetti, Sanjay; Godhane, Alkesh

    2016-01-01

    Aim: The aim of this study is to evaluate the effect of alcohol and nonalcohol containing mouth rinses on the color stability of a nanofilled resin composite restorative material. Materials and Methods: A total of 120 samples of a nanofilled resin composite material (Tetric N-Ceram, Ivoclar Vivadent AG, FL-9494 Schaan/Liechtenstein) were prepared and immersed in distilled water for 24 h. Baseline color values were recorded using Color Spectrophotometer 3600d (Konica Minolta, Japan). Samples were then randomly distributed into six groups: Group I - distilled water (control group), Group II - Listerine, Group III - Eludril, Group IV - Phosflur, Group V - Amflor, and Group VI - Rexidin. The postimmersion color values of the samples were then recorded, respectively. Results: Significant reduction in the mean color value (before and after immersion) was observed in nonalcohol containing mouth rinses (P < 0.001). Conclusion: All mouthrinses tested in the present in-vitro study caused a color shift in the nanofilled resin composite restorative material, but the color shift was dependent on the material and the mouthrinse used. Group VI (Rexidin) showed maximum color change. PMID:27563186

  17. Effects of spermine binding on Taxol-stabilized microtubules

    NASA Astrophysics Data System (ADS)

    Cheng, Shengfeng; Regmi, Chola

    Previous studies have shown that polyamines such as spermine present in cells at physiological concentrations can facilitate the polymerization of tubulins into microtubules (MTs). A recent experiment demonstrates that in the presence of high-concentration spermine, Taxol-stabilized MTs undergo a shape transformation into inverted tubulin tubules (ITTs), the outside surface of which corresponds to the inside surface of a regular MT. However, the molecular mechanism underlying the shape transformation of MTs into ITTs is unclear. We perform all atom molecular dynamics simulations on Taxol-stabilized MT sheets containing two protofilaments surrounded by spermine ions. The spermine concentration is varied from 0 to 25mM to match the range probed experimentally. We identify important spermine binding regions on the MT surface and the influence of the spermine binding on the structure and dynamics of MTs. In contrast to Taxol, our results show that spermine binding seems to decrease the flexibility of tubulin proteins, resulting in weaker tubulin-tubulin contacts and promoting the bending of protofilaments into curved protofilaments, inverted rings, and eventually inverted tubules.

  18. Plant ecology. Anthropogenic environmental changes affect ecosystem stability via biodiversity.

    PubMed

    Hautier, Yann; Tilman, David; Isbell, Forest; Seabloom, Eric W; Borer, Elizabeth T; Reich, Peter B

    2015-04-17

    Human-driven environmental changes may simultaneously affect the biodiversity, productivity, and stability of Earth's ecosystems, but there is no consensus on the causal relationships linking these variables. Data from 12 multiyear experiments that manipulate important anthropogenic drivers, including plant diversity, nitrogen, carbon dioxide, fire, herbivory, and water, show that each driver influences ecosystem productivity. However, the stability of ecosystem productivity is only changed by those drivers that alter biodiversity, with a given decrease in plant species numbers leading to a quantitatively similar decrease in ecosystem stability regardless of which driver caused the biodiversity loss. These results suggest that changes in biodiversity caused by drivers of environmental change may be a major factor determining how global environmental changes affect ecosystem stability. PMID:25883357

  19. Effect of BET Missense Mutations on Bromodomain Function, Inhibitor Binding and Stability.

    PubMed

    Lori, Laura; Pasquo, Alessandra; Lori, Clorinda; Petrosino, Maria; Chiaraluce, Roberta; Tallant, Cynthia; Knapp, Stefan; Consalvi, Valerio

    2016-01-01

    Lysine acetylation is an important epigenetic mark regulating gene transcription and chromatin structure. Acetylated lysine residues are specifically recognized by bromodomains, small protein interaction modules that read these modification in a sequence and acetylation dependent way regulating the recruitment of transcriptional regulators and chromatin remodelling enzymes to acetylated sites in chromatin. Recent studies revealed that bromodomains are highly druggable protein interaction domains resulting in the development of a large number of bromodomain inhibitors. BET bromodomain inhibitors received a lot of attention in the oncology field resulting in the rapid translation of early BET bromodomain inhibitors into clinical studies. Here we investigated the effects of mutations present as polymorphism or found in cancer on BET bromodomain function and stability and the influence of these mutants on inhibitor binding. We found that most BET missense mutations localize to peripheral residues in the two terminal helices. Crystal structures showed that the three dimensional structure is not compromised by these mutations but mutations located in close proximity to the acetyl-lysine binding site modulate acetyl-lysine and inhibitor binding. Most mutations affect significantly protein stability and tertiary structure in solution, suggesting new interactions and an alternative network of protein-protein interconnection as a consequence of single amino acid substitution. To our knowledge this is the first report studying the effect of mutations on bromodomain function and inhibitor binding. PMID:27403962

  20. Effect of BET Missense Mutations on Bromodomain Function, Inhibitor Binding and Stability

    PubMed Central

    Lori, Laura; Pasquo, Alessandra; Lori, Clorinda; Petrosino, Maria; Chiaraluce, Roberta; Tallant, Cynthia; Knapp, Stefan; Consalvi, Valerio

    2016-01-01

    Lysine acetylation is an important epigenetic mark regulating gene transcription and chromatin structure. Acetylated lysine residues are specifically recognized by bromodomains, small protein interaction modules that read these modification in a sequence and acetylation dependent way regulating the recruitment of transcriptional regulators and chromatin remodelling enzymes to acetylated sites in chromatin. Recent studies revealed that bromodomains are highly druggable protein interaction domains resulting in the development of a large number of bromodomain inhibitors. BET bromodomain inhibitors received a lot of attention in the oncology field resulting in the rapid translation of early BET bromodomain inhibitors into clinical studies. Here we investigated the effects of mutations present as polymorphism or found in cancer on BET bromodomain function and stability and the influence of these mutants on inhibitor binding. We found that most BET missense mutations localize to peripheral residues in the two terminal helices. Crystal structures showed that the three dimensional structure is not compromised by these mutations but mutations located in close proximity to the acetyl-lysine binding site modulate acetyl-lysine and inhibitor binding. Most mutations affect significantly protein stability and tertiary structure in solution, suggesting new interactions and an alternative network of protein-protein interconnection as a consequence of single amino acid substitution. To our knowledge this is the first report studying the effect of mutations on bromodomain function and inhibitor binding. PMID:27403962

  1. Stage structure alters how complexity affects stability of ecological networks

    USGS Publications Warehouse

    Rudolf, V.H.W.; Lafferty, Kevin D.

    2011-01-01

    Resolving how complexity affects stability of natural communities is of key importance for predicting the consequences of biodiversity loss. Central to previous stability analysis has been the assumption that the resources of a consumer are substitutable. However, during their development, most species change diets; for instance, adults often use different resources than larvae or juveniles. Here, we show that such ontogenetic niche shifts are common in real ecological networks and that consideration of these shifts can alter which species are predicted to be at risk of extinction. Furthermore, niche shifts reduce and can even reverse the otherwise stabilizing effect of complexity. This pattern arises because species with several specialized life stages appear to be generalists at the species level but act as sequential specialists that are hypersensitive to resource loss. These results suggest that natural communities are more vulnerable to biodiversity loss than indicated by previous analyses.

  2. Mutation of the Zinc-Binding Metalloprotease Motif Affects Bacteroides fragilis Toxin Activity but Does Not Affect Propeptide Processing

    PubMed Central

    Franco, Augusto A.; Buckwold, Simy L.; Shin, Jai W.; Ascon, Miguel; Sears, Cynthia L.

    2005-01-01

    To evaluate the role of the zinc-binding metalloprotease in Bacteroides fragilis toxin (BFT) processing and activity, the zinc-binding consensus sequences (H348, E349, H352, G355, H358, and M366) were mutated by site-directed-mutagenesis. Our results indicated that single point mutations in the zinc-binding metalloprotease motif do not affect BFT processing but do reduce or eliminate BFT biologic activity in vitro. PMID:16041055

  3. Local Perturbations Do Not Affect Stability of Laboratory Fruitfly Metapopulations

    PubMed Central

    Dey, Sutirth; Joshi, Amitabh

    2007-01-01

    Background A large number of theoretical studies predict that the dynamics of spatially structured populations (metapopulations) can be altered by constant perturbations to local population size. However, these studies presume large metapopulations inhabiting noise-free, zero-extinction environments, and their predictions have never been empirically verified. Methodology/Principal Findings Here we report an empirical study on the effects of localized perturbations on global dynamics and stability, using fruitfly metapopulations in the laboratory. We find that constant addition of individuals to a particular subpopulation in every generation stabilizes that subpopulation locally, but does not have any detectable effect on the dynamics and stability of the metapopulation. Simulations of our experimental system using a simple but widely applicable model of population dynamics were able to recover the empirical findings, indicating the generality of our results. We then simulated the possible consequences of perturbing more subpopulations, increasing the strength of perturbations, and varying the rate of migration, but found that none of these conditions were expected to alter the outcomes of our experiments. Finally, we show that our main results are robust to the presence of local extinctions in the metapopulation. Conclusions/Significance Our study shows that localized perturbations are unlikely to affect the dynamics of real metapopulations, a finding that has cautionary implications for ecologists and conservation biologists faced with the problem of stabilizing unstable metapopulations in nature. PMID:17311100

  4. Meningococcal Factor H Binding Protein Vaccine Antigens with Increased Thermal Stability and Decreased Binding of Human Factor H.

    PubMed

    Rossi, Raffaella; Konar, Monica; Beernink, Peter T

    2016-06-01

    Neisseria meningitidis causes cases of bacterial meningitis and sepsis. Factor H binding protein (FHbp) is a component of two licensed meningococcal serogroup B vaccines. FHbp recruits the complement regulator factor H (FH) to the bacterial surface, which inhibits the complement alternative pathway and promotes immune evasion. Binding of human FH impairs the protective antibody responses to FHbp, and mutation of FHbp to decrease binding of FH can increase the protective responses. In a previous study, we identified two amino acid substitutions in FHbp variant group 2 that increased its thermal stability by 21°C and stabilized epitopes recognized by protective monoclonal antibodies (MAbs). Our hypothesis was that combining substitutions to increase stability and decrease FH binding would increase protective antibody responses in the presence of human FH. In the present study, we generated four new FHbp single mutants that decreased FH binding and retained binding of anti-FHbp MAbs and immunogenicity in wild-type mice. From these mutants, we selected two, K219N and G220S, to combine with the stabilized double-mutant FHbp antigen. The two triple mutants decreased FH binding >200-fold, increased the thermal stability of the N-terminal domain by 21°C, and bound better to an anti-FHbp MAb than the wild-type FHbp. In human-FH-transgenic mice, the FHbp triple mutants elicited 8- to 15-fold-higher protective antibody responses than the wild-type FHbp antigen. Collectively, the data suggest that mutations to eliminate binding of human FH and to promote conformational stability act synergistically to optimize FHbp immunogenicity. PMID:27021245

  5. Free-surface stability criterion as affected by velocity distribution

    USGS Publications Warehouse

    Cheng-Lung, Chen

    1995-01-01

    This paper examines how the velocity distribution of flow in open channels affects the kinematic and dynamic wave velocities, from which the various forms of the Vedernikov number V can be formulated. When V >1, disturbances created in open-channel flow will amplify in the form of roll waves; when V <1, some (though not all) disturbances will attenuate. A study of the Vedernikov stability criterion reveals that it can be readily deduced within the framework of the kinematic and dynamic wave theories by comparing the kinematic wave velocity to the corresponding dynamic wave velocity. -from Author

  6. Radioiodination of chicken luteinizing hormone without affecting receptor binding potency

    SciTech Connect

    Kikuchi, M.; Ishii, S. )

    1989-12-01

    By improving the currently used lactoperoxidase method, we were able to obtain radioiodinated chicken luteinizing hormone (LH) that shows high specific binding and low nonspecific binding to a crude plasma membrane fraction of testicular cells of the domestic fowl and the Japanese quail, and to the ovarian granulosa cells of the Japanese quail. The change we made from the original method consisted of (1) using chicken LH for radioiodination that was not only highly purified but also retained a high receptor binding potency; (2) controlling the level of incorporation of radioiodine into chicken LH molecules by employing a short reaction time and low temperature; and (3) fractionating radioiodinated chicken LH further by gel filtration using high-performance liquid chromatography. Specific radioactivity of the final {sup 125}I-labeled chicken LH preparation was 14 microCi/micrograms. When specific binding was 12-16%, nonspecific binding was as low as 2-4% in the gonadal receptors. {sup 125}I-Labeled chicken LH was displaced by chicken LH and ovine LH but not by chicken follicle-stimulating hormone. The equilibrium association constant of quail testicular receptor was 3.6 x 10(9) M-1. We concluded that chicken LH radioiodinated by the present method is useful for studies of avian LH receptors.

  7. Occupation of nucleotide in the binding pocket is critical to the stability of Rab11A.

    PubMed

    Shin, Young-Cheul; Kim, Chang Min; Choi, Jae Young; Jeon, Ju-Hong; Park, Hyun Ho

    2016-04-01

    The Ras superfamily of small G proteins is a family of guanosine triphosphatases (GTPases) and each GTPase has conserved amino acid sequences in the enzymatic active site that are responsible for specific interactions with GDP and GTP molecules. Rab GTPases, which belong to the Ras superfamily, are key regulators of intracellular vesicle trafficking via the recruitment of effector molecules. Here, we purified wild type, active mutant and inactive mutant of Rab11A. In this process, we found that the inactive mutant (Rab11A S25N) had low stability compared with wild type and other mutants. Further analysis revealed that the stability of Rab11A S25N is dependent on the occupation of GDP in the nucleotide binding pocket of the protein. We found that the stability of Rab11A S25N is affected by the presence of GDP, not other nucleotides, and is independent of pH or salt in FPLC buffer. Our results provide a better understanding of how GTPase can be stable under in vitro conditions without effector proteins and how proper substrate/cofactor coordination is crucial to the stability of Rab11A. Successful purification and proposed purification methods will provide a valuable guide for investigation of other small GTPase proteins. PMID:26767484

  8. Factors affecting hazardous waste solidification/stabilization: a review.

    PubMed

    Malviya, Rachana; Chaudhary, Rubina

    2006-09-01

    Solidification/stabilization is accepted as a well-established disposal technique for hazardous waste. As a result many different types of hazardous wastes are treated with different binders. The S/S products have different property from waste and binders individually. The effectiveness of S/S process is studied by physical, chemical and microstructural methods. This paper summarizes the effect of different waste stream such as heavy metals bearing sludge, filter cake, fly ash, and slag on the properties of cement and other binders. The factors affecting strength development is studied using mix designs, including metal bearing waste alters the hydration and setting time of binders. Pore structure depends on relative quantity of the constituents, cement hydration products and their reaction products with admixtures. Carbonation and additives can lead to strength improvement in waste-binder matrix. PMID:16530943

  9. Carboxymethyl modification of konjac glucomannan affects water binding properties.

    PubMed

    Xiao, Man; Dai, Shuhong; Wang, Le; Ni, Xuewen; Yan, Wenli; Fang, Yapeng; Corke, Harold; Jiang, Fatang

    2015-10-01

    The water binding properties of konjac glucomannan (KGM) and carboxymethyl konjac glucomannan (CMKGM) are important for their application in food, pharmaceutical, and chemical engineering fields. The equilibrium moisture content of CMKGM was lower than that of KGM at the relative humidity in the range 30-95% at 25°C. The water absorption and solubility of CMKGM in water solution were lower than that of KGM at 25°C. Carboxymethyl modification of KGM reduces the water adsorption, absorption, and solubility. Both carboxymethylation and deacetylation could confer hydrophobicity for CMKGM. These data provide the basis for expanding CMKGM application. PMID:26076594

  10. Nuclear ubiquitination by FBXL5 modulates Snail1 DNA binding and stability.

    PubMed

    Viñas-Castells, Rosa; Frías, Álex; Robles-Lanuza, Estefanía; Zhang, Kun; Longmore, Gregory D; García de Herreros, Antonio; Díaz, Víctor M

    2014-01-01

    The zinc finger transcription factor Snail1 regulates epithelial to mesenchymal transition, repressing epithelial markers and activating mesenchymal genes. Snail1 is an extremely labile protein degraded by the cytoplasmic ubiquitin-ligases β-TrCP1/FBXW1 and Ppa/FBXL14. Using a short hairpin RNA screening, we have identified FBXL5 as a novel Snail1 ubiquitin ligase. FBXL5 is located in the nucleus where it interacts with Snail1 promoting its polyubiquitination and affecting Snail1 protein stability and function by impairing DNA binding. Snail1 downregulation by FBXL5 is prevented by Lats2, a protein kinase that phosphorylates Snail1 precluding its nuclear export but not its polyubiquitination. Actually, although polyubiquitination by FBXL5 takes place in the nucleus, Snail1 is degraded in the cytosol. Finally, FBXL5 is highly sensitive to stress conditions and is downregulated by iron depletion and γ-irradiation, explaining Snail1 stabilization in these conditions. These results characterize a novel nuclear ubiquitin ligase controlling Snail1 protein stability and provide the molecular basis for understanding how radiotherapy upregulates the epithelial to mesenchymal transition-inducer Snail1. PMID:24157836

  11. Nuclear ubiquitination by FBXL5 modulates Snail1 DNA binding and stability

    PubMed Central

    Viñas-Castells, Rosa; Frías, Álex; Robles-Lanuza, Estefanía; Zhang, Kun; Longmore, Gregory D.; García de Herreros, Antonio; Díaz, Víctor M.

    2014-01-01

    The zinc finger transcription factor Snail1 regulates epithelial to mesenchymal transition, repressing epithelial markers and activating mesenchymal genes. Snail1 is an extremely labile protein degraded by the cytoplasmic ubiquitin-ligases β-TrCP1/FBXW1 and Ppa/FBXL14. Using a short hairpin RNA screening, we have identified FBXL5 as a novel Snail1 ubiquitin ligase. FBXL5 is located in the nucleus where it interacts with Snail1 promoting its polyubiquitination and affecting Snail1 protein stability and function by impairing DNA binding. Snail1 downregulation by FBXL5 is prevented by Lats2, a protein kinase that phosphorylates Snail1 precluding its nuclear export but not its polyubiquitination. Actually, although polyubiquitination by FBXL5 takes place in the nucleus, Snail1 is degraded in the cytosol. Finally, FBXL5 is highly sensitive to stress conditions and is downregulated by iron depletion and γ-irradiation, explaining Snail1 stabilization in these conditions. These results characterize a novel nuclear ubiquitin ligase controlling Snail1 protein stability and provide the molecular basis for understanding how radiotherapy upregulates the epithelial to mesenchymal transition-inducer Snail1. PMID:24157836

  12. Nonconsensus Protein Binding to Repetitive DNA Sequence Elements Significantly Affects Eukaryotic Genomes

    PubMed Central

    Barber-Zucker, Shiran; Gordân, Raluca; Lukatsky, David B.

    2015-01-01

    Recent genome-wide experiments in different eukaryotic genomes provide an unprecedented view of transcription factor (TF) binding locations and of nucleosome occupancy. These experiments revealed that a large fraction of TF binding events occur in regions where only a small number of specific TF binding sites (TFBSs) have been detected. Furthermore, in vitro protein-DNA binding measurements performed for hundreds of TFs indicate that TFs are bound with wide range of affinities to different DNA sequences that lack known consensus motifs. These observations have thus challenged the classical picture of specific protein-DNA binding and strongly suggest the existence of additional recognition mechanisms that affect protein-DNA binding preferences. We have previously demonstrated that repetitive DNA sequence elements characterized by certain symmetries statistically affect protein-DNA binding preferences. We call this binding mechanism nonconsensus protein-DNA binding in order to emphasize the point that specific consensus TFBSs do not contribute to this effect. In this paper, using the simple statistical mechanics model developed previously, we calculate the nonconsensus protein-DNA binding free energy for the entire C. elegans and D. melanogaster genomes. Using the available chromatin immunoprecipitation followed by sequencing (ChIP-seq) results on TF-DNA binding preferences for ~100 TFs, we show that DNA sequences characterized by low predicted free energy of nonconsensus binding have statistically higher experimental TF occupancy and lower nucleosome occupancy than sequences characterized by high free energy of nonconsensus binding. This is in agreement with our previous analysis performed for the yeast genome. We suggest therefore that nonconsensus protein-DNA binding assists the formation of nucleosome-free regions, as TFs outcompete nucleosomes at genomic locations with enhanced nonconsensus binding. In addition, here we perform a new, large-scale analysis using

  13. Balancing Protein Stability and Activity in Cancer: A New Approach for Identifying Driver Mutations Affecting CBL Ubiquitin Ligase Activation.

    PubMed

    Li, Minghui; Kales, Stephen C; Ma, Ke; Shoemaker, Benjamin A; Crespo-Barreto, Juan; Cangelosi, Andrew L; Lipkowitz, Stanley; Panchenko, Anna R

    2016-02-01

    Oncogenic mutations in the monomeric Casitas B-lineage lymphoma (Cbl) gene have been found in many tumors, but their significance remains largely unknown. Several human c-Cbl (CBL) structures have recently been solved, depicting the protein at different stages of its activation cycle and thus providing mechanistic insight underlying how stability-activity tradeoffs in cancer-related proteins-may influence disease onset and progression. In this study, we computationally modeled the effects of missense cancer mutations on structures representing four stages of the CBL activation cycle to identify driver mutations that affect CBL stability, binding, and activity. We found that recurrent, homozygous, and leukemia-specific mutations had greater destabilizing effects on CBL states than random noncancer mutations. We further tested the ability of these computational models, assessing the changes in CBL stability and its binding to ubiquitin-conjugating enzyme E2, by performing blind CBL-mediated EGFR ubiquitination assays in cells. Experimental CBL ubiquitin ligase activity was in agreement with the predicted changes in CBL stability and, to a lesser extent, with CBL-E2 binding affinity. Two thirds of all experimentally tested mutations affected the ubiquitin ligase activity by either destabilizing CBL or disrupting CBL-E2 binding, whereas about one-third of tested mutations were found to be neutral. Collectively, our findings demonstrate that computational methods incorporating multiple protein conformations and stability and binding affinity evaluations can successfully predict the functional consequences of cancer mutations on protein activity, and provide a proof of concept for mutations in CBL. PMID:26676746

  14. Spastin Binds to Lipid Droplets and Affects Lipid Metabolism

    PubMed Central

    Papadopoulos, Chrisovalantis; Orso, Genny; Mancuso, Giuseppe; Herholz, Marija; Gumeni, Sentiljana; Tadepalle, Nimesha; Jüngst, Christian; Tzschichholz, Anne; Schauss, Astrid; Höning, Stefan; Trifunovic, Aleksandra; Daga, Andrea; Rugarli, Elena I.

    2015-01-01

    Mutations in SPAST, encoding spastin, are the most common cause of autosomal dominant hereditary spastic paraplegia (HSP). HSP is characterized by weakness and spasticity of the lower limbs, owing to progressive retrograde degeneration of the long corticospinal axons. Spastin is a conserved microtubule (MT)-severing protein, involved in processes requiring rearrangement of the cytoskeleton in concert to membrane remodeling, such as neurite branching, axonal growth, midbody abscission, and endosome tubulation. Two isoforms of spastin are synthesized from alternative initiation codons (M1 and M87). We now show that spastin-M1 can sort from the endoplasmic reticulum (ER) to pre- and mature lipid droplets (LDs). A hydrophobic motif comprised of amino acids 57 through 86 of spastin was sufficient to direct a reporter protein to LDs, while mutation of arginine 65 to glycine abolished LD targeting. Increased levels of spastin-M1 expression reduced the number but increased the size of LDs. Expression of a mutant unable to bind and sever MTs caused clustering of LDs. Consistent with these findings, ubiquitous overexpression of Dspastin in Drosophila led to bigger and less numerous LDs in the fat bodies and increased triacylglycerol levels. In contrast, Dspastin overexpression increased LD number when expressed specifically in skeletal muscles or nerves. Downregulation of Dspastin and expression of a dominant-negative variant decreased LD number in Drosophila nerves, skeletal muscle and fat bodies, and reduced triacylglycerol levels in the larvae. Moreover, we found reduced amount of fat stores in intestinal cells of worms in which the spas-1 homologue was either depleted by RNA interference or deleted. Taken together, our data uncovers an evolutionarily conserved role of spastin as a positive regulator of LD metabolism and open up the possibility that dysfunction of LDs in axons may contribute to the pathogenesis of HSP. PMID:25875445

  15. Bacterial lipopolysaccharide binding enhances virion stability and promotes environmental fitness of an enteric virus

    PubMed Central

    Robinson, Christopher M.; Jesudhasan, Palmy R.; Pfeiffer, Julie K.

    2014-01-01

    Summary Enteric viruses, including poliovirus and reovirus, encounter a vast microbial community in the mammalian gastrointestinal tract, which has been shown to promote virus replication and pathogenesis. Investigating the underlying mechanisms, we find that poliovirus binds bacterial surface polysaccharides, which enhances virion stability and cell attachment by increasing binding to the viral receptor. Additionally, we identified a poliovirus mutant, VP1-T99K, with reduced lipopolysaccharide (LPS) binding. Although T99K and WT poliovirus cell attachment, replication and pathogenesis in mice are equivalent, following peroral inoculation of mice, VP1-T99K poliovirus was unstable in feces. Consequently, the ratio of mutant virus in feces is reduced following additional cycles of infection in mice. Thus, the mutant virus incurs a fitness cost when environmental stability is a factor. These data suggest that poliovirus binds bacterial surface polysaccharides, enhancing cell attachment and environmental stability, potentially promoting transmission to a new host. PMID:24439896

  16. Arabidopsis AtADF1 is functionally affected by mutations on actin binding sites.

    PubMed

    Dong, Chun-Hai; Tang, Wei-Ping; Liu, Jia-Yao

    2013-03-01

    The plant actin depolymerizing factor (ADF) binds to both monomeric and filamentous actin, and is directly involved in the depolymerization of actin filaments. To better understand the actin binding sites of the Arabidopsis thaliana L. AtADF1, we generated mutants of AtADF1 and investigated their functions in vitro and in vivo. Analysis of mutants harboring amino acid substitutions revealed that charged residues (Arg98 and Lys100) located at the α-helix 3 and forming an actin binding site together with the N-terminus are essential for both G- and F-actin binding. The basic residues on the β-strand 5 (K82/A) and the α-helix 4 (R135/A, R137/A) form another actin binding site that is important for F-actin binding. Using transient expression of CFP-tagged AtADF1 mutant proteins in onion (Allium cepa) peel epidermal cells and transgenic Arabidopsis thaliana L. plants overexpressing these mutants, we analyzed how these mutant proteins regulate actin organization and affect seedling growth. Our results show that the ADF mutants with a lower affinity for actin filament binding can still be functional, unless the affinity for actin monomers is also affected. The G-actin binding activity of the ADF plays an essential role in actin binding, depolymerization of actin polymers, and therefore in the control of actin organization. PMID:23190411

  17. Binding of Gq protein stabilizes the activated state of the muscarinic receptor type 1.

    PubMed

    Tateyama, Michihiro; Kubo, Yoshihiro

    2013-02-01

    Activation of G protein coupled receptors (GPCRs) induces various cellular responses through interactions with G proteins. The key trigger of GPCR activation is agonist binding. It is reportedly known that the agonist-bound active conformation of the GPCRs, such as the muscarinic acetylcholine receptor type 1 (M(1)R), can be affected by the coupling of G proteins and by depolarization of the membrane potential. Here we aimed at investigating their effects on the structural rearrangements of the M(1)Rs between the active and quiescent states, using the fluorescence resonance energy transfer (FRET) technique. For this purpose, fluorescent M(1)R constructs that maintained intact activation of the Gq pathway and interaction with Gq were used. We captured the agonist-induced conformational changes of the M(1)R as the FRET decreases and found that the FRET decreases were enhanced by co-expression of the Gq subunits. In addition, co-expression of the Gq subunits decelerated the recovery of the declined FRET upon removal of the agonists, which was slower than the dissociation of the Gq subunits from the receptor. These results suggested that Gq binding stabilizes the agonist-induced activated conformation of the M(1)R. We also found that depolarization of the membrane potential slightly but significantly enhanced the agonist-induced FRET decrease, by accelerating the agonist-induced conformational changes. Thus, structural rearrangement analyses by FRET revealed that Gq coupling stabilizes the active conformation of the M(1)R and also suggested that depolarization accelerates the transition from quiescent to activation conformation. PMID:23085334

  18. Kinetics of cyanide binding as a probe of local stability/flexibility of cytochrome c.

    PubMed

    Varhac, Rastislav; Tomásková, Natasa; Fabián, Marián; Sedlák, Erik

    2009-09-01

    Effect of anions of the Hofmeister series (thiocyanate, perchlorate, iodide, bromide, nitrate, chloride, sulfate, and phosphate) on local and global stability and flexibility of horse heart ferricytochrome c (cyt c) has been studied. Global stability of cyt c was determined by iso/thermal denaturations monitored by change in ellipticity in the far-UV region and its local stability was determined from absorbance changes in the Soret region. Particularly, relative stability/flexibility of the Met80-heme iron bond has been assessed by analysis of binding of cyanide into the heme iron. Both global and local stabilities of cyt c exhibited monotonous increase induced by a change of anion from chaotropic to kosmotropic species. However, this monotonous dependence was not observed for the rate constants of cyanide association with cyt c. As expected more chaotropic ions induced lower stability of protein and faster binding of cyanide but this correlation was reversed for kosmotropic anions. We propose that the unusual bell-shaped dependence of the rate constant of cyanide association is a result of modulation of Met80-heme iron bond strength and/or flexibility of heme region by Hofmeister anions independently on global stability of cyt c. Further, our results demonstrate sensitivity of cyanide binding to local change in stability/flexibility in the heme region of cyt c. PMID:19545938

  19. Water molecules inside protein structure affect binding of monosaccharides with HIV-1 antibody 2G12.

    PubMed

    Ueno-Noto, Kaori; Takano, Keiko

    2016-10-01

    Water molecules inside biomolecules constitute integral parts of their structure and participate in the functions of the proteins. Some of the X-ray crystallographic data are insufficient for analyzing a series of ligand-protein complexes in the same condition. We theoretically investigated antibody binding abilities of saccharide ligands and the effects of the inner water molecules of ligand-antibody complexes. Classical molecular dynamics and quantum chemical simulations using a model with possible water molecules inside the protein were performed with saccharide ligands and Human Immunodeficiency Virus 1 neutralizing antibody 2G12 complexes to estimate how inner water molecules of the protein affect the dynamics of the complexes as well as the ligand-antibody interaction. Our results indicate the fact that d-fructose's strong affinity to the antibody was partly due to the good retentiveness of solvent water molecules of the ligand and its stability of the ligand's conformation and relative position in the active site. © 2016 Wiley Periodicals, Inc. PMID:27388036

  20. A WD-repeat protein stabilizes ORC binding to chromatin.

    PubMed

    Shen, Zhen; Sathyan, Kizhakke M; Geng, Yijie; Zheng, Ruiping; Chakraborty, Arindam; Freeman, Brian; Wang, Fei; Prasanth, Kannanganattu V; Prasanth, Supriya G

    2010-10-01

    Origin recognition complex (ORC) plays critical roles in the initiation of DNA replication and cell-cycle progression. In metazoans, ORC associates with origin DNA during G1 and with heterochromatin in postreplicated cells. However, what regulates the binding of ORC to chromatin is not understood. We have identified a highly conserved, leucine-rich repeats and WD40 repeat domain-containing protein 1 (LRWD1) or ORC-associated (ORCA) in human cells that interacts with ORC and modulates chromatin association of ORC. ORCA colocalizes with ORC and shows similar cell-cycle dynamics. We demonstrate that ORCA efficiently recruits ORC to chromatin. Depletion of ORCA in human primary cells and embryonic stem cells results in loss of ORC association to chromatin, concomitant reduction of MCM binding, and a subsequent accumulation in G1 phase. Our results suggest ORCA-mediated association of ORC to chromatin is critical to initiate preRC assembly in G1 and chromatin organization in post-G1 cells. PMID:20932478

  1. Functionalization of small platinum nanoparticles with amines and phosphines: Ligand binding modes and particle stability.

    PubMed

    Wand, Patricia; Bartl, Johannes D; Heiz, Ueli; Tschurl, Martin; Cokoja, Mirza

    2016-09-15

    We report the binding mode of amines and phosphines on platinum nanoparticles. Protective ligands comprising different functional groups are systematically studied for the elucidation of ligand binding at different functionalization conditions. From the functionalization conditions it is concluded that the binding of amines to the nanoparticles occurs via the formation of a PtHN moiety or electrostatic interaction, which is supported by spectroscopic evidences. In particular from complex chemistry such a binding mode is surprising, as amines are expected to bind via their electron pair to the metal. Similar results from functionalization are observed for phosphine-protected nanoparticles, which suggest similar binding modes in these systems. In contrast to the strong covalent bond of the protection with thiols, considerable weakly binding systems result. The characteristics of the binding mode are reflected by the stability of the colloids and their catalytic properties. In the selective hydrogenation of 3-hexyne to 3-hexene thiolate-stabilized Pt particles are highly stable, but exhibit the lowest activity. On the other hand, amine- and phosphine-capped platinum nanoparticles show a significantly higher activity, but rapidly agglomerate. PMID:27288572

  2. Upstream regulatory regions required to stabilize binding to the TATA sequence in an adenovirus early promoter.

    PubMed

    Garcia, J; Wu, F; Gaynor, R

    1987-10-26

    Of the five early adenovirus promoters, the early region 3 (E3) promoter is one of the most strongly induced by the E1A protein. To identify cellular proteins involved in both the basal and E1A-induced transcriptional regulation of the E3 promoter, DNase I footprinting using partially purified Hela cell extracts was performed. Four regions of the E3 promoter serve as binding domains for cellular proteins. These regions are found between -156 to -179 (site IV), -83 to -103 (site III), -47 to -67 (site II), and -16 to -37 (site I), relative to the start of transcription. Examination of the DNA sequences in each binding domain suggests that site III likely serves as a binding site for activator protein 1 (AP-1), site II for the cyclic AMP regulatory element binding protein (CREB), and site I for a TATA binding factor. The factors binding to either site II or III were sufficient to stabilize binding to the TATA sequence (site I). Mutagenesis studies indicated that both sites II and III, in addition to site I, are needed for complete basal and E1A-induced transcription. These results suggest that multiple cellular factors are involved in both the basal and E1A-induced transcriptional regulation of the E3 promoter, and that either of two upstream regions are capable of stabilizing factor binding to the TATA sequence. PMID:2959908

  3. Upstream regulatory regions required to stabilize binding to the TATA sequence in an adenovirus early promoter.

    PubMed Central

    Garcia, J; Wu, F; Gaynor, R

    1987-01-01

    Of the five early adenovirus promoters, the early region 3 (E3) promoter is one of the most strongly induced by the E1A protein. To identify cellular proteins involved in both the basal and E1A-induced transcriptional regulation of the E3 promoter, DNase I footprinting using partially purified Hela cell extracts was performed. Four regions of the E3 promoter serve as binding domains for cellular proteins. These regions are found between -156 to -179 (site IV), -83 to -103 (site III), -47 to -67 (site II), and -16 to -37 (site I), relative to the start of transcription. Examination of the DNA sequences in each binding domain suggests that site III likely serves as a binding site for activator protein 1 (AP-1), site II for the cyclic AMP regulatory element binding protein (CREB), and site I for a TATA binding factor. The factors binding to either site II or III were sufficient to stabilize binding to the TATA sequence (site I). Mutagenesis studies indicated that both sites II and III, in addition to site I, are needed for complete basal and E1A-induced transcription. These results suggest that multiple cellular factors are involved in both the basal and E1A-induced transcriptional regulation of the E3 promoter, and that either of two upstream regions are capable of stabilizing factor binding to the TATA sequence. Images PMID:2959908

  4. Phosphorylation in protein-protein binding: effect on stability and function

    PubMed Central

    Nishi, Hafumi; Hashimoto, Kosuke; Panchenko, Anna R.

    2011-01-01

    Summary Post-translational modifications offer a dynamic way to regulate protein activity, subcellular localization and stability. Here we estimate the effect of phosphorylation on protein binding and function for different types of complexes from human proteome. We find that phosphorylation sites have a tendency to be located on binding interfaces in heterooligomeric and weak transient homooligomeric complexes. The analysis of molecular mechanisms of phosphorylation shows that phosphorylation may modulate the strength of interactions directly on interfaces and binding hotspots have a tendency to be phosphorylated in heterooligomers. Although majority of phosphosites do not show significant estimated stability differences upon attaching the phosphate groups, for about one third of all complexes it causes relatively large changes in binding energy. We discuss the cases where phosphorylation mediates the complex formation and regulates the function. We show that phosphorylation sites are not only more likely to be evolutionary conserved than surface residues but even more so than other interfacial residues. PMID:22153503

  5. Thermal stability and binding energetics of thymidylate synthase ThyX.

    PubMed

    Krumova, Sashka; Todinova, Svetla; Tileva, Milena; Bouzhir-Sima, Latifa; Vos, Marten H; Liebl, Ursula; Taneva, Stefka G

    2016-10-01

    The bacterial thymidylate synthase ThyX is a multisubstrate flavoenzyme that takes part in the de novo synthesis of thymidylate in a variety of microorganisms. Herein we study the effect of FAD and dUMP binding on the thermal stability of wild type (WT) ThyX from the mesophilic Paramecium bursaria chlorella virus-1 (PBCV-1) and from the thermophilic bacterium Thermotoga maritima (TmThyX), and from two variants of TmThyX, Y91F and S88W, using differential scanning calorimetry. The energetics underlying these processes was characterized by isothermal titration calorimetry. The PBCV-1 protein is significantly less stable against the thermal challenge than the TmThyX WT. FAD exerted stabilizing effect greater for PBCV-1 than for TmThyX and for both mutants, whereas binding of dUMP to FAD-loaded proteins stabilized further only TmThyX. Different thermodynamic signatures describe the FAD binding to the WT ThyX proteins. While TmThyX binds FAD with a low μM binding affinity in a process characterized by a favorable entropy change, the assembly of PBCV-1 with FAD is governed by a large enthalpy change opposed by an unfavorable entropy change resulting in a relatively strong nM binding. An enthalpy-driven formation of a high affinity ternary ThyX/FAD/dUMP complex was observed only for TmThyX. PMID:27268384

  6. Replacement of Val3 in Human Thymidylate Synthase Affects Its Kinetic Properties and Intracellular Stability

    SciTech Connect

    Huang, Xiao; Gibson, Lydia M.; Bell, Brittnaie J.; Lovelace, Leslie L.; Pea, Maria Marjorette O.; Berger, Franklin G.; Berger, Sondra H.; Lebioda, Lukasz

    2010-11-03

    Human and other mammalian thymidylate synthase (TS) enzymes have an N-terminal extension of {approx}27 amino acids that is not present in bacterial TSs. The extension, which is disordered in all reported crystal structures of TSs, has been considered to play a primary role in protein turnover but not in catalytic activity. In mammalian cells, the variant V3A has a half-life similar to that of wild-type human TS (wt hTS) while V3T is much more stable; V3L, V3F, and V3Y have half-lives approximately half of that for wt hTS. Catalytic turnover rates for most Val3 mutants are only slightly diminished, as expected. However, two mutants, V3L and V3F, have strongly compromised dUMP binding, with K{sub m,app} values increased by factors of 47 and 58, respectively. For V3L, this observation can be explained by stabilization of the inactive conformation of the loop of residues 181-197, which prevents substrate binding. In the crystal structure of V3L, electron density corresponding to a leucine residue is present in a position that stabilizes the loop of residues 181-197 in the inactive conformation. Since this density is not observed in other mutants and all other leucine residues are ordered in this structure, it is likely that this density represents Leu3. In the crystal structure of a V3F {center_dot} FdUMP binary complex, the nucleotide is bound in an alternative mode to that proposed for the catalytic complex, indicating that the high K{sub m,app} value is caused not by stabilization of the inactive conformer but by substrate binding in a nonproductive, inhibitory site. These observations show that the N-terminal extension affects the conformational state of the hTS catalytic region. Each of the mechanisms leading to the high K{sub m,app} values can be exploited to facilitate design of compounds acting as allosteric inhibitors of hTS.

  7. Kinetics of α-Globin Binding to α-Hemoglobin Stabilizing Protein (AHSP) Indicate Preferential Stabilization of Hemichrome Folding Intermediate*

    PubMed Central

    Mollan, Todd L.; Khandros, Eugene; Weiss, Mitchell J.; Olson, John S.

    2012-01-01

    Human α-hemoglobin stabilizing protein (AHSP) is a conserved mammalian erythroid protein that facilitates the production of Hemoglobin A by stabilizing free α-globin. AHSP rapidly binds to ferrous α with association (k′AHSP) and dissociation (kAHSP) rate constants of ≈10 μm−1 s−1 and 0.2 s−1, respectively, at pH 7.4 at 22 °C. A small slow phase was observed when AHSP binds to excess ferrous αCO. This slow phase appears to be due to cis to trans prolyl isomerization of the Asp29-Pro30 peptide bond in wild-type AHSP because it was absent when αCO was mixed with P30A and P30W AHSP, which are fixed in the trans conformation. This slow phase was also absent when met(Fe3+)-α reacted with wild-type AHSP, suggesting that met-α is capable of rapidly binding to either Pro30 conformer. Both wild-type and Pro30-substituted AHSPs drive the formation of a met-α hemichrome conformation following binding to either met- or oxy(Fe2+)-α. The dissociation rate of the met-α·AHSP complex (kAHSP ≈ 0.002 s−1) is ∼100-fold slower than that for ferrous α·AHSP complexes, resulting in a much higher affinity of AHSP for met-α. Thus, in vivo, AHSP acts as a molecular chaperone by rapidly binding and stabilizing met-α hemichrome folding intermediates. The low rate of met-α dissociation also allows AHSP to have a quality control function by kinetically trapping ferric α and preventing its incorporation into less stable mixed valence Hemoglobin A tetramers. Reduction of AHSP-bound met-α allows more rapid release to β subunits to form stable fully, reduced hemoglobin dimers and tetramers. PMID:22298770

  8. DNA Binding of Centromere Protein C (CENPC) Is Stabilized by Single-Stranded RNA

    PubMed Central

    Du, Yaqing; Topp, Christopher N.; Dawe, R. Kelly

    2010-01-01

    Centromeres are the attachment points between the genome and the cytoskeleton: centromeres bind to kinetochores, which in turn bind to spindles and move chromosomes. Paradoxically, the DNA sequence of centromeres has little or no role in perpetuating kinetochores. As such they are striking examples of genetic information being transmitted in a manner that is independent of DNA sequence (epigenetically). It has been found that RNA transcribed from centromeres remains bound within the kinetochore region, and this local population of RNA is thought to be part of the epigenetic marking system. Here we carried out a genetic and biochemical study of maize CENPC, a key inner kinetochore protein. We show that DNA binding is conferred by a localized region 122 amino acids long, and that the DNA-binding reaction is exquisitely sensitive to single-stranded RNA. Long, single-stranded nucleic acids strongly promote the binding of CENPC to DNA, and the types of RNAs that stabilize DNA binding match in size and character the RNAs present on kinetochores in vivo. Removal or replacement of the binding module with HIV integrase binding domain causes a partial delocalization of CENPC in vivo. The data suggest that centromeric RNA helps to recruit CENPC to the inner kinetochore by altering its DNA binding characteristics. PMID:20140237

  9. Structural investigations into the binding mode of novel neolignans Cmp10 and Cmp19 microtubule stabilizers by in silico molecular docking, molecular dynamics, and binding free energy calculations.

    PubMed

    Tripathi, Shubhandra; Kumar, Akhil; Kumar, B Sathish; Negi, Arvind S; Sharma, Ashok

    2016-06-01

    Microtubule stabilizers provide an important mode of treatment via mitotic cell arrest of cancer cells. Recently, we reported two novel neolignans derivatives Cmp10 and Cmp19 showing anticancer activity and working as microtubule stabilizers at micromolar concentrations. In this study, we have explored the binding site, mode of binding, and stabilization by two novel microtubule stabilizers Cmp10 and Cmp19 using in silico molecular docking, molecular dynamics (MD) simulation, and binding free energy calculations. Molecular docking studies were performed to explore the β-tubulin binding site of Cmp10 and Cmp19. Further, MD simulations were used to probe the β-tubulin stabilization mechanism by Cmp10 and Cmp19. Binding affinity was also compared for Cmp10 and Cmp19 using binding free energy calculations. Our docking results revealed that both the compounds bind at Ptxl binding site in β-tubulin. MD simulation studies showed that Cmp10 and Cmp19 binding stabilizes M-loop (Phe272-Val288) residues of β-tubulin and prevent its dynamics, leading to a better packing between α and β subunits from adjacent tubulin dimers. In addition, His229, Ser280 and Gln281, and Arg278, Thr276, and Ser232 were found to be the key amino acid residues forming H-bonds with Cmp10 and Cmp19, respectively. Consequently, binding free energy calculations indicated that Cmp10 (-113.655 kJ/mol) had better binding compared to Cmp19 (-95.216 kJ/mol). This study provides useful insight for better understanding of the binding mechanism of Cmp10 and Cmp19 and will be helpful in designing novel microtubule stabilizers. PMID:26212016

  10. Mg2+ binds to the surface of thymidylate synthase and affects hydride transfer at the interior active site

    PubMed Central

    Wang, Zhen; Sapienza, Paul J.; Abeysinghe, Thelma; Luzum, Calvin; Lee, Andrew L.; Finer-Moore, Janet S.; Stroud, Robert M.; Kohen, Amnon

    2013-01-01

    Thymidylate synthase (TSase) produces the sole intracellular de novo source of thymidine (i.e. the DNA base T) and thus is a common target for antibiotic and anticancer drugs. Mg2+ has been reported to affect TSase activity, but the mechanism of this interaction has not been investigated. Here we show that Mg2+ binds to the surface of Escherichia coli TSase and affects the kinetics of hydride transfer at the interior active site (16 Å away). Examination of the crystal structures identifies a Mg2+ near the glutamyl moiety of the folate cofactor, providing the first structural evidence for Mg2+ binding to TSase. The kinetics and NMR relaxation experiments suggest that the weak binding of Mg2+ to the protein surface stabilizes the closed conformation of the ternary enzyme complex and reduces the entropy of activation on the hydride transfer step. Mg2+ accelerates the hydride transfer by ca. 7-fold but does not affect the magnitude or temperature-dependence of the intrinsic kinetic isotope effect. These results suggest that Mg2+ facilitates the protein motions that bring the hydride donor and acceptor together, but it does not change the tunneling ready state of the hydride transfer. These findings highlight how variations in cellular Mg2+ concentration can modulate enzyme activity through long-range interactions in the protein, rather than binding at the active site. The interaction of Mg2+ with the glutamyl-tail of the folate cofactor and nonconserved residues of bacterial TSase may assist in designing antifolates with poly-glutamyl substitutes as species-specific antibiotic drugs. PMID:23611499

  11. FACTORS AFFECTING DISINFECTION AND STABILIZATION OF SEWAGE SLUDGE

    EPA Science Inventory

    Effective disinfection and stabilization of sewage sludge prior to land application is essential to not only protect human health, but also to convince the public of its benefits and safety. A basic understanding of the key factors involved in producing a stable biosolid product ...

  12. Binding, stability, and antioxidant activity of quercetin with soy protein isolate particles.

    PubMed

    Wang, Yufang; Wang, Xiaoyong

    2015-12-01

    This work is to study the potential of particles fabricated from soy protein isolate (SPI) as a protective carrier for quercetin. When the concentration of SPI particles increases from 0 to 0.35 g/L, quercetin gives a gradually increased fluorescence intensity and fluorescence anisotropy. The addition of quercetin can highly quench the intrinsic fluorescence of SPI particles. These results are explained in terms of the binding of quercetin to the hydrophobic pockets of SPI particles mainly through the hydrophobic force together with the hydrogen bonding. The small difference in the binding constants at 25 and 40 °C suggests the structural stability of SPI particles. The relative changes in values of Gibbs energy, enthalpy, and entropy indicate that the binding of quercetin with SPI particles is spontaneous and hydrophobic interaction is the major force. Furthermore, SPI particles are superior to native SPI for improving the stability and radical scavenging activity of quercetin. PMID:26041159

  13. NADP(+) binding effects tryptophan accessibility, folding and stability of recombinant B. malayi G6PD.

    PubMed

    Verma, Anita; Chandra, Sharat; Suthar, Manish Kumar; Doharey, Pawan Kumar; Siddiqi, Mohammad Imran; Saxena, Jitendra Kumar

    2016-04-01

    Brugia malayi Glucose 6-phosphate dehydrogenase apoenzyme (BmG6PD) was expressed and purified by affinity chromatography to study the differences in kinetic properties of enzyme and the effect of the cofactor NADP(+) binding on enzyme stability. The presence of cofactor NADP(+) influenced the tertiary structure of enzyme due to significant differences in the tryptophan microenvironment. However, NADP(+) binding have no effect on secondary structure of the enzyme. Quenching with acrylamide indicated that two or more tryptophan residues became accessible upon cofactor binding. Unfolding and cross linking study of BmG6PD showed that NADP(+) stabilized the protein in presence of high concentration of urea/GdmCl. A homology model of BmG6PD constructed using human G6PD (PDB id: 2BH9) as a template indicated 34% α-helix, 19% β-sheet and 47% random coil conformations in the predicted model of the enzyme. In the predicted model binding of NADP(+) to BmG6PD was less tight with the structural sites (-10.96kJ/mol binding score) as compared with the coenzyme site (-15.47kJ/mol binding score). PMID:26763177

  14. Molecular Modeling Approaches to Study the Binding Mode on Tubulin of Microtubule Destabilizing and Stabilizing Agents

    NASA Astrophysics Data System (ADS)

    Botta, Maurizio; Forli, Stefano; Magnani, Matteo; Manetti, Fabrizio

    Tubulin targeting agents constitute an important class of anticancer drugs. By acting either as microtubule stabilizers or destabilizers, they disrupt microtubule dynamics, thus inducing mitotic arrest and, ultimately, cell death by apoptosis. Three different binding sites, whose exact location on tubulin has been experimentally detected, have been identified so far for antimitotic compound targeting microtubules, namely the taxoid, the colchicine and the vinka alkaloid binding site. A number of ligand- and structure-based molecular modeling studies in this field has been reported over the years, aimed at elucidating the binding modes of both stabilizing and destabilizing agent, as well as the molecular features responsible for their efficacious interaction with tubulin. Such studies are described in this review, focusing on information provided by different modeling approaches on the structural determinants of antitubulin agents and the interactions with the binding pockets on tubulin emerged as fundamental for antitumor activity.To describe molecular modeling approaches applied to date to molecules known to bind microtubules, this paper has been divided into two main parts: microtubule destabilizing (Part 1) and stabilizing (Part 2) agents. The first part includes structure-based and ligand-based approaches to study molecules targeting colchicine (1.1) and vinca alkaloid (1.2) binding sites, respectively. In the second part, the studies performed on microtubule-stabilizing antimitotic agents (MSAA) are described. Starting from the first representative compound of this class, paclitaxel, molecular modeling studies (quantitative structure-activity relationships - QSAR - and structure-based approaches), performed on natural compounds acting with the same mechanism of action and temptative common pharmacophoric hypotheses for all of these compounds, are reported.

  15. Distinct Pose of Discodermolide in Taxol Binding Pocket Drives a Complementary Mode of Microtubule Stabilization

    PubMed Central

    Khrapunovich-Baine, Marina; Menon, Vilas; Verdier-Pinard, Pascal; Smith, Amos B.; Angeletti, Ruth Hogue; Fiser, Andras; Horwitz, Susan Band; Xiao, Hui

    2010-01-01

    The microtubule cytoskeleton has proven to be an effective target for cancer therapeutics. One class of drugs, known as microtubule stabilizing agents (MSAs), binds to microtubule polymers and stabilizes them against depolymerization. The prototype of this group of drugs, Taxol, is an effective chemotherapeutic agent used extensively in the treatment of human ovarian, breast, and lung carcinomas. Although electron crystallography and photoaffinity labeling experiments determined that the binding site for Taxol is in a hydrophobic pocket in β-tubulin, little was known about the effects of this drug on the conformation of the entire microtubule. A recent study from our laboratory utilizing hydrogen-deuterium exchange (HDX) in concert with various mass spectrometry (MS) techniques has provided new information on the structure of microtubules upon Taxol binding. In the current study we apply this technique to determine the binding mode and the conformational effects on chicken erythrocyte tubulin (CET) of another MSA, discodermolide, whose synthetic analogues may have potential use in the clinic. We confirmed that like Taxol, discodermolide binds to the taxane binding pocket in β-tubulin. However, as opposed to Taxol, which has major interactions with the M-loop, discodermolide orients itself away from this loop and towards the N-terminal H1–S2 loop. Additionally, discodermolide stabilizes microtubules mainly via its effects on interdimer contacts, specifically on the α-tubulin side, and to a lesser extent on interprotofilament contacts between adjacent β-tubulin subunits. Also, our results indicate complementary stabilizing effects of Taxol and discodermolide on the microtubules, which may explain the synergy observed between the two drugs in vivo. PMID:19863156

  16. Investigating the linkage between disease-causing amino acid variants and their effect on protein stability and binding.

    PubMed

    Peng, Yunhui; Alexov, Emil

    2016-02-01

    Single amino acid variations (SAV) occurring in human population result in natural differences between individuals or cause diseases. It is well understood that the molecular effect of SAV can be manifested as changes of the wild type characteristics of the corresponding protein, among which are the protein stability and protein interactions. Typically the effect of SAV on protein stability and interactions was assessed via the changes of the wild type folding and binding free energies. However, in terms of SAV affecting protein functionally and disease susceptibility, one wants to know to what extend the wild type function is perturbed by the SAV. Here it is demonstrated that relative, rather than the absolute, change of the folding and binding free energy serves as a good indicator for SAV association with disease. Using HumVar as a source for disease-causing SAV and experimentally determined free energy changes from ProTherm and SKEMPI databases, correlation coefficients (CC) between the disease index (Pd) and relative folding (Ppr,f) and binding (Ppr,b) probability indexes, respectively, was achieved. The obtained CCs demonstrated the applicability of the proposed approach and it served as good indicator for SAV association with disease. PMID:26650512

  17. Specific potassium binding stabilizes pI258 arsenate reductase from Staphylococcus aureus.

    PubMed

    Lah, Nina; Lah, Jurij; Zegers, Ingrid; Wyns, Lode; Messens, Joris

    2003-07-01

    Arsenate reductase (ArsC) from Staphylococcus aureus plasmid pI258 catalyzes the reduction of arsenate to arsenite and plays a role in bacterial heavy metal resistance. The high resolution x-ray structure of ArsC reveals the atomic details of the K+ binding site situated next to the catalytic P-loop structural motif of this redox enzyme. A full thermodynamic study of the binding characteristics of a series of monovalent cations (Li+, Na+, K+, Rb+, and Cs+) and their influence on the thermal stability of ArsC was performed with isothermal titration calorimetry, circular dichroism spectroscopy, and differential scanning calorimetry. Potassium has the largest affinity with a Ka of 3.8 x 10(3) m(-1), and the effectiveness of stabilization of ArsC by monovalent cations follows the binding affinity order: K+ > Rb+ > Cs+ > Na+ > Li+. A mutagenesis study on the K+ binding side chains showed that Asn-13 and Asp-65 are essential for potassium binding, but the impact on the stability of ArsC was the most extreme when mutating Ser-36. Additionally, the thermal stabilization by K+ is significantly reduced in the case of the ArsC E21A mutant, showing the importance of a Glu-21-coordinated water molecule in its contact with K+. Although potassium is not essential for catalysis, in its presence the kcat/KM increases with a factor of 5. Altogether, the interaction of K+ with specific residues in ArsC is an enthalpydriven process that stabilizes ArsC and increases the specific activity of this redox enzyme. PMID:12682056

  18. Seasonal difference in brain serotonin transporter binding predicts symptom severity in patients with seasonal affective disorder.

    PubMed

    Mc Mahon, Brenda; Andersen, Sofie B; Madsen, Martin K; Hjordt, Liv V; Hageman, Ida; Dam, Henrik; Svarer, Claus; da Cunha-Bang, Sofi; Baaré, William; Madsen, Jacob; Hasholt, Lis; Holst, Klaus; Frokjaer, Vibe G; Knudsen, Gitte M

    2016-05-01

    Cross-sectional neuroimaging studies in non-depressed individuals have demonstrated an inverse relationship between daylight minutes and cerebral serotonin transporter; this relationship is modified by serotonin-transporter-linked polymorphic region short allele carrier status. We here present data from the first longitudinal investigation of seasonal serotonin transporter fluctuations in both patients with seasonal affective disorder and in healthy individuals. Eighty (11)C-DASB positron emission tomography scans were conducted to quantify cerebral serotonin transporter binding; 23 healthy controls with low seasonality scores and 17 patients diagnosed with seasonal affective disorder were scanned in both summer and winter to investigate differences in cerebral serotonin transporter binding across groups and across seasons. The two groups had similar cerebral serotonin transporter binding in the summer but in their symptomatic phase during winter, patients with seasonal affective disorder had higher serotonin transporter than the healthy control subjects (P = 0.01). Compared to the healthy controls, patients with seasonal affective disorder changed their serotonin transporter significantly less between summer and winter (P < 0.001). Further, the change in serotonin transporter was sex- (P = 0.02) and genotype- (P = 0.04) dependent. In the patients with seasonal affective disorder, the seasonal change in serotonin transporter binding was positively associated with change in depressive symptom severity, as indexed by Hamilton Rating Scale for Depression - Seasonal Affective Disorder version scores (P = 0.01). Our findings suggest that the development of depressive symptoms in winter is associated with a failure to downregulate serotonin transporter levels appropriately during exposure to the environmental stress of winter, especially in individuals with high predisposition to affective disorders.media-1vid110.1093/brain/aww043_video_abstractaww043_video

  19. How the spatial variation of tree roots affects slope stability

    NASA Astrophysics Data System (ADS)

    Mao, Zhun; Stokes, A.; Jourdan, C.; Rey, H.; Courbaud, B.; Saint-André, L.

    2010-05-01

    It is now widely recognized that plant roots can reinforce soil against shallow mass movement. Although studies on the interactions between vegetation and slope stability have significantly augmented in recent years, a clear understanding of the spatial dynamics of root reinforcement (through additional cohesion by roots) in subalpine forest is still limited, especially with regard to the roles of different forest management strategies or ecological landscapes. The architecture of root systems is important for soil cohesion, but in reality it is not possible to measure the orientation of each root in a system. Therefore, knowledge on the effect of root orientation and anisotropy on root cohesion on the basis of in situ data is scanty. To determine the effect of root orientation in root cohesion models, we investigated root anisotropy in two mixed, mature, naturally regenerated, subalpine forests of Norway spruce (Picea abies), and Silver fir (Abies alba). Trees were clustered into islands, with open spaces between each group, resulting in strong mosaic heterogeneity within the forest stand. Trenches within and between clusters of trees were dug and root distribution was measured in three dimensions. We then simulated the influence of different values for a root anisotropy correction factor in forests with different ecological structures and soil depths. Using these data, we have carried out simulations of slope stability by calculating the slope factor of safety depending on stand structure. Results should enable us to better estimate the risk of shallow slope failure depending on the type of forest and species.

  20. Specificity of O-glycosylation in enhancing the stability and cellulose binding affinity of Family 1 carbohydrate-binding modules

    PubMed Central

    Chen, Liqun; Drake, Matthew R.; Resch, Michael G.; Greene, Eric R.; Himmel, Michael E.; Chaffey, Patrick K.; Beckham, Gregg T.; Tan, Zhongping

    2014-01-01

    The majority of biological turnover of lignocellulosic biomass in nature is conducted by fungi, which commonly use Family 1 carbohydrate-binding modules (CBMs) for targeting enzymes to cellulose. Family 1 CBMs are glycosylated, but the effects of glycosylation on CBM function remain unknown. Here, the effects of O-mannosylation are examined on the Family 1 CBM from the Trichoderma reesei Family 7 cellobiohydrolase at three glycosylation sites. To enable this work, a procedure to synthesize glycosylated Family 1 CBMs was developed. Subsequently, a library of 20 CBMs was synthesized with mono-, di-, or trisaccharides at each site for comparison of binding affinity, proteolytic stability, and thermostability. The results show that, although CBM mannosylation does not induce major conformational changes, it can increase the thermolysin cleavage resistance up to 50-fold depending on the number of mannose units on the CBM and the attachment site. O-Mannosylation also increases the thermostability of CBM glycoforms up to 16 °C, and a mannose disaccharide at Ser3 seems to have the largest themostabilizing effect. Interestingly, the glycoforms with small glycans at each site displayed higher binding affinities for crystalline cellulose, and the glycoform with a single mannose at each of three positions conferred the highest affinity enhancement of 7.4-fold. Overall, by combining chemical glycoprotein synthesis and functional studies, we show that specific glycosylation events confer multiple beneficial properties on Family 1 CBMs. PMID:24821760

  1. Specificity of O-glycosylation in enhancing the stability and cellulose binding affinity of Family 1 carbohydrate-binding modules.

    PubMed

    Chen, Liqun; Drake, Matthew R; Resch, Michael G; Greene, Eric R; Himmel, Michael E; Chaffey, Patrick K; Beckham, Gregg T; Tan, Zhongping

    2014-05-27

    The majority of biological turnover of lignocellulosic biomass in nature is conducted by fungi, which commonly use Family 1 carbohydrate-binding modules (CBMs) for targeting enzymes to cellulose. Family 1 CBMs are glycosylated, but the effects of glycosylation on CBM function remain unknown. Here, the effects of O-mannosylation are examined on the Family 1 CBM from the Trichoderma reesei Family 7 cellobiohydrolase at three glycosylation sites. To enable this work, a procedure to synthesize glycosylated Family 1 CBMs was developed. Subsequently, a library of 20 CBMs was synthesized with mono-, di-, or trisaccharides at each site for comparison of binding affinity, proteolytic stability, and thermostability. The results show that, although CBM mannosylation does not induce major conformational changes, it can increase the thermolysin cleavage resistance up to 50-fold depending on the number of mannose units on the CBM and the attachment site. O-Mannosylation also increases the thermostability of CBM glycoforms up to 16 °C, and a mannose disaccharide at Ser3 seems to have the largest themostabilizing effect. Interestingly, the glycoforms with small glycans at each site displayed higher binding affinities for crystalline cellulose, and the glycoform with a single mannose at each of three positions conferred the highest affinity enhancement of 7.4-fold. Overall, by combining chemical glycoprotein synthesis and functional studies, we show that specific glycosylation events confer multiple beneficial properties on Family 1 CBMs. PMID:24821760

  2. Autoinhibition of ETV6 DNA Binding Is Established by the Stability of Its Inhibitory Helix.

    PubMed

    De, Soumya; Okon, Mark; Graves, Barbara J; McIntosh, Lawrence P

    2016-04-24

    The ETS transcriptional repressor ETV6 (or TEL) is autoinhibited by an α-helix that sterically blocks its DNA-binding ETS domain. The inhibitory helix is marginally stable and unfolds when ETV6 binds to either specific or non-specific DNA. Using NMR spectroscopy, we show that folding of the inhibitory helix requires a buried charge-dipole interaction with helix H1 of the ETS domain. This interaction also contributes directly to autoinhibition by precluding a highly conserved dipole-enhanced hydrogen bond between the phosphodiester backbone of bound DNA and the N terminus of helix H1. To probe further the thermodynamic basis of autoinhibition, ETV6 variants were generated with amino acid substitutions introduced along the solvent exposed surface of the inhibitory helix. These changes were designed to increase the intrinsic helical propensity of the inhibitory helix without perturbing its packing interactions with the ETS domain. NMR-monitored amide hydrogen exchange measurements confirmed that the stability of the folded inhibitory helix increases progressively with added helix-promoting substitutions. This also results in progressively reinforced autoinhibition and decreased DNA-binding affinity. Surprisingly, locking the inhibitory helix onto the ETS domain by a disulfide bridge severely impairs, but does not abolish DNA binding. Weak interactions still occur via an interface displaced from the canonical ETS domain DNA-binding surface. Collectively, these studies establish a direct thermodynamic linkage between inhibitory helix stability and ETV6 autoinhibition, and demonstrate that helix unfolding does not strictly precede DNA binding. Modulating inhibitory helix stability provides a potential route for the in vivo regulation of ETV6 activity. PMID:26920109

  3. Recent progress with microtubule stabilizers: new compounds, binding modes and cellular activities

    PubMed Central

    Rohena, Cristina C.

    2014-01-01

    Nature has yielded numerous classes of chemically distinct microtubule stabilizers. Several of these, including paclitaxel (Taxol) and docetaxel (Taxotere), are important drugs used in the treatment of cancer. New microtubule stabilizers and novel formulations of these agents continue to provide advances in cancer therapy. In this review we cover recent progress from late 2008 to August 2013 in the chemistry and biology of these diverse microtubule stabilizers focusing on the wide range of organisms that produce these compounds, their mechanisms of inhibiting microtubule-dependent processes, mechanisms of drug resistance, and their interactions with tubulin including their distinct binding sites and modes. A new potential role for microtubule stabilizers in neurodegenerative diseases is reviewed. PMID:24481420

  4. Polyglutamylated Tubulin Binding Protein C1orf96/CSAP Is Involved in Microtubule Stabilization in Mitotic Spindles

    PubMed Central

    Ohta, Shinya; Hamada, Mayako; Sato, Nobuko; Toramoto, Iyo

    2015-01-01

    The centrosome-associated C1orf96/Centriole, Cilia and Spindle-Associated Protein (CSAP) targets polyglutamylated tubulin in mitotic microtubules (MTs). Loss of CSAP causes critical defects in brain development; however, it is unclear how CSAP association with MTs affects mitosis progression. In this study, we explored the molecular mechanisms of the interaction of CSAP with mitotic spindles. Loss of CSAP caused MT instability in mitotic spindles and resulted in mislocalization of Nuclear protein that associates with the Mitotic Apparatus (NuMA), with defective MT dynamics. Thus, CSAP overload in the spindles caused extensive MT stabilization and recruitment of NuMA. Moreover, MT stabilization by CSAP led to high levels of polyglutamylation on MTs. MT depolymerization by cold or nocodazole treatment was inhibited by CSAP binding. Live-cell imaging analysis suggested that CSAP-dependent MT-stabilization led to centrosome-free MT aster formation immediately upon nuclear envelope breakdown without γ-tubulin. We therefore propose that CSAP associates with MTs around centrosomes to stabilize MTs during mitosis, ensuring proper bipolar spindle formation and maintenance. PMID:26562023

  5. Gene size differentially affects the binding of yeast transcription factor tau to two intragenic regions.

    PubMed Central

    Baker, R E; Camier, S; Sentenac, A; Hall, B D

    1987-01-01

    Yeast transcription factor tau (transcription factor IIIC) specifically interacts with tRNA genes, binding to both the A block and the B block elements of the internal promoter. To study the influence of A block-B block spacing, we analyzed the binding of purified tau protein to a series of internally deleted yeast tRNA(3Leu) genes with A and B blocks separated by 0 to 74 base pairs. Optimal binding occurred with genes having A block-B block distances of 30-60 base pairs; the relative helical orientation of the A and B blocks was unimportant. Results from DNase I "footprinting" and lambda exonuclease protection experiments were consistent with these findings and further revealed that changes in A block-B block distance primarily affect the ability of tau to interact with A block sequences; B block interactions are unaltered. When the A block-B block distance is 17 base pairs or less, tau interacts with a sequence located 15 base pairs upstream of the normal A block, and a new RNA initiation site is observed by in vitro transcription. We propose that the initial binding of tau to the B block activates transcription by enhancing its ability to bind at the A block, and that the A block interaction ultimately directs initiation by RNA polymerase III. Images PMID:2827154

  6. Stability Affects of Artificial Viscosity in Detonation Modeling

    SciTech Connect

    Vitello, P; Souers, P C

    2002-06-03

    Accurate multi-dimensional modeling of detonation waves in solid HE materials is a difficult task. To treat applied problems which contain detonation waves one must consider reacting flow with a wide range of length-scales, non-linear equations of state (EOS), and material interfaces at which the detonation wave interacts with other materials. To be useful numerical models of detonation waves must be accurate, stable, and insensitive to details of the modeling such as the mesh spacing, and mesh aspect ratio for multi-dimensional simulations. Studies we have performed show that numerical simulations of detonation waves can be very sensitive to the form of the artificial viscosity term used. The artificial viscosity term is included in our ALE hydrocode to treat shock discontinuities. We show that a monotonic, second order artificial viscosity model derived from an approximate Riemann solver scheme can strongly damp unphysical oscillations in the detonation wave reaction zone, improving the detonation wave boundary wall interaction. These issues are demonstrated in 2D model simulations presented of the 'Bigplate' test. Results using LX-I 7 explosives are compared with numerical simulation results to demonstrate the affects of the artificial viscosity model.

  7. Substrate Binding Tunes Conformational Flexibility and Kinetic Stability of an Amino Acid Antiporter*

    PubMed Central

    Bippes, Christian A.; Zeltina, Antra; Casagrande, Fabio; Ratera, Merce; Palacin, Manuel; Muller, Daniel J.; Fotiadis, Dimitrios

    2009-01-01

    We used single molecule dynamic force spectroscopy to unfold individual serine/threonine antiporters SteT from Bacillus subtilis. The unfolding force patterns revealed interactions and energy barriers that stabilized structural segments of SteT. Substrate binding did not establish strong localized interactions but appeared to be facilitated by the formation of weak interactions with several structural segments. Upon substrate binding, all energy barriers of the antiporter changed thereby describing the transition from brittle mechanical properties of SteT in the unbound state to structurally flexible conformations in the substrate-bound state. The lifetime of the unbound state was much shorter than that of the substrate-bound state. This leads to the conclusion that the unbound state of SteT shows a reduced conformational flexibility to facilitate specific substrate binding and a reduced kinetic stability to enable rapid switching to the bound state. In contrast, the bound state of SteT showed an increased conformational flexibility and kinetic stability such as required to enable transport of substrate across the cell membrane. This result supports the working model of antiporters in which alternate substrate access from one to the other membrane surface occurs in the substrate-bound state. PMID:19419962

  8. Mood stabilizer treatment increases serotonin type 1A receptor binding in bipolar depression

    PubMed Central

    Nugent, Allison C; Carlson, Paul J; Bain, Earle E; Eckelman, William; Herscovitch, Peter; Manji, Husseini; Zarate, Carlos A; Drevets, Wayne C

    2013-01-01

    Abnormal serotonin type 1A (5-HT1A) receptor function and binding have been implicated in the pathophysiology of mood disorders. Preclinical studies have consistently shown that stress decreases the gene expression of 5-HT1A receptors in experimental animals, and that the associated increase in hormone secretion plays a crucial role in mediating this effect. Chronic administration of the mood stabilizers lithium and divalproex (valproate semisodium) reduces glucocorticoid signaling and function in the hippocampus. Lithium has further been shown to enhance 5-HT1A receptor function. To assess whether these effects translate to human subject with bipolar disorder (BD), positron emission tomography (PET) and [18F]trans-4-fluoro-N-(2-[4-(2-methoxyphenyl) piperazino]-ethyl)-N-(2-pyridyl) cyclohexanecarboxamide ([18F]FCWAY) were used to acquire PET images of 5-HT1A receptor binding in 10 subjects with BD, before and after treatment with lithium or divalproex. Mean 5-HT1A binding potential (BPP) significantly increased following mood stabilizer treatment, most prominently in the mesiotemporal cortex (hippocampus plus amygdala). When mood state was also controlled for, treatment was associated with increases in BPP in widespread cortical areas. These preliminary findings are consistent with the hypothesis that these mood stabilizers enhance 5-HT1A receptor expression in BD, which may underscore an important component of these agents' mechanism of action. PMID:23926239

  9. Osteoblastic alkaline phosphatase mRNA is stabilized by binding to vimentin intermediary filaments.

    PubMed

    Schmidt, Yvonne; Biniossek, Martin; Stark, G Björn; Finkenzeller, Günter; Simunovic, Filip

    2015-03-01

    Vascularization is essential in bone tissue engineering and recent research has focused on interactions between osteoblasts (hOBs) and endothelial cells (ECs). It was shown that cocultivation increases the stability of osteoblastic alkaline phosphatase (ALP) mRNA. We investigated the mechanisms behind this observation, focusing on mRNA binding proteins. Using a luciferase reporter assay, we found that the 3'-untranslated region (UTR) of ALP mRNA is necessary for human umbilical vein endothelial cells (HUVEC)-mediated stabilization of osteoblastic ALP mRNA. Using pulldown experiments and nanoflow-HPLC mass spectrometry, vimentin was identified to bind to the 3'-UTR of ALP mRNA. Validation was performed by Western blotting. Functional experiments inhibiting intermediate filaments with iminodipropionitrile and specific inhibition of vimentin by siRNA transfection showed reduced levels of ALP mRNA and protein. Therefore, ALP mRNA binds to and is stabilized by vimentin. This data add to the understanding of intracellular trafficking of ALP mRNA, its function, and have possible implications in tissue engineering applications. PMID:25536665

  10. Gemini surfactants affect the structure, stability, and activity of ribonuclease Sa.

    PubMed

    Amiri, Razieh; Bordbar, Abdol-Khalegh; Laurents, Douglas V

    2014-09-11

    Gemini surfactants have important advantages, e.g., low micromolar CMCs and slow millisecond monomer ↔ micelle kinetics, for membrane mimetics and for delivering nucleic acids for gene therapy or RNA silencing. However, as a prerequisite, it is important to characterize interactions occurring between Gemini surfactants and proteins. Here NMR and CD spectroscopies are employed to investigate the interactions of cationic Gemini surfactants with RNase Sa, a negatively charged ribonuclease. We find that RNase Sa binds Gemini surfactant monomers and micelles at pH values above 4 to form aggregates. Below pH 4, where the protein is positively charged, these aggregates dissolve and interactions are undetectable. Thermal denaturation experiments show that surfactant lowers RNase Sa's conformational stability, suggesting that surfactant binds the protein's denatured state preferentially. Finally, Gemini surfactants were found to bind RNA, leading to the formation of large complexes. Interestingly, Gemini surfactant binding did not prevent RNase Sa from cleaving RNA. PMID:25133582

  11. OMP decarboxylase: phosphodianion binding energy is used to stabilize a vinyl carbanion intermediate.

    PubMed

    Goryanova, Bogdana; Amyes, Tina L; Gerlt, John A; Richard, John P

    2011-05-01

    Orotidine 5'-monophosphate decarboxylase (OMPDC) catalyzes the exchange for deuterium from solvent D(2)O of the C-6 proton of 1-(β-d-erythrofuranosyl)-5-fluorouracil (FEU), a phosphodianion truncated product analog. The deuterium exchange reaction of FEU is accelerated 1.8 × 10(4)-fold by 1 M phosphite dianion (HPO(3)(2-)). This corresponds to a 5.8 kcal/mol stabilization of the vinyl carbanion-like transition state, which is similar to the 7.8 kcal/mol stabilization of the transition state for OMPDC-catalyzed decarboxylation of a truncated substrate analog by bound HPO(3)(2-). These results show that the intrinsic binding energy of phosphite dianion is used in the stabilization of the vinyl carbanion-like transition state common to the decarboxylation and deuterium exchange reactions. PMID:21486036

  12. Poly(zwitterionic)protein conjugates offer increased stability without sacrificing binding affinity or bioactivity

    PubMed Central

    Keefe, Andrew J.; Jiang, Shaoyi

    2013-01-01

    Treatment with therapeutic proteins is an attractive approach to targeting a number of challenging diseases. Unfortunately, the native proteins themselves are often unstable in physiological conditions, reducing bioavailability and therefore increasing the dose that is required. Conjugation with poly(ethylene glycol) (PEG) is often used to increase stability, but this has a detrimental effect on bioactivity. Here, we introduce conjugation with zwitterionic polymers such as poly(carboxybetaine). We show that poly(carboxybetaine) conjugation improves stability in a manner similar to PEGylation, but that the new conjugates retain or even improve the binding affinity as a result of enhanced protein–substrate hydrophobic interactions. This chemistry opens a new avenue for the development of protein therapeutics by avoiding the need to compromise between stability and affinity. PMID:22169873

  13. Stabilization of Cu(I) for binding and calorimetric measurements in aqueous solution†

    PubMed Central

    Johnson, Destinee K.; Stevenson, Michael J.; Almadidy, Zayed A.; Jenkins, Sharon E.; Wilcox, Dean. E.; Grossoehme, Nicholas E.

    2015-01-01

    Conditions have been developed for the comproportionation reaction of Cu2+ and copper metal to prepare aqueous solutions of Cu+ that are stabilized from disproportionation by MeCN and other Cu+-stabilizing ligands. These solutions were then used in ITC measurements to quantify the thermodynamics of formation of a set of Cu+ complexes (CuI(MeCN)3+, CuIMe6Trien+, CuI(BCA)23−, CuI(BCS)23−), which have stabilities ranging over 15 orders of magnitude, for their use in binding and calorimetric measurements of Cu+ interaction with proteins and other biological macromolecules. These complexes were then used to determine the stability and thermodynamics of formation of a 1 : 1 complex of Cu+ with the biologically important tri-peptide glutathione, GSH. These results identify Me6Trien as an attractive Cu+-stabilizing ligand for calorimetric experiments, and suggest that caution should be used with MeCN to stabilize Cu+ due to its potential for participating in unquantifiable ternary interactions. PMID:26327397

  14. LMO2 Oncoprotein Stability in T-Cell Leukemia Requires Direct LDB1 Binding

    PubMed Central

    Layer, Justin H.; Alford, Catherine E.; McDonald, W. Hayes

    2015-01-01

    LMO2 is a component of multisubunit DNA-binding transcription factor complexes that regulate gene expression in hematopoietic stem and progenitor cell development. Enforced expression of LMO2 causes leukemia by inducing hematopoietic stem cell-like features in T-cell progenitor cells, but the biochemical mechanisms of LMO2 function have not been fully elucidated. In this study, we systematically dissected the LMO2/LDB1-binding interface to investigate the role of this interaction in T-cell leukemia. Alanine scanning mutagenesis of the LIM interaction domain of LDB1 revealed a discrete motif, R320LITR, required for LMO2 binding. Most strikingly, coexpression of full-length, wild-type LDB1 increased LMO2 steady-state abundance, whereas coexpression of mutant proteins deficient in LMO2 binding compromised LMO2 stability. These mutant LDB1 proteins also exerted dominant negative effects on growth and transcription in diverse leukemic cell lines. Mass spectrometric analysis of LDB1 binding partners in leukemic lines supports the notion that LMO2/LDB1 function in leukemia occurs in the context of multisubunit complexes, which also protect the LMO2 oncoprotein from degradation. Collectively, these data suggest that the assembly of LMO2 into complexes, via direct LDB1 interaction, is a potential molecular target that could be exploited in LMO2-driven leukemias resistant to existing chemotherapy regimens. PMID:26598604

  15. AbdB-like Hox proteins stabilize DNA binding by the Meis1 homeodomain proteins.

    PubMed Central

    Shen, W F; Montgomery, J C; Rozenfeld, S; Moskow, J J; Lawrence, H J; Buchberg, A M; Largman, C

    1997-01-01

    Recent studies show that Hox homeodomain proteins from paralog groups 1 to 10 gain DNA binding specificity and affinity through cooperative binding with the divergent homeodomain protein Pbx1. However, the AbdB-like Hox proteins from paralogs 11, 12, and 13 do not interact with Pbx1a, raising the possibility of different protein partners. The Meis1 homeobox gene has 44% identity to Pbx within the homeodomain and was identified as a common site of viral integration in myeloid leukemias arising in BXH-2 mice. These integrations result in constitutive activation of Meis1. Furthermore, the Hoxa-9 gene is frequently activated by viral integration in the same BXH-2 leukemias, suggesting a biological synergy between these two distinct classes of homeodomain proteins in causing malignant transformation. We now show that the Hoxa-9 protein physically interacts with Meis1 proteins by forming heterodimeric binding complexes on a DNA target containing a Meis1 site (TGACAG) and an AbdB-like Hox site (TTTTACGAC). Hox proteins from the other AbdB-like paralogs, Hoxa-10, Hoxa-11, Hoxd-12, and Hoxb-13, also form DNA binding complexes with Meis1b, while Hox proteins from other paralogs do not appear to interact with Meis1 proteins. DNA binding complexes formed by Meis1 with Hox proteins dissociate much more slowly than DNA complexes with Meis1 alone, suggesting that Hox proteins stabilize the interactions of Meis1 proteins with their DNA targets. PMID:9343407

  16. AbdB-like Hox proteins stabilize DNA binding by the Meis1 homeodomain proteins.

    PubMed

    Shen, W F; Montgomery, J C; Rozenfeld, S; Moskow, J J; Lawrence, H J; Buchberg, A M; Largman, C

    1997-11-01

    Recent studies show that Hox homeodomain proteins from paralog groups 1 to 10 gain DNA binding specificity and affinity through cooperative binding with the divergent homeodomain protein Pbx1. However, the AbdB-like Hox proteins from paralogs 11, 12, and 13 do not interact with Pbx1a, raising the possibility of different protein partners. The Meis1 homeobox gene has 44% identity to Pbx within the homeodomain and was identified as a common site of viral integration in myeloid leukemias arising in BXH-2 mice. These integrations result in constitutive activation of Meis1. Furthermore, the Hoxa-9 gene is frequently activated by viral integration in the same BXH-2 leukemias, suggesting a biological synergy between these two distinct classes of homeodomain proteins in causing malignant transformation. We now show that the Hoxa-9 protein physically interacts with Meis1 proteins by forming heterodimeric binding complexes on a DNA target containing a Meis1 site (TGACAG) and an AbdB-like Hox site (TTTTACGAC). Hox proteins from the other AbdB-like paralogs, Hoxa-10, Hoxa-11, Hoxd-12, and Hoxb-13, also form DNA binding complexes with Meis1b, while Hox proteins from other paralogs do not appear to interact with Meis1 proteins. DNA binding complexes formed by Meis1 with Hox proteins dissociate much more slowly than DNA complexes with Meis1 alone, suggesting that Hox proteins stabilize the interactions of Meis1 proteins with their DNA targets. PMID:9343407

  17. The Cytoplasmic Domain of Anthrax Toxin Receptor 1 Affects Binding of the Protective Antigen▿

    PubMed Central

    Go, Mandy Y.; Chow, Edith M. C.; Mogridge, Jeremy

    2009-01-01

    The protective antigen (PA) component of anthrax toxin binds the I domain of the receptor ANTXR1. Integrin I domains convert between open and closed conformations that bind ligand with high and low affinities, respectively; this process is regulated by signaling from the cytoplasmic domains. To assess whether intracellular signals might influence the interaction between ANTXR1 and PA, we compared two splice variants of ANTXR1 that differ only in their cytoplasmic domains. We found that cells expressing ANTXR1 splice variant 1 (ANTXR1-sv1) bound markedly less PA than did cells expressing a similar level of the shorter splice variant ANTXR1-sv2. ANTXR1-sv1 but not ANTXR1-sv2 associated with the actin cytoskeleton, although disruption of the cytoskeleton did not affect binding of ANTXR-sv1 to PA. Introduction of a cytoplasmic domain missense mutation found in the related receptor ANTXR2 in a patient with juvenile hyaline fibromatosis impaired actin association and increased binding of PA to ANTXR1-sv1. These results suggest that ANTXR1 has two affinity states that may be modulated by cytoplasmic signals. PMID:18936178

  18. Effect of Na+ binding on the conformation, stability and molecular recognition properties of thrombin

    PubMed Central

    De Filippis, Vincenzo; De Dea, Elisa; Lucatello, Filippo; Frasson, Roberta

    2005-01-01

    In the present work, the effect of Na+ binding on the conformational, stability and molecular recognition properties of thrombin was investigated. The binding of Na+ reduces the CD signal in the far-UV region, while increasing the intensity of the near-UV CD and fluorescence spectra. These spectroscopic changes have been assigned to perturbations in the environment of aromatic residues at the level of the S2 and S3 sites, as a result of global rigidification of the thrombin molecule. Indeed, the Na+-bound form is more stable to urea denaturation than the Na+-free form by ∼2 kcal/mol (1 cal≡4.184 J). Notably, the effects of cation binding on thrombin conformation and stability are specific to Na+ and parallel the affinity order of univalent cations for the enzyme. The Na+-bound form is even more resistant to limited proteolysis by subtilisin, at the level of the 148-loop, which is suggestive of the more rigid conformation this segment assumes in the ‘fast’ form. Finally, we have used hirudin fragment 1–47 as a molecular probe of the conformation of thrombin recognition sites in the fast and ‘slow’ form. From the effects of amino acid substitutions on the affinity of fragment 1–47 for the enzyme allosteric forms, we concluded that the specificity sites of thrombin in the Na+-bound form are in a more open and permissible conformation, compared with the more closed structure they assume in the slow form. Taken together, our results indicate that the binding of Na+ to thrombin serves to stabilize the enzyme into a more open and rigid conformation. PMID:15971999

  19. Dermal nanocrystals from medium soluble actives - physical stability and stability affecting parameters.

    PubMed

    Zhai, Xuezhen; Lademann, Jürgen; Keck, Cornelia M; Müller, Rainer H

    2014-09-01

    Nanocrystals are meanwhile applied to increase the dermal penetration of drugs, but were applied by now only to poorly soluble drugs (e.g. 1-10 μg/ml). As a new concept nanocrystals from medium soluble actives were produced, using caffeine as model compound (solubility 16 mg/ml at 20 °C). Penetration should be increased by (a) further increase in solubility and (b) mainly by increased hair follicle targeting of nanocrystals compared to pure solution. Caffeine nanocrystal production in water lead to pronounced crystal growth. Therefore the stability of nanocrystals in water-ethanol (1:9) and ethanol-propylene glycol (3:7) mixtures with lower dielectric constant D was investigated, using various stabilizers. Both mixtures in combination with Carbopol 981 (non-neutralized) yielded stable nanosuspensions over 2 months at 4 °C and room temperature. Storage at 40 °C lead to crystal growth, attributed to too strong solubility increase, supersaturation and Ostwald ripening effects. Stability of caffeine nanocrystals at lower temperatures could not only be attributed to lower solubility, because the solubilities of caffeine in mixtures and in water are not that much different. Other effects such as quantified by reduced dielectric constant D, and specific interactions between dispersion medium and crystal surface seem to play a role. With the 2 mixtures and Carbopol 981, a basic formulation composition for this type of nanocrystals has been established, to be used in the in vivo proof of principle of the new concept. PMID:25016978

  20. β-lactoglobulin stabilized nanemulsions--Formulation and process factors affecting droplet size and nanoemulsion stability.

    PubMed

    Ali, Ali; Mekhloufi, Ghozlene; Huang, Nicolas; Agnely, Florence

    2016-03-16

    To avoid the toxicological concerns associated to synthetic surfactants, proteins might be an alternative for the stabilization of pharmaceutical nanoemulsions. The present study investigates the use of β-lactoglobulin (β-lg) to stabilize oil in water biocompatible nanoemulsions intended for a pharmaceutical use and prepared by high pressure homogenization (HPH). The effects of composition (nature and weight fraction of oil, β-lg concentration) and of process parameters (pressure and number of cycles) on the droplet size and on the stability of nanoemulsions were thoroughly assessed. The nanoemulsions prepared with β-lg at 1 wt% and with 5 wt% Miglyol 812 (the oil with the lowest viscosity) displayed a relatively small particle size (about 200 nm) and a low polydispersity when a homogenization pressure of 100 MPa was applied for 4 cycles. These nanoemulsions were the most stable formulations over 30 days at least. Emulsification efficiency of β-lg was reduced at higher homogenization pressures (200 MPa and 300 MPa). The effect of HPH process on the interfacial properties of β-lg was evaluated by drop shape analysis. This treatment had an effect neither on the interfacial tension nor on the interfacial dilatational rheology of β-lg at the Miglyol 812/water interface. PMID:26784982

  1. Conformational stability and warfarin-binding properties of human serum albumin studied by recombinant mutants.

    PubMed Central

    Watanabe, H; Kragh-Hansen, U; Tanase, S; Nakajou, K; Mitarai, M; Iwao, Y; Maruyama, T; Otagiri, M

    2001-01-01

    Correctly folded recombinant wild-type human serum albumin and the single-residue mutants K199A, W214A, R218H and H242Q were produced with the use of a yeast expression system. The changes in R218H resulted in a pronounced decrease in intrinsic fluorescence. Thermodynamic parameters for thermal denaturation of the present mutants and of five additional mutants have been determined, showing small increases in stability for two mutants (R218H and H242Q) and a larger decrease in stability for one (W214A). In the last of these, denaturation was a heterogeneous process starting at physiological temperature. The high-affinity binding constant for warfarin at pH 7.4 was determined by fluorescence spectroscopy: there was a significant increase in affinity for binding of warfarin to H242Q and K199A and a smaller decrease in affinity for W214A and R218H. The findings show that Trp-214 is not as essential for the high-affinity binding of warfarin as has previously been thought. PMID:11415459

  2. Peptidyl Prolyl Isomerase PIN1 Directly Binds to and Stabilizes Hypoxia-Inducible Factor-1α.

    PubMed

    Han, Hyeong-Jun; Kwon, Nayoung; Choi, Min-A; Jung, Kyung Oh; Piao, Juan-Yu; Ngo, Hoang Kieu Chi; Kim, Su-Jung; Kim, Do-Hee; Chung, June-Key; Cha, Young-Nam; Youn, Hyewon; Choi, Bu Young; Min, Sang-Hyun; Surh, Young-Joon

    2016-01-01

    Peptidyl prolyl isomerase (PIN1) regulates the functional activity of a subset of phosphoproteins through binding to phosphorylated Ser/Thr-Pro motifs and subsequently isomerization of the phosphorylated bonds. Interestingly, PIN1 is overexpressed in many types of malignancies including breast, prostate, lung and colon cancers. However, its oncogenic functions have not been fully elucidated. Here, we report that PIN1 directly interacts with hypoxia-inducible factor (HIF)-1α in human colon cancer (HCT116) cells. PIN1 binding to HIF-1α occurred in a phosphorylation-dependent manner. We also found that PIN1 interacted with HIF-1α at both exogenous and endogenous levels. Notably, PIN1 binding stabilized the HIF-1α protein, given that their levels were significantly increased under hypoxic conditions. The stabilization of HIF-1α resulted in increased transcriptional activity, consequently upregulating expression of vascular endothelial growth factor, a major contributor to angiogenesis. Silencing of PIN1 or pharmacologic inhibition of its activity abrogated the angiogenesis. By utilizing a bioluminescence imaging technique, we were able to demonstrate that PIN1 inhibition dramatically reduced the tumor volume in a subcutaneous mouse xenograft model and angiogenesis as well as hypoxia-induced transcriptional activity of HIF-1α. These results suggest that PIN1 interacting with HIF-1α is a potential cancer chemopreventive and therapeutic target. PMID:26784107

  3. Peptidyl Prolyl Isomerase PIN1 Directly Binds to and Stabilizes Hypoxia-Inducible Factor-1α

    PubMed Central

    Han, Hyeong-jun; Kwon, Nayoung; Choi, Min-A; Jung, Kyung Oh; Piao, Juan-Yu; Ngo, Hoang Kieu Chi; Kim, Su-Jung; Kim, Do-Hee; Chung, June-Key; Cha, Young-Nam; Youn, Hyewon; Choi, Bu Young; Min, Sang-Hyun; Surh, Young-Joon

    2016-01-01

    Peptidyl prolyl isomerase (PIN1) regulates the functional activity of a subset of phosphoproteins through binding to phosphorylated Ser/Thr-Pro motifs and subsequently isomerization of the phosphorylated bonds. Interestingly, PIN1 is overexpressed in many types of malignancies including breast, prostate, lung and colon cancers. However, its oncogenic functions have not been fully elucidated. Here, we report that PIN1 directly interacts with hypoxia-inducible factor (HIF)-1α in human colon cancer (HCT116) cells. PIN1 binding to HIF-1α occurred in a phosphorylation-dependent manner. We also found that PIN1 interacted with HIF-1α at both exogenous and endogenous levels. Notably, PIN1 binding stabilized the HIF-1α protein, given that their levels were significantly increased under hypoxic conditions. The stabilization of HIF-1α resulted in increased transcriptional activity, consequently upregulating expression of vascular endothelial growth factor, a major contributor to angiogenesis. Silencing of PIN1 or pharmacologic inhibition of its activity abrogated the angiogenesis. By utilizing a bioluminescence imaging technique, we were able to demonstrate that PIN1 inhibition dramatically reduced the tumor volume in a subcutaneous mouse xenograft model and angiogenesis as well as hypoxia-induced transcriptional activity of HIF-1α. These results suggest that PIN1 interacting with HIF-1α is a potential cancer chemopreventive and therapeutic target. PMID:26784107

  4. CFTR gating II: Effects of nucleotide binding on the stability of open states.

    PubMed

    Bompadre, Silvia G; Cho, Jeong Han; Wang, Xiaohui; Zou, Xiaoqin; Sohma, Yoshiro; Li, Min; Hwang, Tzyh-Chang

    2005-04-01

    Previously, we demonstrated that ADP inhibits cystic fibrosis transmembrane conductance regulator (CFTR) opening by competing with ATP for a binding site presumably in the COOH-terminal nucleotide binding domain (NBD2). We also found that the open time of the channel is shortened in the presence of ADP. To further study this effect of ADP on the open state, we have used two CFTR mutants (D1370N and E1371S); both have longer open times because of impaired ATP hydrolysis at NBD2. Single-channel kinetic analysis of DeltaR/D1370N-CFTR shows unequivocally that the open time of this mutant channel is decreased by ADP. DeltaR/E1371S-CFTR channels can be locked open by millimolar ATP with a time constant of approximately 100 s, estimated from current relaxation upon nucleotide removal. ADP induces a shorter locked-open state, suggesting that binding of ADP at a second site decreases the locked-open time. To test the functional consequence of the occupancy of this second nucleotide binding site, we changed the [ATP] and performed similar relaxation analysis for E1371S-CFTR channels. Two locked-open time constants can be discerned and the relative distribution of each component is altered by changing [ATP] so that increasing [ATP] shifts the relative distribution to the longer locked-open state. Single-channel kinetic analysis for DeltaR/E1371S-CFTR confirms an [ATP]-dependent shift of the distribution of two locked-open time constants. These results support the idea that occupancy of a second ATP binding site stabilizes the locked-open state. This binding site likely resides in the NH2-terminal nucleotide binding domain (NBD1) because introducing the K464A mutation, which decreases ATP binding affinity at NBD1, into E1371S-CFTR shortens the relaxation time constant. These results suggest that the binding energy of nucleotide at NBD1 contributes to the overall energetics of the open channel conformation. PMID:15767296

  5. Identifying Sequential Substrate Binding at the Single-Molecule Level by Enzyme Mechanical Stabilization

    PubMed Central

    Rivas-Pardo, Jaime Andrés; Alegre-Cebollada, Jorge; Ramírez-Sarmiento, César A.; Fernandez, Julio M.; Guixé, Victoria

    2015-01-01

    Enzyme-substrate binding is a dynamic process intimately coupled to protein structural changes, which in turn changes the unfolding energy landscape. By the use of single molecule force spectroscopy (SMFS), we characterize the open-to-closed conformational transition experienced by the hyperthermophilic ADP-dependent glucokinase from Thermococcus litoralis triggered by the sequential binding of substrates. In the absence of substrates, the mechanical unfolding of TlGK shows an intermediate I, which is stabilized in the presence of Mg·ADP-, the first substrate to bind to the enzyme. However, in the presence of this substrate, an additional unfolding event is observed, intermediate-1*. Finally, in the presence of both substrates, the unfolding force of intermediates-1 and -1*, increases as a consequence of the domain closure. These results show that SMFS could be used as a powerful experimental tool to investigate binding mechanisms of different enzymes with more than one ligand, expanding the repertoire of protocols traditionally used in enzymology. PMID:25840594

  6. Directed evolution of a G protein-coupled receptor for expression, stability, and binding selectivity

    PubMed Central

    Sarkar, Casim A.; Dodevski, Igor; Kenig, Manca; Dudli, Stefan; Mohr, Anja; Hermans, Emmanuel; Plückthun, Andreas

    2008-01-01

    We outline a powerful method for the directed evolution of integral membrane proteins in the inner membrane of Escherichia coli. For a mammalian G protein-coupled receptor, we arrived at a sequence with an order-of-magnitude increase in functional expression that still retains the biochemical properties of wild type. This mutant also shows enhanced heterologous expression in eukaryotes (12-fold in Pichia pastoris and 3-fold in HEK293T cells) and greater stability when solubilized and purified, indicating that the biophysical properties of the protein had been under the pressure of selection. These improvements arise from multiple small contributions, which would be difficult to assemble by rational design. In a second screen, we rapidly pinpointed a single amino acid substitution in wild type that abolishes antagonist binding while retaining agonist-binding affinity. These approaches may alleviate existing bottlenecks in structural studies of these targets by providing sufficient quantities of stable variants in defined conformational states. PMID:18812512

  7. The Tubulin Binding Mode of Microtubule Stabilizing Agents Studied by Electron Crystallography

    NASA Astrophysics Data System (ADS)

    Nettles, James H.; Downing, Kenneth H.

    Since tubulin was discovered in 1967, drug probes have been used to manipulate mechanisms of microtubule polymerization and disassembly. In parallel, advances in optical imagery, electron microscopy, along with both electron and X-ray diffraction have provided ability to "see" the molecular underpinning of these machines. Nanoscale mapping of different tubulin polymers formed in the presence of different drugs and cofactors provide a context for examining the dynamic features relevant to their biological activity. Models built from EM maps have been used to understand the binding of stabilizing drugs such as taxanes and epothilones, to predict more effective molecules, and to explain mutation based resistance. Here, we discuss drug binding in the context of different polymeric forms and propose a trigger mechanism associated with microtubules' dynamic instability.

  8. Role of the glutamic acid 54 residue in transthyretin stability and thyroxine binding.

    PubMed

    Miyata, Masanori; Sato, Takashi; Mizuguchi, Mineyuki; Nakamura, Teruya; Ikemizu, Shinji; Nabeshima, Yuko; Susuki, Seiko; Suwa, Yoshiaki; Morioka, Hiroshi; Ando, Yukio; Suico, Mary Ann; Shuto, Tsuyoshi; Koga, Tomoaki; Yamagata, Yuriko; Kai, Hirofumi

    2010-01-12

    Transthyretin (TTR) is a tetrameric protein associated with amyloidosis caused by tetramer dissociation and monomer misfolding. The structure of two TTR variants (E54G and E54K) with Glu54 point mutation that cause clinically aggressive amyloidosis remains unclear, although amyloidogenicity of artificial triple mutations (residues 53-55) in beta-strand D had been investigated. Here we first analyzed the crystal structures and biochemical and biophysical properties of E54G and E54K TTRs. The direction of the Lys15 side chain in E54K TTR and the surface electrostatic potential in the edge region in both variants were different from those of wild-type TTR. The presence of Lys54 leads to destabilization of tetramer structure due to enhanced electrostatic repulsion between Lys15 of two monomers. Consistent with structural data, the biochemical analyses demonstrated that E54G and E54K TTRs were more unstable than wild-type TTR. Furthermore, the entrance of the thyroxine (T(4)) binding pocket in TTR was markedly narrower in E54K TTR and wider in E54G TTR compared with wild-type TTR. The tetramer stabilization and amyloid fibril formation assays in the presence of T(4) showed lower tetramer stability and more fibril formation in E54K and E54G TTRs than in wild-type TTR, suggesting decreased T(4) binding to the TTR variants. These findings indicate that structural modification by Glu54 point mutation may sufficiently alter tetramer stability and T(4) binding. PMID:19950966

  9. Enhanced exo-inulinase activity and stability by fusion of an inulin-binding module.

    PubMed

    Zhou, Shun-Hua; Liu, Yuan; Zhao, Yu-Juan; Chi, Zhe; Chi, Zhen-Ming; Liu, Guang-Lei

    2016-09-01

    In this study, an inulin-binding module from Bacillus macerans was successfully fused to an exo-inulinase from Kluyveromyces marxianus, creating a hybrid functional enzyme. The recombinant exo-inulinase (rINU), the hybrid enzyme (rINUIBM), and the recombinant inulin-binding module (rIBM) were, respectively, heterologously expressed and biochemically characterized. It was found that both the inulinase activity and the catalytic efficiency (k cat/K m(app)) of the rINUIBM were considerably higher than those of rINU. Though the rINU and the rINUIBM shared the same optimum pH of 4.5, the optimum temperature of the rINUIBM (60 °C) was 5 °C higher than that of the rINU. Notably, the fused IBM significantly enhanced both the pH stability and the thermostability of the rINUIBM, suggesting that the rINUIBM obtained would have more extensive potential applications. Furthermore, the fusion of the IBM could substantially improve the inulin-binding capability of the rINUIBM, which was consistent with the determination of the K m(app). This meant that the fused IBM could play a critical role in the recognition of polysaccharides and enhanced the hydrolase activity of the associated inulinase by increasing enzyme-substrate proximity. Besides, the extra supplement of the independent non-catalytic rIBM could also improve the inulinase activity of the rINU. However, this improvement was much better in case of the fusion. Consequently, the IBM could be designated as a multifunctional domain that was responsible for the activity enhancement, the stabilization, and the substrate binding of the rINUIBM. All these features obtained in this study make the rINUIBM become an attractive candidate for an efficient inulin hydrolysis. PMID:27164865

  10. Haptoglobin Binding Stabilizes Hemoglobin Ferryl Iron and the Globin Radical on Tyrosine β145

    PubMed Central

    Schaer, Dominik J.; Buehler, Paul W.; Wilson, Michael T.; Reeder, Brandon J.; Silkstone, Gary; Svistunenko, Dimitri A.; Bulow, Leif; Alayash, Abdu I.

    2013-01-01

    Abstract Aim: Hemoglobin (Hb) becomes toxic when released from the erythrocyte. The acute phase protein haptoglobin (Hp) binds avidly to Hb and decreases oxidative damage to Hb itself and to the surrounding proteins and lipids. However, the molecular mechanism underpinning Hp protection is to date unclear. The aim of this study was to use electron paramagnetic resonance (EPR) spectroscopy, stopped flow optical spectrophotometry, and site-directed mutagenesis to explore the mechanism and specifically the role of specific tyrosine residues in this protection. Results: Following peroxide challenge Hb produces reactive oxidative intermediates in the form of ferryl heme and globin free radicals. Hp binding increases the steady state level of ferryl formation during Hb-catalyzed lipid peroxidation, while at the same time dramatically inhibiting the overall reaction rate. This enhanced ferryl stability is also seen in the absence of lipids and in the presence of external reductants. Hp binding is not accompanied by a decrease in the pK of ferryl protonation; the protonated ferryl species still forms, but is intrinsically less reactive. Ferryl stabilization is accompanied by a significant increase in the concentration of the peroxide-induced tyrosine free radical. EPR spectral parameters and mutagenesis studies suggest that this radical is located on tyrosine 145, the penultimate C-terminal amino acid on the beta Hb subunit. Innovation: Hp binding decreases both the ferryl iron and free radical reactivity of Hb. Conclusion: Hp protects against Hb-induced damage in the vasculature, not by preventing the primary reactivity of heme oxidants, but by rendering the resultant protein products less damaging. Antioxid. Redox Signal. 18, 2264–2273. PMID:22702311

  11. GABA(B) receptor subunit 1 binds to proteins affected in 22q11 deletion syndrome.

    PubMed

    Zunner, Dagmar; Deschermeier, Christina; Kornau, Hans-Christian

    2010-03-01

    GABA(B) receptors mediate slow inhibitory effects of the neurotransmitter gamma-aminobutyric acid (GABA) on synaptic transmission in the central nervous system. They function as heterodimeric G-protein-coupled receptors composed of the seven-transmembrane domain proteins GABA(B1) and GABA(B2), which are linked through a coiled-coil interaction. The ligand-binding subunit GABA(B1) is at first retained in the endoplasmic reticulum and is transported to the cell surface only upon assembly with GABA(B2). Here, we report that GABA(B1), via the coiled-coil domain, can also bind to soluble proteins of unknown function, that are affected in 22q11 deletion/DiGeorge syndrome and are therefore referred to as DiGeorge critical region 6 (DGCR6). In transfected neurons the GABA(B1)-DGCR6 association resulted in a redistribution of both proteins into intracellular clusters. Furthermore, the C-terminus of GABA(B2) interfered with the novel interaction, consistent with heterodimer formation overriding transient DGCR6-binding to GABA(B1). Thus, sequential coiled-coil interactions may direct GABA(B1) into functional receptors. PMID:20036641

  12. Hand proximity differentially affects visual working memory for color and orientation in a binding task.

    PubMed

    Kelly, Shane P; Brockmole, James R

    2014-01-01

    Observers determined whether two sequentially presented arrays of six lines were the same or different. Differences, when present, involved either a swap in the color of two lines or a swap in the orientation of two lines. Thus, accurate change detection required the binding of color and orientation information for each line within visual working memory. Holding viewing distance constant, the proximity of the arrays to the hands was manipulated. Placing the hands near the to-be-remembered array decreased participants' ability to remember color information, but increased their ability to remember orientation information. This pair of results indicates that hand proximity differentially affects the processing of various types of visual information, a conclusion broadly consistent with functional and anatomical differences in the magnocellular and parvocellular pathways. It further indicates that hand proximity affects the likelihood that various object features will be encoded into integrated object files. PMID:24795671

  13. Binding energy and mechanical stability of single- and multi-walled carbon nanotube serpentines.

    PubMed

    Zhao, Junhua; Lu, Lixin; Rabczuk, Timon

    2014-05-28

    Recently, Geblinger et al. [Nat. Nanotechnol. 3, 195 (2008)] and Machado et al. [Phys. Rev. Lett. 110, 105502 (2013)] reported the experimental and molecular dynamics realization of S-like shaped single-walled carbon nanotubes (CNTs), the so-called CNT serpentines. We reported here results from continuum modeling of the binding energy γ between different single- and multi-walled CNT serpentines and substrates as well as the mechanical stability of the CNT serpentine formation. The critical length for the mechanical stability and adhesion of different CNT serpentines are determined in dependence of EiIi, d, and γ, where EiIi and d are the CNT bending stiffness and distance of the CNT translation period. Our continuum model is validated by comparing its solution to full-atom molecular dynamics calculations. The derived analytical solutions are of great importance for understanding the interaction mechanism between different single- and multi-walled CNT serpentines and substrates. PMID:24880308

  14. Protein stability induced by ligand binding correlates with changes in protein flexibility

    PubMed Central

    Celej, María Soledad; Montich, Guillermo G.; Fidelio, Gerardo D.

    2003-01-01

    The interaction between ligands and proteins usually induces changes in protein thermal stability with modifications in the midpoint denaturation temperature, enthalpy of unfolding, and heat capacity. These modifications are due to the coupling of unfolding with binding equilibrium. Furthermore, they can be attained by changes in protein structure and conformational flexibility induced by ligand interaction. To study these effects we have used bovine serum albumin (BSA) interacting with three different anilinonaphthalene sulfonate derivatives (ANS). These ligands have different effects on protein stability, conformation, and dynamics. Protein stability was studied by differential scanning calorimetry and fluorescence spectroscopy, whereas conformational changes were detected by circular dichroism and infrared spectroscopy including kinetics of hydrogen/deuterium exchange. The order of calorimetric midpoint of denaturation was: 1,8-ANS-BSA > 2,6-ANS-BSA > free BSA >> (nondetected) bis-ANS-BSA. Both 1,8-ANS and 2,6-ANS did not substantially modify the secondary structure of BSA, whereas bis-ANS induced a distorted α-helix conformation with an increase of disordered structure. Protein flexibility followed the order: 1,8-ANS-BSA < 2,6-ANS-BSA < free BSA << bis-ANS-BSA, indicating a clear correlation between stability and conformational flexibility. The structure induced by an excess of bis-ANS to BSA is compatible with a molten globule-like state. Within the context of the binding landscape model, we have distinguished five conformers (identified by subscript): BSA1,8-ANS, BSA2,6-ANS, BSAfree, BSAbis-ANS, and BSAunfolded among the large number of possible states of the conformational dynamic ensemble. The relative population of each distinguishable conformer depends on the type and concentration of ligand and the temperature of the system. PMID:12824495

  15. Haemoglobin polymorphisms affect the oxygen-binding properties in Atlantic cod populations

    PubMed Central

    Andersen, Øivind; Wetten, Ola Frang; De Rosa, Maria Cristina; Andre, Carl; Carelli Alinovi, Cristiana; Colafranceschi, Mauro; Brix, Ole; Colosimo, Alfredo

    2008-01-01

    A major challenge in evolutionary biology is to identify the genes underlying adaptation. The oxygen-transporting haemoglobins directly link external conditions with metabolic needs and therefore represent a unique system for studying environmental effects on molecular evolution. We have discovered two haemoglobin polymorphisms in Atlantic cod populations inhabiting varying temperature and oxygen regimes in the North Atlantic. Three-dimensional modelling of the tetrameric haemoglobin structure demonstrated that the two amino acid replacements Met55β1Val and Lys62β1Ala are located at crucial positions of the α1β1 subunit interface and haem pocket, respectively. The replacements are proposed to affect the oxygen-binding properties by modifying the haemoglobin quaternary structure and electrostatic feature. Intriguingly, the same molecular mechanism for facilitating oxygen binding is found in avian species adapted to high altitudes, illustrating convergent evolution in water- and air-breathing vertebrates to reduction in environmental oxygen availability. Cod populations inhabiting the cold Arctic waters and the low-oxygen Baltic Sea seem well adapted to these conditions by possessing the high oxygen affinity Val55–Ala62 haplotype, while the temperature-insensitive Met55–Lys62 haplotype predominates in the southern populations. The distinct distributions of the functionally different haemoglobin variants indicate that the present biogeography of this ecologically and economically important species might be seriously affected by global warming. PMID:19033139

  16. Biglycan is an extracellular MuSK binding protein important for synapse stability

    PubMed Central

    Amenta, A.R.; Creely, H.E.; Mercado, M.L.; Hagiwara, H.; McKechnie, B. A.; Lechner, B.E.; Rossi, S. G.; Wang, Q.; Owens, R. T.; Marrero, E.; Mei, L.; Hoch, W.; Young, M. F.; McQuillan, D. J.; Rotundo, R. L.; Fallon, J.R.

    2012-01-01

    The receptor tyrosine kinase MuSK is indispensable for nerve-muscle synapse formation and maintenance. MuSK is necessary for pre-patterning of the endplate zone anlage and as a signaling receptor for agrin-mediated postsynaptic differentiation. MuSK-associated proteins such as Dok7, LRP4, and Wnt11r are involved in these early events in neuromuscular junction formation. However, the mechanisms regulating synapse stability are poorly understood. Here we examine a novel role for the extracellular matrix protein biglycan in synapse stability. Synaptic development in fetal and early postnatal biglycan null (bgn-/o) muscle is indistinguishable from wild type controls. However, by 5 wks after birth nerve-muscle synapses in bgn-/o mice are abnormal as judged by the presence of perijunctional folds, increased segmentation and focal misalignment of acetylcholinesterase and AChRs. These observations indicate that previously occupied pre- and post- synaptic territory has been vacated. Biglycan binds MuSK and the levels of this receptor tyrosine kinase are selectively reduced at bgn-/o synapses. In bgn-/o myotubes, the initial stages of agrin-induced MuSK phosphorylation and AChR clustering are normal, but the AChR clusters are unstable. This stability defect can be substantially rescued by the addition of purified biglycan. Together, these results indicate that biglycan is an extracellular ligand for MuSK that is important for synapse stability. PMID:22396407

  17. Structure and stability of recombinant bovine odorant-binding protein: II. Unfolding of the monomeric forms.

    PubMed

    Stepanenko, Olga V; Roginskii, Denis O; Stepanenko, Olesya V; Kuznetsova, Irina M; Uversky, Vladimir N; Turoverov, Konstantin K

    2016-01-01

    In a family of monomeric odorant-binding proteins (OBPs), bovine OBP (bOBP), that lacks conserved disulfide bond found in other OBPs, occupies unique niche because of its ability to form domain-swapped dimers. In this study, we analyzed conformational stabilities of the recombinant bOBP and its monomeric variants, the bOBP-Gly121+ mutant containing an additional glycine residue after the residue 121 of the bOBP, and the GCC-bOBP mutant obtained from the bOBP-Gly121+ form by introduction of the Trp64Cys/His155Cys double mutation to restore the canonical disulfide bond. We also analyzed the effect of the natural ligand binding on the conformational stabilities of these bOBP variants. Our data are consistent with the conclusion that the unfolding-refolding pathways of the recombinant bOBP and its mutant monomeric forms bOBP-Gly121+ and GCC-bOBP are similar and do not depend on the oligomeric status of the protein. This clearly shows that the information on the unfolding-refolding mechanism is encoded in the structure of the bOBP monomers. However, the process of the bOBP unfolding is significantly complicated by the formation of the domain-swapped dimer, and the rates of the unfolding-refolding reactions essentially depend on the conditions in which the protein is located. PMID:27114857

  18. Structural insights into how the MIDAS ion stabilizes integrin binding to an RGD peptide under force.

    PubMed

    Craig, David; Gao, Mu; Schulten, Klaus; Vogel, Viola

    2004-11-01

    Integrin alpha(V)beta(3) binds to extracellular matrix proteins through the tripeptide Arg-Gly-Asp (RGD), forming a shallow crevice rather than a deep binding pocket. A dynamic picture of how the RGD-alpha(V)beta(3) complex resists dissociation by mechanical force is derived here from steered molecular dynamic (SMD) simulations in which the major force peak correlates with the breaking of the contact between Asp(RGD) and the MIDAS ion. SMD predicts that the RGD-alpha(V)beta(3) complex is stabilized from dissociation by a single water molecule tightly coordinated to the divalent MIDAS ion, thereby blocking access of free water molecules to the most critical force-bearing interaction. The MIDAS motif is common to many other proteins that contain the phylogenetically ancient von Willebrand A (vWA) domain. The functional role of single water molecules tightly coordinated to the MIDAS ion might reflect a general strategy for the stabilization of protein-protein adhesion against cell-derived forces through divalent cations. PMID:15530369

  19. Structure and stability of recombinant bovine odorant-binding protein: II. Unfolding of the monomeric forms

    PubMed Central

    Stepanenko, Olga V.; Roginskii, Denis O.; Stepanenko, Olesya V.; Kuznetsova, Irina M.

    2016-01-01

    In a family of monomeric odorant-binding proteins (OBPs), bovine OBP (bOBP), that lacks conserved disulfide bond found in other OBPs, occupies unique niche because of its ability to form domain-swapped dimers. In this study, we analyzed conformational stabilities of the recombinant bOBP and its monomeric variants, the bOBP-Gly121+ mutant containing an additional glycine residue after the residue 121 of the bOBP, and the GCC-bOBP mutant obtained from the bOBP-Gly121+ form by introduction of the Trp64Cys/His155Cys double mutation to restore the canonical disulfide bond. We also analyzed the effect of the natural ligand binding on the conformational stabilities of these bOBP variants. Our data are consistent with the conclusion that the unfolding-refolding pathways of the recombinant bOBP and its mutant monomeric forms bOBP-Gly121+ and GCC-bOBP are similar and do not depend on the oligomeric status of the protein. This clearly shows that the information on the unfolding-refolding mechanism is encoded in the structure of the bOBP monomers. However, the process of the bOBP unfolding is significantly complicated by the formation of the domain-swapped dimer, and the rates of the unfolding-refolding reactions essentially depend on the conditions in which the protein is located. PMID:27114857

  20. Effect of Mutation and Substrate Binding on the Stability of Cytochrome P450BM3 Variants.

    PubMed

    Geronimo, Inacrist; Denning, Catherine A; Rogers, W Eric; Othman, Thaer; Huxford, Tom; Heidary, David K; Glazer, Edith C; Payne, Christina M

    2016-06-28

    Cytochrome P450BM3 is a heme-containing enzyme from Bacillus megaterium that exhibits high monooxygenase activity and has a self-sufficient electron transfer system in the full-length enzyme. Its potential synthetic applications drive protein engineering efforts to produce variants capable of oxidizing nonnative substrates such as pharmaceuticals and aromatic pollutants. However, promiscuous P450BM3 mutants often exhibit lower stability, thereby hindering their industrial application. This study demonstrated that the heme domain R47L/F87V/L188Q/E267V/F81I pentuple mutant (PM) is destabilized because of the disruption of hydrophobic contacts and salt bridge interactions. This was directly observed from crystal structures of PM in the presence and absence of ligands (palmitic acid and metyrapone). The instability of the tertiary structure and heme environment of substrate-free PM was confirmed by pulse proteolysis and circular dichroism, respectively. Binding of the inhibitor, metyrapone, significantly stabilized PM, but the presence of the native substrate, palmitic acid, had no effect. On the basis of high-temperature molecular dynamics simulations, the lid domain, β-sheet 1, and Cys ligand loop (a β-bulge segment connected to the heme) are the most labile regions and, thus, potential sites for stabilizing mutations. Possible approaches to stabilization include improvement of hydrophobic packing interactions in the lid domain and introduction of new salt bridges into β-sheet 1 and the heme region. An understanding of the molecular factors behind the loss of stability of P450BM3 variants therefore expedites site-directed mutagenesis studies aimed at developing thermostability. PMID:27267136

  1. Stabilization of Poliovirus Polymerase by NTP Binding and Fingers-Thumb Interactions

    PubMed Central

    Thompson, Aaron A.; Albertini, Rebecca A.; Peersen, Olve B.

    2007-01-01

    The viral RNA-dependent RNA polymerases show a conserved structure where the fingers domain interacts with the top of the thumb domain to create a tunnel through which nucleotide triphosphates reach the active site. We have solved the crystal structures of poliovirus polymerase (3Dpol) in complex with all four NTPs, showing that they all bind in a common preinsertion site where the phosphates are not yet positioned over the active site. The NTPs interact with both the fingers and palm domains, forming bridging interactions that explain the increased thermal stability of 3Dpol in the presence of NTPs. We have also examined the importance of the fingers-thumb domain interaction for the function and structural stability of 3Dpol. Results from thermal denaturation experiments using circular dichroism and 2-aniliino-6-napthaline-sulfonate (ANS) fluorescence show that 3Dpol has a melting temperature of only ∼40°C. NTP binding stabilizes the protein and increases the melting by 5-6 °C while mutations in the fingers-thumb domain interface destabilize the protein and reduce the melting point by as much as 6 °C. In particular, the burial of Phe30 and Phe34 from the tip of the index finger into a pocket at the top of the thumb and the presence of Trp403 on the thumb domain are key interactions required to maintain the structural integrity of the polymerase. The data suggest the fingers domain has significant conformational flexibility and exists in a highly dynamic molten globule state at physiological temperature. The role of the enclosed active site motif as a structural scaffold for constraining the fingers domain and accommodating conformational changes in 3Dpol and other viral polymerases during the catalytic cycle is discussed. PMID:17223130

  2. Mutations in the putative calcium-binding domain of polyomavirus VP1 affect capsid assembly

    NASA Technical Reports Server (NTRS)

    Haynes, J. I. 2nd; Chang, D.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    Calcium ions appear to play a major role in maintaining the structural integrity of the polyomavirus and are likely involved in the processes of viral uncoating and assembly. Previous studies demonstrated that a VP1 fragment extending from Pro-232 to Asp-364 has calcium-binding capabilities. This fragment contains an amino acid stretch from Asp-266 to Glu-277 which is quite similar in sequence to the amino acids that make up the calcium-binding EF hand structures found in many proteins. To assess the contribution of this domain to polyomavirus structural integrity, the effects of mutations in this region were examined by transfecting mutated viral DNA into susceptible cells. Immunofluorescence studies indicated that although viral protein synthesis occurred normally, infective viral progeny were not produced in cells transfected with polyomavirus genomes encoding either a VP1 molecule lacking amino acids Thr-262 through Gly-276 or a VP1 molecule containing a mutation of Asp-266 to Ala. VP1 molecules containing the deletion mutation were unable to bind 45Ca in an in vitro assay. Upon expression in Escherichia coli and purification by immunoaffinity chromatography, wild-type VP1 was isolated as pentameric, capsomere-like structures which could be induced to form capsid-like structures upon addition of CaCl2, consistent with previous studies. However, although VP1 containing the point mutation was isolated as pentamers which were indistinguishable from wild-type VP1 pentamers, addition of CaCl2 did not result in their assembly into capsid-like structures. Immunogold labeling and electron microscopy studies of transfected mammalian cells provided in vivo evidence that a mutation in this region affects the process of viral assembly.

  3. Stabilization/solidification of heavy metals in sludge ceramsite and leachability affected by oxide substances.

    PubMed

    Xu, Guoren; Zou, Jinlong; Li, Guibai

    2009-08-01

    To investigate stabilization of heavy metals in ceramsite made from wastewater treatment sludge (WWTS) and drinking water treatment sludge (DWTS), leaching tests were conducted to find out the effect of SiO2:Al2O3, acidic oxides (SiO2 and Al2O3), Fe2O3: CaO:MgO, and basic oxides (Fe2O3, CaO, and MgO) on the binding ability of heavy metals. Results show that as ratios of SiO2: Al2O3 decrease, leaching contents of Cu and Pb increase, while leaching contents of Cd and Cr first decrease and then increase; under the variation of Fe2O3:CaO:MgO (Fe2O3 contents decrease), leaching contents of Cd, Cu, and Pb increase, while leaching contents of Cr decrease. Acidic and basic oxide leaching results show that higher contents of Al2O3, Fe2O3, and MgO are advantageous to improve the stability of heavy metals, while the binding capacity for Cd, Cu, and Pb is significantly reduced at higher contents of SiO2 and CaO. The solidifying efficiencies of heavy metals are improved by crystallization, and the main compounds in ceramsite are crocoite, chrome oxide, cadmium silicate, and copper oxide. These results can be considered as a basic understanding for new technologies of stabilization of heavy metals in heavily polluted WWTS. PMID:19731695

  4. Intermonomer Interactions in Hemagglutinin Subunits HA1 and HA2 Affecting Hemagglutinin Stability and Influenza Virus Infectivity

    PubMed Central

    DeFeo, Christopher J.; Alvarado-Facundo, Esmeralda; Vassell, Russell

    2015-01-01

    ABSTRACT Influenza virus hemagglutinin (HA) mediates virus entry by binding to cell surface receptors and fusing the viral and endosomal membranes following uptake by endocytosis. The acidic environment of endosomes triggers a large-scale conformational change in the transmembrane subunit of HA (HA2) involving a loop (B loop)-to-helix transition, which releases the fusion peptide at the HA2 N terminus from an interior pocket within the HA trimer. Subsequent insertion of the fusion peptide into the endosomal membrane initiates fusion. The acid stability of HA is influenced by residues in the fusion peptide, fusion peptide pocket, coiled-coil regions of HA2, and interactions between the surface (HA1) and HA2 subunits, but details are not fully understood and vary among strains. Current evidence suggests that the HA from the circulating pandemic 2009 H1N1 influenza A virus [A(H1N1)pdm09] is less stable than the HAs from other seasonal influenza virus strains. Here we show that residue 205 in HA1 and residue 399 in the B loop of HA2 (residue 72, HA2 numbering) in different monomers of the trimeric A(H1N1)pdm09 HA are involved in functionally important intermolecular interactions and that a conserved histidine in this pair helps regulate HA stability. An arginine-lysine pair at this location destabilizes HA at acidic pH and mediates fusion at a higher pH, while a glutamate-lysine pair enhances HA stability and requires a lower pH to induce fusion. Our findings identify key residues in HA1 and HA2 that interact to help regulate H1N1 HA stability and virus infectivity. IMPORTANCE Influenza virus hemagglutinin (HA) is the principal antigen in inactivated influenza vaccines and the target of protective antibodies. However, the influenza A virus HA is highly variable, necessitating frequent vaccine changes to match circulating strains. Sequence changes in HA affect not only antigenicity but also HA stability, which has important implications for vaccine production, as well

  5. Post-translational Modifications Differentially Affect IgG1 Conformation and Receptor Binding*

    PubMed Central

    Houde, Damian; Peng, Yucai; Berkowitz, Steven A.; Engen, John R.

    2010-01-01

    Post-translational modifications (PTMs) can have profound effects on protein structure and protein dynamics and thereby can influence protein function. To understand and connect PTM-induced functional differences with any resulting conformational changes, the conformational changes must be detected and localized to specific parts of the protein. We illustrate these principles here with a study of the functional and conformational changes that accompany modifications to a monoclonal immunoglobulin γ1 (IgG1) antibody. IgG1s are large and heterogeneous proteins capable of incorporating a multiplicity of PTMs both in vivo and in vitro. For many IgG1s, these PTMs can play a critical role in affecting conformation, biological function, and the ability of the antibody to initiate a potential adverse biological response. We investigated the impact of differential galactosylation, methionine oxidation, and fucosylation on solution conformation using hydrogen/deuterium exchange mass spectrometry and probed the effects of IgG1 binding to the FcγRIIIa receptor. The results showed that methionine oxidation and galactosylation both impact IgG1 conformation, whereas fucosylation appears to have little or no impact to the conformation. FcγRIIIa binding was strongly influenced by both the glycan structure/composition (namely galactose and fucose) and conformational changes that were induced by some of the modifications. PMID:20103567

  6. High-Density Lipoprotein Binds to Mycobacterium avium and Affects the Infection of THP-1 Macrophages.

    PubMed

    Ichimura, Naoya; Sato, Megumi; Yoshimoto, Akira; Yano, Kouji; Ohkawa, Ryunosuke; Kasama, Takeshi; Tozuka, Minoru

    2016-01-01

    High-density lipoprotein (HDL) is involved in innate immunity toward various infectious diseases. Concerning bacteria, HDL is known to bind to lipopolysaccharide (LPS) and to neutralize its physiological activity. On the other hand, cholesterol is known to play an important role in mycobacterial entry into host cells and in survival in the intracellular environment. However, the pathogenicity of Mycobacterium avium (M. avium) infection, which tends to increase worldwide, remains poorly studied. Here we report that HDL indicated a stronger interaction with M. avium than that with other Gram-negative bacteria containing abundant LPS. A binding of apolipoprotein (apo) A-I, the main protein component of HDL, with a specific lipid of M. avium might participate in this interaction. HDL did not have a direct bactericidal activity toward M. avium but attenuated the engulfment of M. avium by THP-1 macrophages. HDL also did not affect bacterial killing after ingestion of live M. avium by THP-1 macrophage. Furthermore, HDL strongly promoted the formation of lipid droplets in M. avium-infected THP-1 macrophages. These observations provide new insights into the relationship between M. avium infection and host lipoproteins, especially HDL. Thus, HDL may help M. avium to escape from host innate immunity. PMID:27516907

  7. High-Density Lipoprotein Binds to Mycobacterium avium and Affects the Infection of THP-1 Macrophages

    PubMed Central

    Ichimura, Naoya; Sato, Megumi; Yoshimoto, Akira; Yano, Kouji; Ohkawa, Ryunosuke; Kasama, Takeshi

    2016-01-01

    High-density lipoprotein (HDL) is involved in innate immunity toward various infectious diseases. Concerning bacteria, HDL is known to bind to lipopolysaccharide (LPS) and to neutralize its physiological activity. On the other hand, cholesterol is known to play an important role in mycobacterial entry into host cells and in survival in the intracellular environment. However, the pathogenicity of Mycobacterium avium (M. avium) infection, which tends to increase worldwide, remains poorly studied. Here we report that HDL indicated a stronger interaction with M. avium than that with other Gram-negative bacteria containing abundant LPS. A binding of apolipoprotein (apo) A-I, the main protein component of HDL, with a specific lipid of M. avium might participate in this interaction. HDL did not have a direct bactericidal activity toward M. avium but attenuated the engulfment of M. avium by THP-1 macrophages. HDL also did not affect bacterial killing after ingestion of live M. avium by THP-1 macrophage. Furthermore, HDL strongly promoted the formation of lipid droplets in M. avium-infected THP-1 macrophages. These observations provide new insights into the relationship between M. avium infection and host lipoproteins, especially HDL. Thus, HDL may help M. avium to escape from host innate immunity. PMID:27516907

  8. The stability and the metal ions binding properties of mutant A85M of CopC.

    PubMed

    Song, Zhen; Dong, Jinlong; Yuan, Wen; Zhang, Caifeng; Ren, Yuehong; Yang, Binsheng

    2016-08-01

    In this work, the mutant A85M of CopC was obtained. The stability of mutant A85M of CopC and the binding properties of metal ions were clarified through various spectroscopic techniques. The binding capacity of A85M to metal ions was measured by fluorescence spectroscopy and UV differential absorbance. The results suggested that Cu(2+) can bind with A85M in 1:1 form, and the constant of A85M was nearly the same as that of CopC. Ag(+) can occupy the Cu(+) binding site located at C-terminal, and the binding constant was (2.64±0.48)×10(6)L/mol. Hg(2+) not only can occupy the Cu(+) binding site located at C-terminal, but also can occupy the Cu(2+) binding site located at N-terminal. The stability of A85M was measured by chemical unfolding experiment. The intermediate was observed in the unfolding pathway of A85M-Cu(2+) induced by urea. In addition, the interaction of SDS with A85M also can result in the formation of the intermediate. The effect of metal ions on the stability of intermediate suggested that the C terminal region of intermediate was unfolded and the N terminal region suffered few effects. Compared with CopC, the stability of A85M was decreased. The main reason was the lower stability of N terminal region. The results of molecular dynamic simulation suggested that when the alanine at 85 site was mutated to methionine, the hydrophobic almost unchanged, but the distance between the phenylalanine at 25 site and tryptophan at 83 site increased because of the spatial effect. And it made the stacking interaction of aromatic rings decreased, which was the main reason for the decreasing stability of N terminal region for A85M. PMID:27309682

  9. Binding-induced Stabilization and Assembly of the Phage P22 Tail Accessory Factor gp4

    SciTech Connect

    Olia,A.; Al-Bassam, J.; Winn-Stapley, D.; Joss, L.; Casjens, S.; Cingolani, G.

    2006-01-01

    To infect and replicate, bacteriophage P22 injects its 43 kbp genome across the cell wall of Salmonella enterica serovar Typhimurium. The attachment of phage P22 to the host cell as well as the injection of the viral DNA into the host is mediated by the virion's tail complex. This 2.8 MDa molecular machine is formed by five proteins, which include the portal protein gp1, the adhesion tailspike protein gp9, and three tail accessory factors: gp4, gp10, gp26. We have isolated the tail accessory factor gp4 and characterized its structure and binding interactions with portal protein. Interestingly, gp4 exists in solution as a monomer, which displays an exceedingly low structural stability (T{sub m} 34 {sup o}C). Unfolded gp4 is prone to aggregation within a narrow range of temperatures both in vitro and in Salmonella extracts. In the virion the thermal unfolding of gp4 is prevented by the interaction with the dodecameric portal protein, which stabilizes the structure of gp4 and suppresses unfolded gp4 from irreversibly aggregating in the Salmonella milieu. The structural stabilization of gp4 is accompanied by the concomitant oligomerization of the protein to form a ring of 12 subunits bound to the lower end of the portal ring. The interaction of gp4 with portal protein is complex and likely involves the distinct binding of two non-equivalent sets of six gp4 proteins. Binding of the first set of six gp4 equivalents to dodecameric portal protein yields a gp(1){sub 12}:gp(4){sub 6} assembly intermediate, which is stably populated at 30 {sup o}C and can be resolved by native gel electrophoresis. The final product of the assembly reaction is a bi-dodecameric gp(1){sub 12}:gp(4){sub 12} complex, which appears hollow by electron microscopy, suggesting that gp4 does not physically plug the DNA entry/exit channel, but acts as a structural adaptor for the other tail accessory factors: gp10 and gp26.

  10. Diabetes may affect intracranial aneurysm stabilization in older patients: Analysis based on intraoperative findings

    PubMed Central

    Song, Jihye; Shin, Yong Sam

    2016-01-01

    Background: Only a small proportion of aneurysms progress to rupture. Previous studies have focused on predicting the rupture risk of intracranial aneurysms. Atherosclerotic aneurysm wall appears resistant to rupture. The purpose of this study was to evaluate clinical and morphological factors affecting atherosclerosis of an aneurysm and identify the parameters that predict aneurysm stabilization. Methods: We conducted a retrospective analysis of 253 consecutive patients with 291 unruptured aneurysms who underwent clipping surgery in a single institution between January 2012 and October 2013. Aneurysms were categorized based on intraoperative video findings and assessed morphologic and demographic data. Aneurysms which had the atherosclerotic wall without any super thin and transparent portion were defined as stabilized group and the others as a not-stabilized group. Results: Of the 207 aneurysms, 176 (85.0%) were assigned to the not-stabilized group and 31 (15.0%) to the stabilized group. The relative proportion of stabilized aneurysms increased significantly as the age increased (P < 0.001). Univariate logistic analysis showed that age ≥65 years (P < 0.001), hypertension (P = 0.012), diabetes (P = 0.007), and height ≥3 mm (P = 0.007) were correlated with stabilized aneurysms. Multivariate logistic analysis showed that age ≥65 years (P = 0.009) and hypertension (P = 0.041) were strongly correlated with stable aneurysms. In older patients (≥65 years of age), multivariate logistic regression revealed that only diabetes was associated with stabilized aneurysms (P = 0.027). Conclusions: In patients ≥65 years of age, diabetes mellitus may highly predict the stabilized aneurysms. These results provide useful information in determining treatment and follow-up strategies, especially in older patients. PMID:27313965

  11. Systematic reconstruction of binding and stability landscapes of the fluorogenic aptamer spinach.

    PubMed

    Ketterer, Simon; Fuchs, David; Weber, Wilfried; Meier, Matthias

    2015-10-30

    Fluorogenic RNAs that are based on the complex formed by 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) derivatives and the RNA aptamer named Spinach were used to engineer a new generation of in vitro and in vivo sensors for bioanalytics. With the resolved crystal structure of the RNA/small molecule complex, the engineering map becomes available, but comprehensive information regarding the thermodynamic profile of the molecule is missing. Here, we reconstructed the full thermodynamic binding and stability landscapes between DFHBI and a truncated sequence of first-generation Spinach. For this purpose, we established a systematic screening procedure for single- and double-point mutations on a microfluidic large-scale integrated chip platform for 87-nt long RNAs. The thermodynamic profile with single base resolution was used to engineer an improved fluorogenic spinach generation via a directed rather than evolutional approach. PMID:26400180

  12. Systematic reconstruction of binding and stability landscapes of the fluorogenic aptamer spinach

    PubMed Central

    Ketterer, Simon; Fuchs, David; Weber, Wilfried; Meier, Matthias

    2015-01-01

    Fluorogenic RNAs that are based on the complex formed by 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) derivatives and the RNA aptamer named Spinach were used to engineer a new generation of in vitro and in vivo sensors for bioanalytics. With the resolved crystal structure of the RNA/small molecule complex, the engineering map becomes available, but comprehensive information regarding the thermodynamic profile of the molecule is missing. Here, we reconstructed the full thermodynamic binding and stability landscapes between DFHBI and a truncated sequence of first-generation Spinach. For this purpose, we established a systematic screening procedure for single- and double-point mutations on a microfluidic large-scale integrated chip platform for 87-nt long RNAs. The thermodynamic profile with single base resolution was used to engineer an improved fluorogenic spinach generation via a directed rather than evolutional approach. PMID:26400180

  13. The dynamin-binding domains of Dap160/intersectin affect bulk membrane retrieval in synapses

    PubMed Central

    Winther, Åsa M. E.; Jiao, Wei; Vorontsova, Olga; Rees, Kathryn A.; Koh, Tong-Wey; Sopova, Elena; Schulze, Karen L.; Bellen, Hugo J.; Shupliakov, Oleg

    2013-01-01

    Summary Dynamin-associated protein 160 kDa (Dap160)/intersectin interacts with several synaptic proteins and affects endocytosis and synapse development. The functional role of the different protein interaction domains is not well understood. Here we show that Drosophila Dap160 lacking the dynamin-binding SH3 domains does not affect the development of the neuromuscular junction but plays a key role in synaptic vesicle recycling. dap160 mutants lacking dynamin-interacting domains no longer accumulate dynamin properly at the periactive zone, and it becomes dispersed in the bouton during stimulation. This is accompanied by a reduction in uptake of the dye FM1-43 and an accumulation of large vesicles and membrane invaginations. However, we do not observe an increase in the number of clathrin-coated intermediates. We also note a depression in evoked excitatory junction potentials (EJPs) during high-rate stimulation, accompanied by aberrantly large miniature EJPs. The data reveal the important role of Dap160 in the targeting of dynamin to the periactive zone, where it is required to suppress bulk synaptic vesicle membrane retrieval during high-frequency activity. PMID:23321638

  14. Effects of ligand binding on the mechanical stability of protein GB1 studied by steered molecular dynamics simulation.

    PubMed

    Su, Ji-Guo; Zhao, Shu-Xin; Wang, Xiao-Feng; Li, Chun-Hua; Li, Jing-Yuan

    2016-08-01

    Regulation of the mechanical properties of proteins plays an important role in many biological processes, and sheds light on the design of biomaterials comprised of protein. At present, strategies to regulate protein mechanical stability focus mainly on direct modulation of the force-bearing region of the protein. Interestingly, the mechanical stability of GB1 can be significantly enhanced by the binding of Fc fragments of human IgG antibody, where the binding site is distant from the force-bearing region of the protein. The mechanism of this long-range allosteric control of protein mechanics is still elusive. In this work, the impact of ligand binding on the mechanical stability of GB1 was investigated using steered molecular dynamics simulation, and a mechanism underlying the enhanced protein mechanical stability is proposed. We found that the external force causes deformation of both force-bearing region and ligand binding site. In other words, there is a long-range coupling between these two regions. The binding of ligand restricts the distortion of the binding site and reduces the deformation of the force-bearing region through a long-range allosteric communication, which thus improves the overall mechanical stability of the protein. The simulation results are very consistent with previous experimental observations. Our studies thus provide atomic-level insights into the mechanical unfolding process of GB1, and explain the impact of ligand binding on the mechanical properties of the protein through long-range allosteric regulation, which should facilitate effective modulation of protein mechanical properties. PMID:27444879

  15. Cyclophilin A stabilizes the HIV-1 capsid through a novel non-canonical binding site

    PubMed Central

    Liu, Chuang; Perilla, Juan R.; Ning, Jiying; Lu, Manman; Hou, Guangjin; Ramalho, Ruben; Himes, Benjamin A.; Zhao, Gongpu; Bedwell, Gregory J.; Byeon, In-Ja; Ahn, Jinwoo; Gronenborn, Angela M.; Prevelige, Peter E.; Rousso, Itay; Aiken, Christopher; Polenova, Tatyana; Schulten, Klaus; Zhang, Peijun

    2016-01-01

    The host cell factor cyclophilin A (CypA) interacts directly with the HIV-1 capsid and regulates viral infectivity. Although the crystal structure of CypA in complex with the N-terminal domain of the HIV-1 capsid protein (CA) has been known for nearly two decades, how CypA interacts with the viral capsid and modulates HIV-1 infectivity remains unclear. We determined the cryoEM structure of CypA in complex with the assembled HIV-1 capsid at 8-Å resolution. The structure exhibits a distinct CypA-binding pattern in which CypA selectively bridges the two CA hexamers along the direction of highest curvature. EM-guided all-atom molecular dynamics simulations and solid-state NMR further reveal that the CypA-binding pattern is achieved by single-CypA molecules simultaneously interacting with two CA subunits, in different hexamers, through a previously uncharacterized non-canonical interface. These results provide new insights into how CypA stabilizes the HIV-1 capsid and is recruited to facilitate HIV-1 infection. PMID:26940118

  16. Stabilization of integrin-linked kinase by binding to Hsp90

    SciTech Connect

    Aoyagi, Yumiko; Fujita, Naoya; Tsuruo, Takashi; E-mail: ttsuruo@iam.u-tokyo.ac.jp

    2005-06-17

    Integrin-linked kinase (ILK) is a serine/threonine kinase that interacts with the cytoplasmic domain of {beta}-integrins and growth factor receptors in response to extracellular signals. It is a key molecule in cell adhesion, proliferation, and cell survival. We found that treating cells with specific inhibitors of the heat shock protein 90 (Hsp90) caused rapid cell detachment. Screening the responsible proteins revealed a decreased amount of ILK in Hsp90 inhibitor-treated cells. ILK was identified as a new Hsp90 client protein because it formed a complex with Hsp90 and Cdc37, and binding was suppressed by Hsp90 inhibitors. Experiments with a series of ILK-deletion mutants revealed that the amino acid residues 377-406 were required for Hsp90 binding. Dissociation of ILK from Hsp90 shortened its half-life by promoting proteasome-dependent degradation. These results indicate that Hsp90 plays an important role in the stability of ILK in cells.

  17. Cyclophilin A stabilizes the HIV-1 capsid through a novel non-canonical binding site.

    PubMed

    Liu, Chuang; Perilla, Juan R; Ning, Jiying; Lu, Manman; Hou, Guangjin; Ramalho, Ruben; Himes, Benjamin A; Zhao, Gongpu; Bedwell, Gregory J; Byeon, In-Ja; Ahn, Jinwoo; Gronenborn, Angela M; Prevelige, Peter E; Rousso, Itay; Aiken, Christopher; Polenova, Tatyana; Schulten, Klaus; Zhang, Peijun

    2016-01-01

    The host cell factor cyclophilin A (CypA) interacts directly with the HIV-1 capsid and regulates viral infectivity. Although the crystal structure of CypA in complex with the N-terminal domain of the HIV-1 capsid protein (CA) has been known for nearly two decades, how CypA interacts with the viral capsid and modulates HIV-1 infectivity remains unclear. We determined the cryoEM structure of CypA in complex with the assembled HIV-1 capsid at 8-Å resolution. The structure exhibits a distinct CypA-binding pattern in which CypA selectively bridges the two CA hexamers along the direction of highest curvature. EM-guided all-atom molecular dynamics simulations and solid-state NMR further reveal that the CypA-binding pattern is achieved by single-CypA molecules simultaneously interacting with two CA subunits, in different hexamers, through a previously uncharacterized non-canonical interface. These results provide new insights into how CypA stabilizes the HIV-1 capsid and is recruited to facilitate HIV-1 infection. PMID:26940118

  18. Structural Stability and Binding Strength of a Designed Peptide-Carbon Nanotube Hybrid

    PubMed Central

    Roxbury, Daniel; Zhang, Shao-Qing; Mittal, Jeetain; DeGrado, William F.; Jagota, Anand

    2014-01-01

    Biological polymers hybridized with single-walled carbon nanotubes (SWCNTs) have elicited much interest recently for applications in SWCNT-based sorting as well as biomedical imaging, sensing, and drug delivery. Recently, de novo designed peptides forming a coiled-coil structure have been engineered to selectively disperse SWCNT of a certain diameter. Here we report on a study of the binding strength and structural stability of the hybrid between such a “HexCoil-Ala” peptide and the (6,5)-SWCNT. Using the competitive binding of a surfactant, we find that affinity strength of the peptide ranks in comparison to that of two single-stranded DNA sequences as (GT)30-DNA > HexCoil-Ala > (TAT)4T-DNA. Further, using replica exchange molecular dynamics (REMD), we show that the hexamer peptide complex has both similarities with and differences from the original design. While one of two distinct helix-helix interfaces of the original model was largely retained, a second interface showed much greater variability. These conformational differences allowed an aromatic tyrosine residue designed to lie along the solvent-exposed surface of the protein instead to penetrate between the two helices and directly contact the SWCNT. These insights will inform future designs of SWCNT-interacting peptides. PMID:24466357

  19. Cyclophilin A stabilizes the HIV-1 capsid through a novel non-canonical binding site

    NASA Astrophysics Data System (ADS)

    Liu, Chuang; Perilla, Juan R.; Ning, Jiying; Lu, Manman; Hou, Guangjin; Ramalho, Ruben; Himes, Benjamin A.; Zhao, Gongpu; Bedwell, Gregory J.; Byeon, In-Ja; Ahn, Jinwoo; Gronenborn, Angela M.; Prevelige, Peter E.; Rousso, Itay; Aiken, Christopher; Polenova, Tatyana; Schulten, Klaus; Zhang, Peijun

    2016-03-01

    The host cell factor cyclophilin A (CypA) interacts directly with the HIV-1 capsid and regulates viral infectivity. Although the crystal structure of CypA in complex with the N-terminal domain of the HIV-1 capsid protein (CA) has been known for nearly two decades, how CypA interacts with the viral capsid and modulates HIV-1 infectivity remains unclear. We determined the cryoEM structure of CypA in complex with the assembled HIV-1 capsid at 8-Å resolution. The structure exhibits a distinct CypA-binding pattern in which CypA selectively bridges the two CA hexamers along the direction of highest curvature. EM-guided all-atom molecular dynamics simulations and solid-state NMR further reveal that the CypA-binding pattern is achieved by single-CypA molecules simultaneously interacting with two CA subunits, in different hexamers, through a previously uncharacterized non-canonical interface. These results provide new insights into how CypA stabilizes the HIV-1 capsid and is recruited to facilitate HIV-1 infection.

  20. Introducing folding stability into the score function for computational design of RNA-binding peptides boosts the probability of success.

    PubMed

    Xiao, Xingqing; Agris, Paul F; Hall, Carol K

    2016-05-01

    A computational strategy that integrates our peptide search algorithm with atomistic molecular dynamics simulation was used to design rational peptide drugs that recognize and bind to the anticodon stem and loop domain (ASL(Lys3) ) of human tRNAUUULys3 for the purpose of interrupting HIV replication. The score function of the search algorithm was improved by adding a peptide stability term weighted by an adjustable factor λ to the peptide binding free energy. The five best peptide sequences associated with five different values of λ were determined using the search algorithm and then input in atomistic simulations to examine the stability of the peptides' folded conformations and their ability to bind to ASL(Lys3) . Simulation results demonstrated that setting an intermediate value of λ achieves a good balance between optimizing the peptide's binding ability and stabilizing its folded conformation during the sequence evolution process, and hence leads to optimal binding to the target ASL(Lys3) . Thus, addition of a peptide stability term significantly improves the success rate for our peptide design search. Proteins 2016; 84:700-711. © 2016 Wiley Periodicals, Inc. PMID:26914059

  1. A STRIPAK component Strip regulates neuronal morphogenesis by affecting microtubule stability

    PubMed Central

    Sakuma, Chisako; Okumura, Misako; Umehara, Tomoki; Miura, Masayuki; Chihara, Takahiro

    2015-01-01

    During neural development, regulation of microtubule stability is essential for proper morphogenesis of neurons. Recently, the striatin-interacting phosphatase and kinase (STRIPAK) complex was revealed to be involved in diverse cellular processes. However, there is little evidence that STRIPAK components regulate microtubule dynamics, especially in vivo. Here, we show that one of the core STRIPAK components, Strip, is required for microtubule organization during neuronal morphogenesis. Knockdown of Strip causes a decrease in the level of acetylated α-tubulin in Drosophila S2 cells, suggesting that Strip influences the stability of microtubules. We also found that Strip physically and genetically interacts with tubulin folding cofactor D (TBCD), an essential regulator of α- and β-tubulin heterodimers. Furthermore, we demonstrate the genetic interaction between strip and Down syndrome cell adhesion molecule (Dscam), a cell surface molecule that is known to work with TBCD. Thus, we propose that Strip regulates neuronal morphogenesis by affecting microtubule stability. PMID:26644129

  2. Prep1 and Meis1 competition for Pbx1 binding regulates protein stability and tumorigenesis.

    PubMed

    Dardaei, Leila; Longobardi, Elena; Blasi, Francesco

    2014-03-11

    Pbx-regulating protein-1 (Prep1) is a tumor suppressor, whereas myeloid ecotropic viral integration site-1 (Meis1) is an oncogene. We show that, to perform these activities in mouse embryonic fibroblasts, both proteins competitively heterodimerize with pre-B-cell leukemia homeobox-1 (Pbx1). Meis1 alone transforms Prep1-deficient fibroblasts, whereas Prep1 overexpression inhibits Meis1 tumorigenicity. Pbx1 can, therefore, alternatively act as an oncogene or tumor suppressor. Prep1 posttranslationally controls the level of Meis1, decreasing its stability by sequestering Pbx1. The different levels of Meis1 and the presence of Prep1 are followed at the transcriptional level by the induction of specific transcriptional signatures. The decrease of Meis1 prevents Meis1 interaction with Ddx3x and Ddx5, which are essential for Meis1 tumorigenesis, and modifies the growth-promoting DNA binding landscape of Meis1 to the growth-controlling landscape of Prep1. Hence, the key feature of Prep1 tumor-inhibiting activity is the control of Meis1 stability. PMID:24578510

  3. Centrally truncated and stabilized porcine neuropeptide Y analogs: design, synthesis, and mouse brain receptor binding.

    PubMed Central

    Krstenansky, J L; Owen, T J; Buck, S H; Hagaman, K A; McLean, L R

    1989-01-01

    Porcine neuropeptide Y (pNPY) has been proposed to form an intramolecularly stabilized structure characterized by N- and C-terminal helical regions arranged antiparallel due to a central turn region. Analogs based on this structural model that have the central turn region and various amounts of the helical regions removed, yet retain the N and C termini in a similar spatial orientation were designed. The gap formed by removal of the central residues (residues 8-17 or 7-20) was spanned with a single 8-aminooctanoic acid residue (Aoc) and the structure was further stabilized by the introduction of a disulfide bridge. [D-Cys7,Aoc8-17,Cys20]pNPY and [Cys5,Aoc7-20,D-Cys24]pNPY were synthesized and found to have receptor binding affinities of 2.3 nM and 150 nM, respectively, in mouse brain membranes (pNPY affinity is 3.6 nM in this assay). It is proposed that the central region (residues 7-17) of pNPY serves a structural role in the peptide and is not involved in direct receptor interaction. PMID:2543973

  4. Binding energy and mechanical stability of single- and multi-walled carbon nanotube serpentines

    SciTech Connect

    Zhao, Junhua E-mail: timon.rabczuk@uni-weimar.de; Lu, Lixin; Rabczuk, Timon E-mail: timon.rabczuk@uni-weimar.de

    2014-05-28

    Recently, Geblinger et al. [Nat. Nanotechnol. 3, 195 (2008)] and Machado et al. [Phys. Rev. Lett. 110, 105502 (2013)] reported the experimental and molecular dynamics realization of S-like shaped single-walled carbon nanotubes (CNTs), the so-called CNT serpentines. We reported here results from continuum modeling of the binding energy γ between different single- and multi-walled CNT serpentines and substrates as well as the mechanical stability of the CNT serpentine formation. The critical length for the mechanical stability and adhesion of different CNT serpentines are determined in dependence of E{sub i}I{sub i}, d, and γ, where E{sub i}I{sub i} and d are the CNT bending stiffness and distance of the CNT translation period. Our continuum model is validated by comparing its solution to full-atom molecular dynamics calculations. The derived analytical solutions are of great importance for understanding the interaction mechanism between different single- and multi-walled CNT serpentines and substrates.

  5. Centromere binding and a conserved role in chromosome stability for SUMO-dependent ubiquitin ligases.

    PubMed

    van de Pasch, Loes A L; Miles, Antony J; Nijenhuis, Wilco; Brabers, Nathalie A C H; van Leenen, Dik; Lijnzaad, Philip; Brown, Markus K; Ouellet, Jimmy; Barral, Yves; Kops, Geert J P L; Holstege, Frank C P

    2013-01-01

    The Saccharomyces cerevisiae Slx5/8 complex is the founding member of a recently defined class of SUMO-targeted ubiquitin ligases (STUbLs). Slx5/8 has been implicated in genome stability and transcription, but the precise contribution is unclear. To characterise Slx5/8 function, we determined genome-wide changes in gene expression upon loss of either subunit. The majority of mRNA changes are part of a general stress response, also exhibited by mutants of other genome integrity pathways and therefore indicative of an indirect effect on transcription. Genome-wide binding analysis reveals a uniquely centromeric location for Slx5. Detailed phenotype analyses of slx5Δ and slx8Δ mutants show severe mitotic defects that include aneuploidy, spindle mispositioning, fish hooks and aberrant spindle kinetics. This is associated with accumulation of the PP2A regulatory subunit Rts1 at centromeres prior to entry into anaphase. Knockdown of the human STUbL orthologue RNF4 also results in chromosome segregation errors due to chromosome bridges. The study shows that STUbLs have a conserved role in maintenance of chromosome stability and links SUMO-dependent ubiquitination to a centromere-specific function during mitosis. PMID:23785440

  6. Host alkaloids differentially affect developmental stability and wing vein canalization in cactophilic Drosophila buzzatii.

    PubMed

    Padró, J; Carreira, V; Corio, C; Hasson, E; Soto, I M

    2014-12-01

    Host shifts cause drastic consequences on fitness in cactophilic species of Drosophila. It has been argued that changes in the nutritional values accompanying host shifts may elicit these fitness responses, but they may also reflect the presence of potentially toxic secondary compounds that affect resource quality. Recent studies reported that alkaloids extracted from the columnar cactus Trichocereus terscheckii are toxic for the developing larvae of Drosophila buzzatii. In this study, we tested the effect of artificial diets including increasing doses of host alkaloids on developmental stability and wing morphology in D. buzzatii. We found that alkaloids disrupt normal wing venation patterning and affect viability, wing size and fluctuating asymmetry, suggesting the involvement of stress-response mechanisms. Theoretical implications are discussed in the context of developmental stability, stress, fitness and their relationship with robustness, canalization and phenotypic plasticity. PMID:25366093

  7. Stability junction at a common mutation site in the collagenous domain of the mannose binding lectin.

    PubMed

    Mohs, Angela; Li, Yingjie; Doss-Pepe, Ellen; Baum, Jean; Brodsky, Barbara

    2005-02-15

    Missense mutations in the collagen triple-helix that replace one of the required Gly residues in the (Gly-Xaa-Yaa)(n)() repeating sequence have been implicated in various disorders. Although most hereditary collagen disorders are rare, a common occurrence of a Gly replacement mutation is found in the collagenous domain of mannose binding lectin (MBL). A Gly --> Asp mutation at position 54 in MBL is found at a frequency as high as 30% in certain populations and leads to increased susceptibility to infections. The structural and energetic consequences of this mutation are investigated by comparing a triple-helical peptide containing the N-terminal Gly-X-Y units of MBL with the homologous peptide containing the Gly to Asp replacement. The mutation leads to a loss of triple-helix content but only a small decrease in the stability of the triple-helix (DeltaT(m) approximately 2 degrees C) and no change in the calorimetric enthalpy. NMR studies on specifically labeled residues indicate the portion of the peptide C-terminal to residue 54 is in a highly ordered triple-helix in both peptides, while residues N-terminal to the mutation site have a weak triple-helical signal in the parent peptide and are completely disordered in the mutant peptide. These results suggest that the N-terminal triplet residues are contributing little to the stability of this peptide, a hypothesis confirmed by the stability and enthalpy of shorter peptides containing only the region C-terminal to the mutation site. The Gly to Asp replacement at position 54 in MBL occurs at the boundary of a highly stable triple-helix region and a very unstable sequence. The junctional position of this mutation minimizes its destabilizing effect, in contrast with the significant destabilization seen for Gly replacements in peptides modeling collagen diseases. PMID:15697204

  8. Structure and stability of recombinant bovine odorant-binding protein: I. Design and analysis of monomeric mutants

    PubMed Central

    Stepanenko, Olga V.; Roginskii, Denis O.; Stepanenko, Olesya V.; Kuznetsova, Irina M.

    2016-01-01

    Bovine odorant-binding protein (bOBP) differs from other lipocalins by lacking the conserved disulfide bond and for being able to form the domain-swapped dimers. To identify structural features responsible for the formation of the bOBP unique dimeric structure and to understand the role of the domain swapping on maintaining the native structure of the protein, structural properties of the recombinant wild type bOBP and its mutant that cannot dimerize via the domain swapping were analyzed. We also looked at the effect of the disulfide bond by designing a monomeric bOBPs with restored disulfide bond which is conserved in other lipocalins. Finally, to understand which features in the microenvironment of the bOBP tryptophan residues play a role in the defining peculiarities of the intrinsic fluorescence of this protein we designed and investigated single-tryptophan mutants of the monomeric bOBP. Our analysis revealed that the insertion of the glycine after the residue 121 of the bOBP prevents domain swapping and generates a stable monomeric protein bOBP-Gly121+. We also show that the restored disulfide bond in the GCC-bOBP mutant leads to the noticeable stabilization of the monomeric structure. Structural and functional analysis revealed that none of the amino acid substitutions introduced to the bOBP affected functional activity of the protein and that the ligand binding leads to the formation of a more compact and stable state of the recombinant bOBP and its mutant monomeric forms. Finally, analysis of the single-tryptophan mutants of the monomeric bOBP gave us a unique possibility to find peculiarities of the microenvironment of tryptophan residues which were not previously described. PMID:27114880

  9. Nanog RNA-binding proteins YBX1 and ILF3 affect pluripotency of embryonic stem cells.

    PubMed

    Guo, Chuanliang; Xue, Yan; Yang, Guanheng; Yin, Shang; Shi, Wansheng; Cheng, Yan; Yan, Xiaoshuang; Fan, Shuyue; Zhang, Huijun; Zeng, Fanyi

    2016-08-01

    Nanog is a well-known transcription factor that plays a fundamental role in stem cell self-renewal and the maintenance of their pluripotent cell identity. There remains a large data gap with respect to the spectrum of the key pluripotency transcription factors' interaction partners. Limited information is available concerning Nanog-associated RNA-binding proteins (RBPs), and the intrinsic protein-RNA interactions characteristic of the regulatory activities of Nanog. Herein, we used an improved affinity protocol to purify Nanog-interacting RBPs from mouse embryonic stem cells (ESCs), and 49 RBPs of Nanog were identified. Among them, the interaction of YBX1 and ILF3 with Nanog mRNA was further confirmed by in vitro assays, such as Western blot, RNA immunoprecipitation (RIP), and ex vivo methods, such as immunofluorescence staining and fluorescent in situ hybridization (FISH), MS2 in vivo biotin-tagged RNA affinity purification (MS2-BioTRAP). Interestingly, RNAi studies revealed that YBX1 and ILF3 positively affected the expression of Nanog and other pluripotency-related genes. Particularly, downregulation of YBX1 or ILF3 resulted in high expression of mesoderm markers. Thus, a reduction in the expression of YBX1 and ILF3 controls the expression of pluripotency-related genes in ESCs, suggesting their roles in further regulation of the pluripotent state of ESCs. PMID:26289635

  10. Honey bee odorant-binding protein 14: effects on thermal stability upon odorant binding revealed by FT-IR spectroscopy and CD measurements.

    PubMed

    Schwaighofer, Andreas; Kotlowski, Caroline; Araman, Can; Chu, Nam; Mastrogiacomo, Rosa; Becker, Christian; Pelosi, Paolo; Knoll, Wolfgang; Larisika, Melanie; Nowak, Christoph

    2014-03-01

    In the present work, we study the effect of odorant binding on the thermal stability of honey bee (Apis mellifera L.) odorant-binding protein 14. Thermal denaturation of the protein in the absence and presence of different odorant molecules was monitored by Fourier transform infrared spectroscopy (FT-IR) and circular dichroism (CD). FT-IR spectra show characteristic bands for intermolecular aggregation through the formation of intermolecular β-sheets during the heating process. Transition temperatures in the FT-IR spectra were evaluated using moving-window 2D correlation maps and confirmed by CD measurements. The obtained results reveal an increase of the denaturation temperature of the protein when bound to an odorant molecule. We could also discriminate between high- and low-affinity odorants by determining transition temperatures, as demonstrated independently by the two applied methodologies. The increased thermal stability in the presence of ligands is attributed to a stabilizing effect of non-covalent interactions between odorant-binding protein 14 and the odorant molecule. PMID:24362824

  11. The Chromatin-binding Protein HMGN1 Regulates the Expression of Methyl CpG-binding Protein 2 (MECP2) and Affects the Behavior of Mice*

    PubMed Central

    Abuhatzira, Liron; Shamir, Alon; Schones, Dustin E.; Schäffer, Alejandro A.; Bustin, Michael

    2011-01-01

    High mobility group N1 protein (HMGN1), a nucleosomal-binding protein that affects the structure and function of chromatin, is encoded by a gene located on chromosome 21 and is overexpressed in Down syndrome, one of the most prevalent genomic disorders. Misexpression of HMGN1 affects the cellular transcription profile; however, the biological function of this protein is still not fully understood. We report that HMGN1 modulates the expression of methyl CpG-binding protein 2 (MeCP2), a DNA-binding protein known to affect neurological functions including autism spectrum disorders, and whose alterations in HMGN1 levels affect the behavior of mice. Quantitative PCR and Western analyses of cell lines and brain tissues from mice that either overexpress or lack HMGN1 indicate that HMGN1 is a negative regulator of MeCP2 expression. Alterations in HMGN1 levels lead to changes in chromatin structure and histone modifications in the MeCP2 promoter. Behavior analyses by open field test, elevated plus maze, Reciprocal Social Interaction, and automated sociability test link changes in HMGN1 levels to abnormalities in activity and anxiety and to social deficits in mice. Targeted analysis of the Autism Genetic Resource Exchange genotype collection reveals a non-random distribution of genotypes within 500 kbp of HMGN1 in a region affecting its expression in families predisposed to autism spectrum disorders. Our results reveal that HMGN1 affects the behavior of mice and suggest that epigenetic changes resulting from altered HMGN1 levels could play a role in the etiology of neurodevelopmental disorders. PMID:22009741

  12. A single-nucleotide variation in a p53-binding site affects nutrient-sensitive human SIRT1 expression

    PubMed Central

    Naqvi, Asma; Hoffman, Timothy A.; DeRicco, Jeremy; Kumar, Ajay; Kim, Cuk-Seong; Jung, Saet-Byel; Yamamori, Tohru; Kim, Young-Rae; Mehdi, Fardeen; Kumar, Santosh; Rankinen, Tuomo; Ravussin, Eric; Irani, Kaikobad

    2010-01-01

    The SIRTUIN1 (SIRT1) deacetylase responds to changes in nutrient availability and regulates mammalian physiology and metabolism. Human and mouse SIRT1 are transcriptionally repressed by p53 via p53 response elements in their proximal promoters. Here, we identify a novel p53-binding sequence in the distal human SIRT1 promoter that is required for nutrient-sensitive SIRT1 transcription. In addition, we show that a common single-nucleotide (C/T) variation in this sequence affects nutrient deprivation-induced SIRT1 transcription, and calorie restriction-induced SIRT1 expression. The p53-binding sequence lies in a region of the SIRT1 promoter that also binds the transcriptional repressor Hypermethylated-In-Cancer-1 (HIC1). Nutrient deprivation increases occupancy by p53, while decreasing occupancy by HIC1, of this region of the promoter. HIC1 and p53 compete with each other for promoter occupancy. In comparison with the T variation, the C variation disrupts the mirror image symmetry of the p53-binding sequence, resulting in decreased binding to p53, decreased nutrient sensitivity of the promoter and impaired calorie restriction-stimulated tissue expression of SIRT1 and SIRT1 target genes AMPKα2 and PGC-1β. Thus, a common SNP in a novel p53-binding sequence in the human SIRT1 promoter affects nutrient-sensitive SIRT1 expression, and could have a significant impact on calorie restriction-induced, SIRT1-mediated, changes in human metabolism and physiology. PMID:20693263

  13. The role of the second binding loop of the cysteine protease inhibitor, cystatin A (stefin A), in stabilizing complexes with target proteases is exerted predominantly by Leu73.

    PubMed

    Pavlova, Alona; Björk, Ingemar

    2002-11-01

    The aim of this work was to elucidate the roles of individual residues within the flexible second binding loop of human cystatin A in the inhibition of cysteine proteases. Four recombinant variants of the inhibitor, each with a single mutation, L73G, P74G, Q76G or N77G, in the most exposed part of this loop were generated by PCR-based site-directed mutagenesis. The binding of these variants to papain, cathepsin L, and cathepsin B was characterized by equilibrium and kinetic methods. Mutation of Leu73 decreased the affinity for papain, cathepsin L and cathepsin B by approximately 300-fold, >10-fold and approximately 4000-fold, respectively. Mutation of Pro74 decreased the affinity for cathepsin B by approximately 10-fold but minimally affected the affinity for the other two enzymes. Mutation of Gln76 and Asn77 did not alter the affinity of cystatin A for any of the proteases studied. The decreased affinities were caused exclusively by increased dissociation rate constants. These results show that the second binding loop of cystatin A plays a major role in stabilizing the complexes with proteases by retarding their dissociation. In contrast with cystatin B, only one amino-acid residue of the loop, Leu73, is of principal importance for this effect, Pro74 assisting to a minor extent only in the case of cathepsin B binding. The contribution of the second binding loop of cystatin A to protease binding varies with the protease, being largest, approximately 45% of the total binding energy, for inhibition of cathepsin B. PMID:12423365

  14. Runx1 Phosphorylation by Src Increases Trans-activation via Augmented Stability, Reduced Histone Deacetylase (HDAC) Binding, and Increased DNA Affinity, and Activated Runx1 Favors Granulopoiesis.

    PubMed

    Leong, Wan Yee; Guo, Hong; Ma, Ou; Huang, Hui; Cantor, Alan B; Friedman, Alan D

    2016-01-01

    Src phosphorylates Runx1 on one central and four C-terminal tyrosines. We find that activated Src synergizes with Runx1 to activate a Runx1 luciferase reporter. Mutation of the four Runx1 C-terminal tyrosines to aspartate or glutamate to mimic phosphorylation increases trans-activation of the reporter in 293T cells and allows induction of Cebpa or Pu.1 mRNAs in 32Dcl3 myeloid cells, whereas mutation of these residues to phenylalanine to prevent phosphorylation obviates these effects. Three mechanisms contribute to increased Runx1 activity upon tyrosine modification as follows: increased stability, reduced histone deacetylase (HDAC) interaction, and increased DNA binding. Mutation of the five modified Runx1 tyrosines to aspartate markedly reduced co-immunoprecipitation with HDAC1 and HDAC3, markedly increased stability in cycloheximide or in the presence of co-expressed Cdh1, an E3 ubiquitin ligase coactivator, with reduced ubiquitination, and allowed DNA-binding in gel shift assay similar to wild-type Runx1. In contrast, mutation of these residues to phenylalanine modestly increased HDAC interaction, modestly reduced stability, and markedly reduced DNA binding in gel shift assays and as assessed by chromatin immunoprecipitation with the -14-kb Pu.1 or +37-kb Cebpa enhancers after stable expression in 32Dcl3 cells. Affinity for CBFβ, the Runx1 DNA-binding partner, was not affected by these tyrosine modifications, and in vitro translated CBFβ markedly increased DNA affinity of both the translated phenylalanine and aspartate Runx1 variants. Finally, further supporting a positive role for Runx1 tyrosine phosphorylation during granulopoiesis, mutation of the five Src-modified residues to aspartate but not phenylalanine allows Runx1 to increase Cebpa and granulocyte colony formation by Runx1-deleted murine marrow. PMID:26598521

  15. Stability of mRNA/DNA and DNA/DNA Duplexes Affects mRNA Transcription

    PubMed Central

    Kraeva, Rayna I.; Krastev, Dragomir B.; Roguev, Assen; Ivanova, Anna; Nedelcheva-Veleva, Marina N.; Stoynov, Stoyno S.

    2007-01-01

    Nucleic acids, due to their structural and chemical properties, can form double-stranded secondary structures that assist the transfer of genetic information and can modulate gene expression. However, the nucleotide sequence alone is insufficient in explaining phenomena like intron-exon recognition during RNA processing. This raises the question whether nucleic acids are endowed with other attributes that can contribute to their biological functions. In this work, we present a calculation of thermodynamic stability of DNA/DNA and mRNA/DNA duplexes across the genomes of four species in the genus Saccharomyces by nearest-neighbor method. The results show that coding regions are more thermodynamically stable than introns, 3′-untranslated regions and intergenic sequences. Furthermore, open reading frames have more stable sense mRNA/DNA duplexes than the potential antisense duplexes, a property that can aid gene discovery. The lower stability of the DNA/DNA and mRNA/DNA duplexes of 3′-untranslated regions and the higher stability of genes correlates with increased mRNA level. These results suggest that the thermodynamic stability of DNA/DNA and mRNA/DNA duplexes affects mRNA transcription. PMID:17356699

  16. Increases thermal stability and cellulose-binding capacity of Cryptococcus sp. S-2 lipase by fusion of cellulose binding domain derived from Trichoderma reesei

    SciTech Connect

    Thongekkaew, Jantaporn; Ikeda, Hiroko; Iefuji, Haruyuki

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer The CSLP and fusion enzyme were successfully expressed in the Pichia pastoris. Black-Right-Pointing-Pointer The fusion enzyme was stable at 80 Degree-Sign C for 120-min. Black-Right-Pointing-Pointer The fusion enzyme was responsible for cellulose-binding capacity. Black-Right-Pointing-Pointer The fusion enzyme has an attractive applicant for enzyme immobilization. -- Abstract: To improve the thermal stability and cellulose-binding capacity of Cryptococcus sp. S-2 lipase (CSLP), the cellulose-binding domain originates from Trichoderma reesei cellobiohydrolase I was engineered into C-terminal region of the CSLP (CSLP-CBD). The CSLP and CSLP-CBD were successfully expressed in the Pichia pastoris using the strong methanol inducible alcohol oxidase 1 (AOX1) promoter and the secretion signal sequence from Saccharomyces cerevisiae ({alpha} factor). The recombinant CSLP and CSLP-CBD were secreted into culture medium and estimated by SDS-PAGE to be 22 and 27 kDa, respectively. The fusion enzyme was stable at 80 Degree-Sign C and retained more than 80% of its activity after 120-min incubation at this temperature. Our results also found that the fusion of fungal exoglucanase cellulose-binding domain to CSLP is responsible for cellulose-binding capacity. This attribute should make it an attractive applicant for enzyme immobilization.

  17. Stabilization of the glucan-binding lectin of Streptococcus sobrinus by specific ligand.

    PubMed

    Denson, A M; Doyle, R J

    1998-01-01

    Cell suspensions of Streptococcus sobrinus can be aggregated by high molecular-weight alpha-1,6 glucans. The aggregation depends on the fidelity of a cell wall-bound, glucan-binding lectin (GBL). It is thought that the lectin may play a part in the sucrose-dependent accretion of streptococci in dental plaques. Results showed that the anionic detergent, sodium dodecyl sulphate (SDS) was a potent inhibitor of the lectin. When cells were incubated in SDS and washed to remove the detergent, lectin activity was diminished. Following incubation of the cells with SDS in the presence of glucan T-10, a low molecular-weight alpha-1,6 glucan, the loss of activity was less pronounced, suggesting that the glucan afforded partial protection against denaturation. Urea and guanidine hydrochloride were good inhibitors of the lectin, but, unlike SDS, were not able to inhibit it irreversibly, except at very high concentrations. Cationic detergents, such as cetylpyridinium bromide (and chloride), also irreversibly denatured the streptococcal lectin, but were not as effective as SDS in abolishing its activity. The results suggest that alpha-1,6 glucan stabilizes the GBL of S. sobrinus, rendering it more resistant to the effect of chaotropes. This may be one reason why dental plaques tend to resist detergents in dentrifices. PMID:9569988

  18. TRAIP is a PCNA-binding ubiquitin ligase that protects genome stability after replication stress

    PubMed Central

    Hoffmann, Saskia; Smedegaard, Stine; Nakamura, Kyosuke; Mortuza, Gulnahar B.; Räschle, Markus; Ibañez de Opakua, Alain; Oka, Yasuyoshi; Feng, Yunpeng; Blanco, Francisco J.; Mann, Matthias; Montoya, Guillermo; Groth, Anja; Bekker-Jensen, Simon

    2016-01-01

    Cellular genomes are highly vulnerable to perturbations to chromosomal DNA replication. Proliferating cell nuclear antigen (PCNA), the processivity factor for DNA replication, plays a central role as a platform for recruitment of genome surveillance and DNA repair factors to replication forks, allowing cells to mitigate the threats to genome stability posed by replication stress. We identify the E3 ubiquitin ligase TRAIP as a new factor at active and stressed replication forks that directly interacts with PCNA via a conserved PCNA-interacting peptide (PIP) box motif. We show that TRAIP promotes ATR-dependent checkpoint signaling in human cells by facilitating the generation of RPA-bound single-stranded DNA regions upon replication stress in a manner that critically requires its E3 ligase activity and is potentiated by the PIP box. Consequently, loss of TRAIP function leads to enhanced chromosomal instability and decreased cell survival after replication stress. These findings establish TRAIP as a PCNA-binding ubiquitin ligase with an important role in protecting genome integrity after obstacles to DNA replication. PMID:26711499

  19. Bile acid salt binding with colesevelam HCl is not affected by suspension in common beverages.

    PubMed

    Hanus, Martin; Zhorov, Eugene

    2006-12-01

    It has been previously reported that anions in common beverages may bind to bile acid sequestrants (BAS), reducing their capacity for binding bile acid salts. This study examined the ability of the novel BAS colesevelam hydrochloride (HCl), in vitro, to bind bile acid sodium salts following suspension in common beverages. Equilibrium binding was evaluated under conditions of constant time and varying concentrations of bile acid salts in simulated intestinal fluid (SIF). A stock solution of sodium salts of glycochenodeoxycholic acid (GCDC), taurodeoxycholic acid (TDC), and glycocholic acid (GC), was added to each prepared sample of colesevelam HCl. Bile acid salt binding was calculated by high-performance liquid chromatography (HPLC) analysis. Kinetics experiments were conducted using constant initial bile acid salt concentrations and varying binding times. The affinity, capacity, and kinetics of colesevelam HCl binding for GCDC, TDC, and GC were not significantly altered after suspension in water, carbonated water, Coca-Cola, Sprite, grape juice, orange juice, tomato juice, or Gatorade. The amount of bile acid sodium salt bound as a function of time was unchanged by pretreatment with any beverage tested. The in vitro binding characteristics of colesevelam HCl are unchanged by suspension in common beverages. PMID:16937334

  20. Binding of manganese(II) to a tertiary stabilized hammerhead ribozyme as studied by electron paramagnetic resonance spectroscopy

    PubMed Central

    KISSELEVA, NATALIA; KHVOROVA, ANASTASIA; WESTHOF, ERIC; SCHIEMANN, OLAV

    2005-01-01

    Electron paramagnetic resonance (EPR) spectroscopy is used to study the binding of MnII ions to a tertiary stabilized hammer-head ribozyme (tsHHRz) and to compare it with the binding to the minimal hammerhead ribozyme (mHHRz). Continuous wave EPR measurements show that the tsHHRz possesses a single high-affinity MnII binding site with a KD of ≤10 nM at an NaCl concentration of 0.1 M. This dissociation constant is at least two orders of magnitude smaller than the KD determined previously for the single high-affinity MnII site in the mHHRz. In addition, whereas the high-affinity MnII is displaced from the mHHRz upon binding of the aminoglycoside antibiotic neomycin B, it is not from the tsHHRz. Despite these pronounced differences in binding, a comparison between the electron spin echo envelope modulation and hyperfine sublevel correlation spectra of the minimal and tertiary stabilized HHRz demonstrates that the structure of both binding sites is very similar. This suggests that the MnII is located in both ribozymes between the bases A9 and G10.1 of the sheared G · A tandem base pair, as shown previously and in detail for the mHHRz. Thus, the much stronger MnII binding in the tsHHRz is attributed to the interaction between the two external loops, which locks in the RNA fold, trapping the MnII in the tightly bound conformation, whereas the absence of long-range loop–loop interactions in the mHHRz leads to more dynamical and open conformations, decreasing MnII binding. PMID:15611296

  1. Nucleobases bind to and stabilize aggregates of a prebiotic amphiphile, providing a viable mechanism for the emergence of protocells.

    PubMed

    Black, Roy A; Blosser, Matthew C; Stottrup, Benjamin L; Tavakley, Ravi; Deamer, David W; Keller, Sarah L

    2013-08-13

    Primordial cells presumably combined RNAs, which functioned as catalysts and carriers of genetic information, with an encapsulating membrane of aggregated amphiphilic molecules. Major questions regarding this hypothesis include how the four bases and the sugar in RNA were selected from a mixture of prebiotic compounds and colocalized with such membranes, and how the membranes were stabilized against flocculation in salt water. To address these questions, we explored the possibility that aggregates of decanoic acid, a prebiotic amphiphile, interact with the bases and sugar found in RNA. We found that these bases, as well as some but not all related bases, bind to decanoic acid aggregates. Moreover, both the bases and ribose inhibit flocculation of decanoic acid by salt. The extent of inhibition by the bases correlates with the extent of their binding, and ribose inhibits to a greater extent than three similar sugars. Finally, the stabilizing effects of a base and ribose are additive. Thus, aggregates of a prebiotic amphiphile bind certain heterocyclic bases and sugars, including those found in RNA, and this binding stabilizes the aggregates against salt. These mutually reinforcing mechanisms might have driven the emergence of protocells. PMID:23901105

  2. Nucleobases bind to and stabilize aggregates of a prebiotic amphiphile, providing a viable mechanism for the emergence of protocells

    PubMed Central

    Black, Roy A.; Blosser, Matthew C.; Stottrup, Benjamin L.; Tavakley, Ravi; Deamer, David W.; Keller, Sarah L.

    2013-01-01

    Primordial cells presumably combined RNAs, which functioned as catalysts and carriers of genetic information, with an encapsulating membrane of aggregated amphiphilic molecules. Major questions regarding this hypothesis include how the four bases and the sugar in RNA were selected from a mixture of prebiotic compounds and colocalized with such membranes, and how the membranes were stabilized against flocculation in salt water. To address these questions, we explored the possibility that aggregates of decanoic acid, a prebiotic amphiphile, interact with the bases and sugar found in RNA. We found that these bases, as well as some but not all related bases, bind to decanoic acid aggregates. Moreover, both the bases and ribose inhibit flocculation of decanoic acid by salt. The extent of inhibition by the bases correlates with the extent of their binding, and ribose inhibits to a greater extent than three similar sugars. Finally, the stabilizing effects of a base and ribose are additive. Thus, aggregates of a prebiotic amphiphile bind certain heterocyclic bases and sugars, including those found in RNA, and this binding stabilizes the aggregates against salt. These mutually reinforcing mechanisms might have driven the emergence of protocells. PMID:23901105

  3. Milk protein composition and stability changes affected by iron in water sources.

    PubMed

    Wang, Aili; Duncan, Susan E; Knowlton, Katharine F; Ray, William K; Dietrich, Andrea M

    2016-06-01

    Water makes up more than 80% of the total weight of milk. However, the influence of water chemistry on the milk proteome has not been extensively studied. The objective was to evaluate interaction of water-sourced iron (low, medium, and high levels) on milk proteome and implications on milk oxidative state and mineral content. Protein composition, oxidative stability, and mineral composition of milk were investigated under conditions of iron ingestion through bovine drinking water (infused) as well as direct iron addition to commercial milk in 2 studies. Four ruminally cannulated cows each received aqueous infusions (based on water consumption of 100L) of 0, 2, 5, and 12.5mg/L Fe(2+) as ferrous lactate, resulting in doses of 0, 200, 500 or 1,250mg of Fe/d, in a 4×4Latin square design for a 14-d period. For comparison, ferrous sulfate solution was directly added into commercial retail milk at the same concentrations: control (0mg of Fe/L), low (2mg of Fe/L), medium (5mg of Fe/L), and high (12.5mg of Fe/L). Two-dimensional electrophoresis coupled with matrix-assisted laser desorption/ionization-tandem time-of-flight (MALDI-TOF/TOF) high-resolution tandem mass spectrometry analysis was applied to characterize milk protein composition. Oxidative stability of milk was evaluated by the thiobarbituric acid reactive substances (TBARS) assay for malondialdehyde, and mineral content was measured by inductively coupled plasma mass spectrometry. For milk from both abomasal infusion of ferrous lactate and direct addition of ferrous sulfate, an iron concentration as low as 2mg of Fe/L was able to cause oxidative stress in dairy cattle and infused milk, respectively. Abomasal infusion affected both caseins and whey proteins in the milk, whereas direct addition mainly influenced caseins. Although abomasal iron infusion did not significantly affect oxidation state and mineral balance (except iron), it induced oxidized off-flavor and partial degradation of whey proteins. Direct

  4. Mapping the Binding Site of the Inhibitor Tariquidar That Stabilizes the First Transmembrane Domain of P-glycoprotein.

    PubMed

    Loo, Tip W; Clarke, David M

    2015-12-01

    ABC (ATP-binding cassette) transporters are clinically important because drug pumps like P-glycoprotein (P-gp, ABCB1) confer multidrug resistance and mutant ABC proteins are responsible for many protein-folding diseases such as cystic fibrosis. Identification of the tariquidar-binding site has been the subject of intensive molecular modeling studies because it is the most potent inhibitor and corrector of P-gp. Tariquidar is a unique P-gp inhibitor because it locks the pump in a conformation that blocks drug efflux but activates ATPase activity. In silico docking studies have identified several potential tariquidar-binding sites. Here, we show through cross-linking studies that tariquidar most likely binds to sites within the transmembrane (TM) segments located in one wing or at the interface between the two wings (12 TM segments form 2 divergent wings). We then introduced arginine residues at all positions in the 12 TM segments (223 mutants) of P-gp. The rationale was that a charged residue in the drug-binding pocket would disrupt hydrophobic interaction with tariquidar and inhibit its ability to rescue processing mutants or stimulate ATPase activity. Arginines introduced at 30 positions significantly inhibited tariquidar rescue of a processing mutant and activation of ATPase activity. The results suggest that tariquidar binds to a site within the drug-binding pocket at the interface between the TM segments of both structural wings. Tariquidar differed from other drug substrates, however, as it stabilized the first TM domain. Stabilization of the first TM domain appears to be a key mechanism for high efficiency rescue of ABC processing mutants that cause disease. PMID:26507655

  5. Mapping the Binding Site of the Inhibitor Tariquidar That Stabilizes the First Transmembrane Domain of P-glycoprotein*

    PubMed Central

    Loo, Tip W.; Clarke, David M.

    2015-01-01

    ABC (ATP-binding cassette) transporters are clinically important because drug pumps like P-glycoprotein (P-gp, ABCB1) confer multidrug resistance and mutant ABC proteins are responsible for many protein-folding diseases such as cystic fibrosis. Identification of the tariquidar-binding site has been the subject of intensive molecular modeling studies because it is the most potent inhibitor and corrector of P-gp. Tariquidar is a unique P-gp inhibitor because it locks the pump in a conformation that blocks drug efflux but activates ATPase activity. In silico docking studies have identified several potential tariquidar-binding sites. Here, we show through cross-linking studies that tariquidar most likely binds to sites within the transmembrane (TM) segments located in one wing or at the interface between the two wings (12 TM segments form 2 divergent wings). We then introduced arginine residues at all positions in the 12 TM segments (223 mutants) of P-gp. The rationale was that a charged residue in the drug-binding pocket would disrupt hydrophobic interaction with tariquidar and inhibit its ability to rescue processing mutants or stimulate ATPase activity. Arginines introduced at 30 positions significantly inhibited tariquidar rescue of a processing mutant and activation of ATPase activity. The results suggest that tariquidar binds to a site within the drug-binding pocket at the interface between the TM segments of both structural wings. Tariquidar differed from other drug substrates, however, as it stabilized the first TM domain. Stabilization of the first TM domain appears to be a key mechanism for high efficiency rescue of ABC processing mutants that cause disease. PMID:26507655

  6. Protein stability, conformational change and binding mechanism of human serum albumin upon binding of embelin and its role in disease control.

    PubMed

    Yeggoni, Daniel Pushparaju; Rachamallu, Aparna; Subramanyam, Rajagopal

    2016-07-01

    Here, we present the inclusive binding mode of phytochemical embelin, an anticancer drug with human serum albumin (HSA) established under physiological condition. Also, to understand the pharmacological role of embelin molecule, here, we have studied the anti-cancer activity of embelin on human cervical cancer cell line (HeLa cell line), which revealed that embelin showed dose dependent inhibition in the growth of cancer cells and also induces 26.3% of apoptosis at an IC50 value of 29μM. Further, embelin was titrated with HSA and the fluorescence emission quenching of HSA due to the formation of the HSA-embelin complex was observed. The binding constant of this complex is 5.9±.01×10(4)M(-1) and the number of bound embelin molecules is approximately 1.0. Consequently, molecular displacement and computational docking experiments show that the embelin is binding to subdomain IB to HSA. Further evidence from microTOF-Q mass spectrometry showed an increase in mass from 66,563Da to 66,857Da observed for free HSA and HSA+embelin complex, signifying that there is robust binding of embelin with HSA. In addition, the variations of HSA secondary structural elements in presence of embelin were confirmed by circular dichroism which indicates partial unfolding of protein. Furthermore, the transmission electron micrographs established that complex formation leads to aggregation of HSA plus embelin. Molecular dynamics simulations revealed that the stability of the HSA-embelin complexes and results suggests that at around 3500ps the complex reaches equilibration state which clearly contributes to the understanding of the stability of the HSA-embelin complexes. PMID:27130964

  7. Creating Prebiotic Sanctuary: Self-Assembling Supramolecular Peptide Structures Bind and Stabilize RNA

    NASA Astrophysics Data System (ADS)

    Carny, Ohad; Gazit, Ehud

    2011-04-01

    Any attempt to uncover the origins of life must tackle the known `blind watchmaker problem'. That is to demonstrate the likelihood of the emergence of a prebiotic system simple enough to be formed spontaneously and yet complex enough to allow natural selection that will lead to Darwinistic evolution. Studies of short aromatic peptides revealed their ability to self-assemble into ordered and stable structures. The unique physical and chemical characteristics of these peptide assemblies point out to their possible role in the origins of life. We have explored mechanisms by which self-assembling short peptides and RNA fragments could interact together and go through a molecular co-evolution, using diphenylalanine supramolecular assemblies as a model system. The spontaneous formation of these self-assembling peptides under prebiotic conditions, through the salt-induced peptide formation (SIPF) pathway was demonstrated. These peptide assemblies possess the ability to bind and stabilize ribonucleotides in a sequence-depended manner, thus increase their relative fitness. The formation of these peptide assemblies is dependent on the homochirality of the peptide monomers: while homochiral peptides (L-Phe-L-Phe and D-Phe-D-Phe) self-assemble rapidly in aqueous environment, heterochiral diastereoisomers (L-Phe-D-Phe and D-Phe-L-Phe) do not tend to self-assemble. This characteristic consists with the homochirality of all living matter. Finally, based on these findings, we propose a model for the role of short self-assembling peptides in the prebiotic molecular evolution and the origin of life.

  8. Cloning and mutational analysis of the gamma gene from Azotobacter vinelandii defines a new family of proteins capable of metallocluster binding and protein stabilization.

    PubMed

    Rubio, Luis M; Rangaraj, Priya; Homer, Mary J; Roberts, Gary P; Ludden, Paul W

    2002-04-19

    Dinitrogenase is a heterotetrameric (alpha(2)beta(2)) enzyme that catalyzes the reduction of dinitrogen to ammonium and contains the iron-molybdenum cofactor (FeMo-co) at its active site. Certain Azotobacter vinelandii mutant strains unable to synthesize FeMo-co accumulate an apo form of dinitrogenase (lacking FeMo-co), with a subunit composition alpha(2)beta(2)gamma(2), which can be activated in vitro by the addition of FeMo-co. The gamma protein is able to bind FeMo-co or apodinitrogenase independently, leading to the suggestion that it facilitates FeMo-co insertion into the apoenzyme. In this work, the non-nif gene encoding the gamma subunit (nafY) has been cloned, sequenced, and found to encode a NifY-like protein. This finding, together with a wealth of knowledge on the biochemistry of proteins involved in FeMo-co and FeV-co biosyntheses, allows us to define a new family of iron and molybdenum (or vanadium) cluster-binding proteins that includes NifY, NifX, VnfX, and now gamma. In vitro FeMo-co insertion experiments presented in this work demonstrate that gamma stabilizes apodinitrogenase in the conformation required to be fully activable by the cofactor. Supporting this conclusion, we show that strains containing mutations in both nafY and nifX are severely affected in diazotrophic growth and extractable dinitrogenase activity when cultured under conditions that are likely to occur in natural environments. This finding reveals the physiological importance of the apodinitrogenase-stabilizing role of which both proteins are capable. The relationship between the metal cluster binding capabilities of this new family of proteins and the ability of some of them to stabilize an apoenzyme is still an open matter. PMID:11823455

  9. Arabidopsis CROLIN1, a Novel Plant Actin-binding Protein, Functions in Cross-linking and Stabilizing Actin Filaments*

    PubMed Central

    Jia, Honglei; Li, Jisheng; Zhu, Jingen; Fan, Tingting; Qian, Dong; Zhou, Yuelong; Wang, Jiaojiao; Ren, Haiyun; Xiang, Yun; An, Lizhe

    2013-01-01

    Higher order actin filament structures are necessary for cytoplasmic streaming, organelle movement, and other physiological processes. However, the mechanism by which the higher order cytoskeleton is formed in plants remains unknown. In this study, we identified a novel actin-cross-linking protein family (named CROLIN) that is well conserved only in the plant kingdom. There are six isovariants of CROLIN in the Arabidopsis genome, with CROLIN1 specifically expressed in pollen. In vitro biochemical analyses showed that CROLIN1 is a novel actin-cross-linking protein with binding and stabilizing activities. Remarkably, CROLIN1 can cross-link actin bundles into actin networks. CROLIN1 loss of function induces pollen germination and pollen tube growth hypersensitive to latrunculin B. All of these results demonstrate that CROLIN1 may play an important role in stabilizing and remodeling actin filaments by binding to and cross-linking actin filaments. PMID:24072702

  10. Identification of VPS13C as a Galectin-12-Binding Protein That Regulates Galectin-12 Protein Stability and Adipogenesis

    PubMed Central

    Yang, Ri-Yao; Xue, Huiting; Yu, Lan; Velayos-Baeza, Antonio; Monaco, Anthony P.; Liu, Fu-Tong

    2016-01-01

    Galectin-12, a member of the galectin family of β-galactoside-binding animal lectins, is preferentially expressed in adipocytes and required for adipocyte differentiation in vitro. This protein was recently found to regulate lipolysis, whole body adiposity, and glucose homeostasis in vivo. Here we identify VPS13C, a member of the VPS13 family of vacuolar protein sorting-associated proteins highly conserved throughout eukaryotic evolution, as a major galectin-12-binding protein. VPS13C is upregulated during adipocyte differentiation, and is required for galectin-12 protein stability. Knockdown of Vps13c markedly reduces the steady-state levels of galectin-12 by promoting its degradation through primarily the lysosomal pathway, and impairs adipocyte differentiation. Our studies also suggest that VPS13C may have a broader role in protein quality control. The regulation of galectin-12 stability by VPS13C could potentially be exploited for therapeutic intervention of obesity and related metabolic diseases. PMID:27073999

  11. Specific Fluorine Labeling of the HyHEL10 Antibody Affects Antigen Binding and Dynamics

    PubMed Central

    Acchione, Mauro; Lee, Yi-Chien; DeSantis, Morgan E.; Lipschultz, Claudia A.; Wlodawer, Alexander; Li, Mi; Shanmuganathan, Aranganathan; Walter, Richard L.; Smith-Gill, Sandra; Barchi, Joseph J.

    2012-01-01

    To more fully understand the molecular mechanisms responsible for variations in binding affinity with antibody maturation, we explored the use of site specific fluorine labeling and 19F nuclear magnetic resonance (NMR). Several single-chain (scFv) antibodies, derived from an affinity-matured series of anti-hen egg white lysozyme (HEL) mouse IgG1, were constructed with either complete or individual replacement of tryptophan residues with 5-fluorotryptophan (5FW). An array of biophysical techniques was used to gain insight into the impact of fluorine substitution on the overall protein structure and antigen binding. SPR measurements indicated that 5FW incorporation lowered binding affinity for the HEL antigen. The degree of analogue impact was residue-dependent, and the greatest decrease in affinity was observed when 5FW was substituted for residues near the binding interface. In contrast, corresponding crystal structures in complex with HEL were essentially indistinguishable from the unsubstituted antibody. 19F NMR analysis showed severe overlap of signals in the free fluorinated protein that was resolved upon binding to antigen, suggesting very distinct chemical environments for each 5FW in the complex. Preliminary relaxation analysis suggested the presence of chemical exchange in the antibody–antigen complex that could not be observed by X-ray crystallography. These data demonstrate that fluorine NMR can be an extremely useful tool for discerning structural changes in scFv antibody–antigen complexes with altered function that may not be discernible by other biophysical techniques. PMID:22769726

  12. Degenerate in vitro genetic selection reveals mutations that diminish alfalfa mosaic virus RNA replication without affecting coat protein binding.

    PubMed

    Rocheleau, Gail; Petrillo, Jessica; Guogas, Laura; Gehrke, Lee

    2004-08-01

    The alfalfa mosaic virus (AMV) RNAs are infectious only in the presence of the viral coat protein; however, the mechanisms describing coat protein's role during replication are disputed. We reasoned that mechanistic details might be revealed by identifying RNA mutations in the 3'-terminal coat protein binding domain that increased or decreased RNA replication without affecting coat protein binding. Degenerate (doped) in vitro genetic selection, based on a pool of randomized 39-mers, was used to select 30 variant RNAs that bound coat protein with high affinity. AUGC sequences that are conserved among AMV and ilarvirus RNAs were among the invariant nucleotides in the selected RNAs. Five representative clones were analyzed in functional assays, revealing diminished viral RNA expression resulting from apparent defects in replication and/or translation. These data identify a set of mutations, including G-U wobble pairs and nucleotide mismatches in the 5' hairpin, which affect viral RNA functions without significant impact on coat protein binding. Because the mutations associated with diminished function were scattered over the 3'-terminal nucleotides, we considered the possibility that RNA conformational changes rather than disruption of a precise motif might limit activity. Native polyacrylamide gel electrophoresis experiments showed that the 3' RNA conformation was indeed altered by nucleotide substitutions. One interpretation of the data is that coat protein binding to the AUGC sequences determines the orientation of the 3' hairpins relative to one another, while local structural features within these hairpins are also critical determinants of functional activity. PMID:15254175

  13. The Study of Stability of Compression-Loaded Multispan Composite Panel Upon Failure of Elements Binding it to Panel Supports

    NASA Technical Reports Server (NTRS)

    Zamula, G. N.; Ierusalimsky, K. M.; Fomin, V. P.; Grishin, V. I.; Kalmykova, G. S.

    1999-01-01

    The present document is a final technical report carried out within co-operation between United States'NASA Langley RC and Russia's Goskomoboronprom in aeronautics, and continues similar programs, accomplished in 1996, 1997, and 1998, respectively). The report provides results of "The study of stability of compression-loaded multispan composite panels upon failure of elements binding it to panel supports"; these comply with requirements established at TsAGI on 24 March 1998 and at NASA on 15 September 1998.

  14. Phosphorylation of Measles Virus Nucleoprotein Affects Viral Growth by Changing Gene Expression and Genomic RNA Stability

    PubMed Central

    Sugai, Akihiro; Sato, Hiroki; Yoneda, Misako

    2013-01-01

    The measles virus (MV) nucleoprotein associates with the viral RNA genome to form the N-RNA complex, providing a template for viral RNA synthesis. In our previous study, major phosphorylation sites of the nucleoprotein were identified as S479 and S510. However, the functions of these phosphorylation sites have not been clarified. In this study, we rescued recombinant MVs (rMVs) whose phosphorylation sites in the nucleoprotein were substituted (rMV-S479A, rMV-S510A, and rMV-S479A/S510A) by reverse genetics and used them in subsequent analyses. In a one-step growth experiment, rMVs showed rapid growth kinetics compared with wild-type MV, although the peak titer of the wild-type MV was the same as or slightly higher than those of the rMVs. Time course analysis of nucleoprotein accumulation also revealed that viral gene expression of rMV was enhanced during the early phase of infection. These findings suggest that nucleoprotein phosphorylation has an important role in controlling viral growth rate through the regulation of viral gene expression. Conversely, multistep growth curves revealed that nucleoprotein-phosphorylation intensity inversely correlated with viral titer at the plateau phase. Additionally, the phosphorylation intensity of the wild-type nucleoprotein in infected cells was significantly reduced through nucleoprotein-phosphoprotein binding. Excessive nucleoprotein-phosphorylation resulted in lower stability against RNase and faster turnover of viral genomic RNA. These results suggest that nucleoprotein-phosphorylation is also involved in viral genomic RNA stability. PMID:23966404

  15. FLiK: a direct-binding assay for the identification and kinetic characterization of stabilizers of inactive kinase conformations.

    PubMed

    Simard, Jeffrey R; Rauh, Daniel

    2014-01-01

    Despite the hundreds of kinase inhibitors currently in discovery and preclinical phases, the number of FDA-approved kinase inhibitors remains very low by comparison, a discrepancy which reflects the challenges which accompanies kinase inhibitor development. Targeting protein kinases with ATP-competitive inhibitors has been the classical approach to inhibit kinase activity, but the highly conserved nature of the ATP-binding site often contributes to the poor inhibitor selectivity. To address this problem, we developed a high-throughput screening technology that can discriminate for inhibitors, which stabilize inactive kinase conformations by binding within allosteric pockets in the kinase domain. Here, we describe how to use the Fluorescence Labels in Kinases approach to measure the K(d) of ligands as well as how to kinetically characterize the binding and dissociation of ligands to the kinase. We also describe how this technology can be used to rapidly screen small molecule libraries in high throughput. PMID:25399645

  16. Direct Measurement of the Nanomechanical Stability of a Redox Protein Active Site and Its Dependence upon Metal Binding.

    PubMed

    Giannotti, Marina I; Cabeza de Vaca, Israel; Artés, Juan M; Sanz, Fausto; Guallar, Victor; Gorostiza, Pau

    2015-09-10

    The structural basis of the low reorganization energy of cupredoxins has long been debated. These proteins reconcile a conformationally heterogeneous and exposed metal-chelating site with the highly rigid copper center required for efficient electron transfer. Here we combine single-molecule mechanical unfolding experiments with statistical analysis and computer simulations to show that the metal-binding region of apo-azurin is mechanically flexible and that high mechanical stability is imparted by copper binding. The unfolding pathway of the metal site depends on the pulling residue and suggests that partial unfolding of the metal-binding site could be facilitated by the physical interaction with certain regions of the redox protein. PMID:26305718

  17. Membrane binding of Escherichia coli RNase E catalytic domain stabilizes protein structure and increases RNA substrate affinity.

    PubMed

    Murashko, Oleg N; Kaberdin, Vladimir R; Lin-Chao, Sue

    2012-05-01

    RNase E plays an essential role in RNA processing and decay and tethers to the cytoplasmic membrane in Escherichia coli; however, the function of this membrane-protein interaction has remained unclear. Here, we establish a mechanistic role for the RNase E-membrane interaction. The reconstituted highly conserved N-terminal fragment of RNase E (NRne, residues 1-499) binds specifically to anionic phospholipids through electrostatic interactions. The membrane-binding specificity of NRne was confirmed using circular dichroism difference spectroscopy; the dissociation constant (K(d)) for NRne binding to anionic liposomes was 298 nM. E. coli RNase G and RNase E/G homologs from phylogenetically distant Aquifex aeolicus, Haemophilus influenzae Rd, and Synechocystis sp. were found to be membrane-binding proteins. Electrostatic potentials of NRne and its homologs were found to be conserved, highly positive, and spread over a large surface area encompassing four putative membrane-binding regions identified in the "large" domain (amino acids 1-400, consisting of the RNase H, S1, 5'-sensor, and DNase I subdomains) of E. coli NRne. In vitro cleavage assay using liposome-free and liposome-bound NRne and RNA substrates BR13 and GGG-RNAI showed that NRne membrane binding altered its enzymatic activity. Circular dichroism spectroscopy showed no obvious thermotropic structural changes in membrane-bound NRne between 10 and 60 °C, and membrane-bound NRne retained its normal cleavage activity after cooling. Thus, NRne membrane binding induced changes in secondary protein structure and enzymatic activation by stabilizing the protein-folding state and increasing its binding affinity for its substrate. Our results demonstrate that RNase E-membrane interaction enhances the rate of RNA processing and decay. PMID:22509045

  18. Membrane binding of Escherichia coli RNase E catalytic domain stabilizes protein structure and increases RNA substrate affinity

    PubMed Central

    Murashko, Oleg N.; Kaberdin, Vladimir R.; Lin-Chao, Sue

    2012-01-01

    RNase E plays an essential role in RNA processing and decay and tethers to the cytoplasmic membrane in Escherichia coli; however, the function of this membrane–protein interaction has remained unclear. Here, we establish a mechanistic role for the RNase E–membrane interaction. The reconstituted highly conserved N-terminal fragment of RNase E (NRne, residues 1–499) binds specifically to anionic phospholipids through electrostatic interactions. The membrane-binding specificity of NRne was confirmed using circular dichroism difference spectroscopy; the dissociation constant (Kd) for NRne binding to anionic liposomes was 298 nM. E. coli RNase G and RNase E/G homologs from phylogenetically distant Aquifex aeolicus, Haemophilus influenzae Rd, and Synechocystis sp. were found to be membrane-binding proteins. Electrostatic potentials of NRne and its homologs were found to be conserved, highly positive, and spread over a large surface area encompassing four putative membrane-binding regions identified in the “large” domain (amino acids 1–400, consisting of the RNase H, S1, 5′-sensor, and DNase I subdomains) of E. coli NRne. In vitro cleavage assay using liposome-free and liposome-bound NRne and RNA substrates BR13 and GGG-RNAI showed that NRne membrane binding altered its enzymatic activity. Circular dichroism spectroscopy showed no obvious thermotropic structural changes in membrane-bound NRne between 10 and 60 °C, and membrane-bound NRne retained its normal cleavage activity after cooling. Thus, NRne membrane binding induced changes in secondary protein structure and enzymatic activation by stabilizing the protein-folding state and increasing its binding affinity for its substrate. Our results demonstrate that RNase E–membrane interaction enhances the rate of RNA processing and decay. PMID:22509045

  19. Disruption of NAD+ binding site in glyceraldehyde 3-phosphate dehydrogenase affects its intranuclear interactions

    PubMed Central

    Phadke, Manali; Krynetskaia, Natalia; Mishra, Anurag; Barrero, Carlos; Merali, Salim; Gothe, Scott A; Krynetskiy, Evgeny

    2015-01-01

    AIM: To characterize phosphorylation of human glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and mobility of GAPDH in cancer cells treated with chemotherapeutic agents. METHODS: We used proteomics analysis to detect and characterize phosphorylation sites within human GAPDH. Site-specific mutagenesis and alanine scanning was then performed to evaluate functional significance of phosphorylation sites in the GAPDH polypeptide chain. Enzymatic properties of mutated GAPDH variants were assessed using kinetic studies. Intranuclear dynamics parameters (diffusion coefficient and the immobile fraction) were estimated using fluorescence recovery after photobleaching (FRAP) experiments and confocal microscopy. Molecular modeling experiments were performed to estimate the effects of mutations on NAD+ cofactor binding. RESULTS: Using MALDI-TOF analysis, we identified novel phosphorylation sites within the NAD+ binding center of GAPDH at Y94, S98, and T99. Using polyclonal antibody specific to phospho-T99-containing peptide within GAPDH, we demonstrated accumulation of phospho-T99-GAPDH in the nuclear fractions of A549, HCT116, and SW48 cancer cells after cytotoxic stress. We performed site-mutagenesis, and estimated enzymatic properties, intranuclear distribution, and intranuclear mobility of GAPDH mutated variants. Site-mutagenesis at positions S98 and T99 in the NAD+ binding center reduced enzymatic activity of GAPDH due to decreased affinity to NAD+ (Km = 741 ± 257 μmol/L in T99I vs 57 ± 11.1 µmol/L in wild type GAPDH. Molecular modeling experiments revealed the effect of mutations on NAD+ binding with GAPDH. FRAP (fluorescence recovery after photo bleaching) analysis showed that mutations in NAD+ binding center of GAPDH abrogated its intranuclear interactions. CONCLUSION: Our results suggest an important functional role of phosphorylated amino acids in the NAD+ binding center in GAPDH interactions with its intranuclear partners. PMID:26629320

  20. Influenza A viruses suppress cyclooxygenase-2 expression by affecting its mRNA stability

    PubMed Central

    Dudek, Sabine Eva; Nitzsche, Katja; Ludwig, Stephan; Ehrhardt, Christina

    2016-01-01

    Infection with influenza A viruses (IAV) provokes activation of cellular defence mechanisms contributing to the innate immune and inflammatory response. In this process the cyclooxygenase-2 (COX-2) plays an important role in the induction of prostaglandin-dependent inflammation. While it has been reported that COX-2 is induced upon IAV infection, in the present study we observed a down-regulation at later stages of infection suggesting a tight regulation of COX-2 by IAV. Our data indicate the pattern-recognition receptor RIG-I as mediator of the initial IAV-induced COX-2 synthesis. Nonetheless, during on-going IAV replication substantial suppression of COX-2 mRNA and protein synthesis could be detected, accompanied by a decrease in mRNA half-life. Interestingly, COX-2 mRNA stability was not only imbalanced by IAV replication but also by stimulation of cells with viral RNA. Our results reveal tristetraprolin (TTP), which is known to bind COX-2 mRNA and promote its rapid degradation, as regulator of COX-2 expression in IAV infection. During IAV replication and viral RNA accumulation TTP mRNA synthesis was induced, resulting in reduced COX-2 levels. Accordingly, the down-regulation of TTP resulted in increased COX-2 protein expression after IAV infection. These findings indicate a novel IAV-regulated cellular mechanism, contributing to the repression of host defence and therefore facilitating viral replication. PMID:27265729

  1. Harvest date affects aronia juice polyphenols, sugars, and antioxidant activity, but not anthocyanin stability.

    PubMed

    Bolling, Bradley W; Taheri, Rod; Pei, Ruisong; Kranz, Sarah; Yu, Mo; Durocher, Shelley N; Brand, Mark H

    2015-11-15

    The goal of this work was to characterize how the date of harvest of 'Viking' aronia berry impacts juice pigmentation, sugars, and antioxidant activity. Aronia juice anthocyanins doubled at the fifth week of the harvest, and then decreased. Juice hydroxycinnamic acids decreased 33% from the first week, while proanthocyanidins increased 64%. Juice fructose and glucose plateaued at the fourth week, but sorbitol increased 40% to the seventh harvest week. Aronia juice pigment density increased due to anthocyanin concentration, and polyphenol copigmentation did not significantly affect juice pigmentation. Anthocyanin stability at pH 4.5 was similar between weeks. However, addition of quercetin, sorbitol, and chlorogenic acid to aronia anthocyanins inhibited pH-induced loss of color. Sorbitol and citric acid may be partially responsible for weekly variation in antioxidant activity, as addition of these agents inhibited DPPH scavenging 13-30%. Thus, aronia polyphenol and non-polyphenol components contribute to its colorant and antioxidant functionality. PMID:25977015

  2. Radiation Power Affected by Current and Wall Radius in Water Cooled Vortex Wall-stabilized Arc

    NASA Astrophysics Data System (ADS)

    Iwao, Toru; Nakamura, Takaya; Yanagi, Kentaro; Yamamoto, Shinji

    2015-11-01

    The arc lighting to obtain the environment to evacuate, save the life, keep the safety and be comfortable are focus on. The lack of radiation intensity and color rendering is problem because of inappropriate energy balance. Some researchers have researched the arc lamp mixed with metal vapor for improvement of color rendering spectrum. The metal vapor can emit the high intense radiation. In addition, the radiation is derived from the high temperature medium. Because the arc temperature can be controlled by current and arc radius, the radiation can be controlled by the current and arc radius. This research elucidates the radiation power affected by the current and wall radius in wall-stabilized arc of water-cooled vortex type. As a result, the radiation power increases with increasing the square of current / square of wall radius because of the temperature distribution which is derived from the current density at the simulation.

  3. The Binding Ring Illusion: assimilation affects the perceived size of a circular array

    PubMed Central

    Caplovitz, Gideon P

    2013-01-01

    Our perception of an object’s size arises from the integration of multiple sources of visual information including retinal size, perceived distance and its size relative to other objects in the visual field. This constructive process is revealed through a number of classic size illusions such as the Delboeuf Illusion, the Ebbinghaus Illusion and others illustrating size constancy. Here we present a novel variant of the Delbouef and Ebbinghaus size illusions that we have named the Binding Ring Illusion. The illusion is such that the perceived size of a circular array of elements is underestimated when superimposed by a circular contour – a binding ring – and overestimated when the binding ring slightly exceeds the overall size of the array. Here we characterize the stimulus conditions that lead to the illusion, and the perceptual principles that underlie it. Our findings indicate that the perceived size of an array is susceptible to the assimilation of an explicitly defined superimposed contour. Our results also indicate that the assimilation process takes place at a relatively high level in the visual processing stream, after different spatial frequencies have been integrated and global shape has been constructed. We hypothesize that the Binding Ring Illusion arises due to the fact that the size of an array of elements is not explicitly defined and therefore can be influenced (through a process of assimilation) by the presence of a superimposed object that does have an explicit size. PMID:24555042

  4. Size and molecular flexibility affect the binding of ellagitannins to bovine serum albumin.

    PubMed

    Dobreva, Marina A; Green, Rebecca J; Mueller-Harvey, Irene; Salminen, Juha-Pekka; Howlin, Brendan J; Frazier, Richard A

    2014-09-17

    Binding to bovine serum albumin of monomeric (vescalagin and pedunculagin) and dimeric ellagitannins (roburin A, oenothein B, and gemin A) was investigated by isothermal titration calorimetry and fluorescence spectroscopy, which indicated two types of binding sites. Stronger and more specific sites exhibited affinity constants, K1, of 10(4)-10(6) M(-1) and stoichiometries, n1, of 2-13 and dominated at low tannin concentrations. Weaker and less-specific binding sites had K2 constants of 10(3)-10(5) M(-1) and stoichiometries, n2, of 16-30 and dominated at higher tannin concentrations. Binding to stronger sites appeared to be dependent on tannin flexibility and the presence of free galloyl groups. Positive entropies for all but gemin A indicated that hydrophobic interactions dominated during complexation. This was supported by an exponential relationship between the affinity, K1, and the modeled hydrophobic accessible surface area and by a linear relationship between K1 and the Stern-Volmer quenching constant, K(SV). PMID:25162485

  5. Purified recombinant human prosaposin forms oligomers that bind procathepsin D and affect its autoactivation

    PubMed Central

    2004-01-01

    Before delivery to endosomes, portions of proCD (procathepsin D) and proSAP (prosaposin) are assembled into complexes. We demonstrate that such complexes are also present in secretions of cultured cells. To study the formation and properties of the complexes, we purified proCD and proSAP from culture media of Spodoptera frugiperda cells that were infected with baculoviruses bearing the respective cDNAs. The biological activity of proCD was demonstrated by its pH-dependent autoactivation to pseudocathepsin D and that of proSAP was demonstrated by feeding to saposin-deficient cultured cells that corrected the storage of radioactive glycolipids. In gel filtration, proSAP behaved as an oligomer and proCD as a monomer. ProSAP altered the elution of proCD such that the latter was shifted into proSAP-containing fractions. ProSAP did not change the elution of mature cathepsin D. Using surface plasmon resonance and an immobilized biotinylated proCD, binding of proSAP was demonstrated under neutral and weakly acidic conditions. At pH 6.8, specific binding appeared to involve more than one binding site on a proSAP oligomer. The dissociation of the first site was characterized by a KD1 of 5.8±2.9×10−8 M−1 (calculated for the monomer). ProSAP stimulated the autoactivation of proCD and also the activity of pseudocathepsin D. Concomitant with the activation, proSAP behaved as a substrate yielding tri- and disaposins and smaller fragments. Our results demonstrate that proSAP forms oligomers that are capable of binding proCD spontaneously and independent of the mammalian type N-glycosylation but not capable of binding mature cathepsin D. In addition to binding proSAP, proCD behaves as an autoactivable and processing enzyme and its binding partner as an activator and substrate. PMID:15255780

  6. Heterozygous screen in Saccharomyces cerevisiae identifies dosage-sensitive genes that affect chromosome stability.

    PubMed

    Strome, Erin D; Wu, Xiaowei; Kimmel, Marek; Plon, Sharon E

    2008-03-01

    Current techniques for identifying mutations that convey a small increased cancer risk or those that modify cancer risk in carriers of highly penetrant mutations are limited by the statistical power of epidemiologic studies, which require screening of large populations and candidate genes. To identify dosage-sensitive genes that mediate genomic stability, we performed a genomewide screen in Saccharomyces cerevisiae for heterozygous mutations that increase chromosome instability in a checkpoint-deficient diploid strain. We used two genome stability assays sensitive enough to detect the impact of heterozygous mutations and identified 172 heterozygous gene disruptions that affected chromosome fragment (CF) loss, 45% of which also conferred modest but statistically significant instability of endogenous chromosomes. Analysis of heterozygous deletion of 65 of these genes demonstrated that the majority increased genomic instability in both checkpoint-deficient and wild-type backgrounds. Strains heterozygous for COMA kinetochore complex genes were particularly unstable. Over 50% of the genes identified in this screen have putative human homologs, including CHEK2, ERCC4, and TOPBP1, which are already associated with inherited cancer susceptibility. These findings encourage the incorporation of this orthologous gene list into cancer epidemiology studies and suggest further analysis of heterozygous phenotypes in yeast as models of human disease resulting from haplo-insufficiency. PMID:18245329

  7. Plant species richness and functional traits affect community stability after a flood event.

    PubMed

    Fischer, Felícia M; Wright, Alexandra J; Eisenhauer, Nico; Ebeling, Anne; Roscher, Christiane; Wagg, Cameron; Weigelt, Alexandra; Weisser, Wolfgang W; Pillar, Valério D

    2016-05-19

    Climate change is expected to increase the frequency and magnitude of extreme weather events. It is therefore of major importance to identify the community attributes that confer stability in ecological communities during such events. In June 2013, a flood event affected a plant diversity experiment in Central Europe (Jena, Germany). We assessed the effects of plant species richness, functional diversity, flooding intensity and community means of functional traits on different measures of stability (resistance, resilience and raw biomass changes from pre-flood conditions). Surprisingly, plant species richness reduced community resistance in response to the flood. This was mostly because more diverse communities grew more immediately following the flood. Raw biomass increased over the previous year; this resulted in decreased absolute value measures of resistance. There was no clear response pattern for resilience. We found that functional traits drove these changes in raw biomass: communities with a high proportion of late-season, short-statured plants with dense, shallow roots and small leaves grew more following the flood. Late-growing species probably avoided the flood, whereas greater root length density might have allowed species to better access soil resources brought from the flood, thus growing more in the aftermath. We conclude that resource inputs following mild floods may favour the importance of traits related to resource acquisition and be less associated with flooding tolerance. PMID:27114578

  8. Nectar vs. pollen loading affects the tradeoff between flight stability and maneuverability in bumblebees.

    PubMed

    Mountcastle, Andrew M; Ravi, Sridhar; Combes, Stacey A

    2015-08-18

    Bumblebee foragers spend a significant portion of their lives transporting nectar and pollen, often carrying loads equivalent to more than half their body mass. Whereas nectar is stored in the abdomen near the bee's center of mass, pollen is carried on the hind legs, farther from the center of mass. We examine how load position changes the rotational moment of inertia in bumblebees and whether this affects their flight maneuverability and/or stability. We applied simulated pollen or nectar loads of equal mass to Bombus impatiens bumblebees and examined flight performance in a wind tunnel under three conditions: flight in unsteady flow, tracking an oscillating flower in smooth flow, and flower tracking in unsteady flow. Using an inertial model, we estimated that carrying a load on the legs rather than in the abdomen increases a bee's moment of inertia about the roll and yaw axes but not the pitch axis. Consistent with these predictions, we found that bees carrying a load on their legs displayed slower rotations about their roll and yaw axes, regardless of whether these rotations were driven by external perturbations or self-initiated steering maneuvers. This allowed pollen-loaded bees to maintain a more stable body orientation and higher median flight speed in unsteady flow but reduced their performance when tracking a moving flower, supporting the concept of a tradeoff between stability and maneuverability. These results demonstrate that the types of resources collected by bees affect their flight performance and energetics and suggest that wind conditions may influence resource selection. PMID:26240364

  9. Nectar vs. pollen loading affects the tradeoff between flight stability and maneuverability in bumblebees

    PubMed Central

    Mountcastle, Andrew M.; Combes, Stacey A.

    2015-01-01

    Bumblebee foragers spend a significant portion of their lives transporting nectar and pollen, often carrying loads equivalent to more than half their body mass. Whereas nectar is stored in the abdomen near the bee’s center of mass, pollen is carried on the hind legs, farther from the center of mass. We examine how load position changes the rotational moment of inertia in bumblebees and whether this affects their flight maneuverability and/or stability. We applied simulated pollen or nectar loads of equal mass to Bombus impatiens bumblebees and examined flight performance in a wind tunnel under three conditions: flight in unsteady flow, tracking an oscillating flower in smooth flow, and flower tracking in unsteady flow. Using an inertial model, we estimated that carrying a load on the legs rather than in the abdomen increases a bee’s moment of inertia about the roll and yaw axes but not the pitch axis. Consistent with these predictions, we found that bees carrying a load on their legs displayed slower rotations about their roll and yaw axes, regardless of whether these rotations were driven by external perturbations or self-initiated steering maneuvers. This allowed pollen-loaded bees to maintain a more stable body orientation and higher median flight speed in unsteady flow but reduced their performance when tracking a moving flower, supporting the concept of a tradeoff between stability and maneuverability. These results demonstrate that the types of resources collected by bees affect their flight performance and energetics and suggest that wind conditions may influence resource selection. PMID:26240364

  10. Phosphorylation Modulates the Mechanical Stability of the Cardiac Myosin-Binding Protein C Motif

    PubMed Central

    Michalek, Arthur J.; Howarth, Jack W.; Gulick, James; Previs, Michael J.; Robbins, Jeffrey; Rosevear, Paul R.; Warshaw, David M.

    2013-01-01

    Cardiac myosin-binding protein C (cMyBP-C) is a thick-filament-associated protein that modulates cardiac contractility through interactions of its N-terminal immunoglobulin (Ig)-like C0-C2 domains with actin and/or myosin. These interactions are modified by the phosphorylation of at least four serines located within the motif linker between domains C1 and C2. We investigated whether motif phosphorylation alters its mechanical properties by characterizing force-extension relations using atomic force spectroscopy of expressed mouse N-terminal cMyBP-C fragments (i.e., C0-C3). Protein kinase A phosphorylation or serine replacement with aspartic acids did not affect persistence length (0.43 ± 0.04 nm), individual Ig-like domain unfolding forces (118 ± 3 pN), or Ig extension due to unfolding (30 ± 0.38 nm). However, phosphorylation did significantly decrease the C0-C3 mean contour length by 24 ± 2 nm. These results suggest that upon phosphorylation, the motif, which is freely extensible in the nonphosphorylated state, adopts a more stable and/or different structure. Circular dichroism and dynamic light scattering data for shorter expressed C1-C2 fragments with all four serines replaced by aspartic acids confirmed that the motif did adopt a more stable structure that was not apparent in the nonphosphorylated motif. These biophysical data provide both a mechanical and structural basis for cMyBP-C regulation by motif phosphorylation. PMID:23442866

  11. Structure, stability, and IgE binding of the peach allergen Peamaclein (Pru p 7).

    PubMed

    Tuppo, Lisa; Spadaccini, Roberta; Alessandri, Claudia; Wienk, Hans; Boelens, Rolf; Giangrieco, Ivana; Tamburrini, Maurizio; Mari, Adriano; Picone, Delia; Ciardiello, Maria Antonietta

    2014-09-01

    Knowledge of the structural properties of allergenic proteins is a necessary prerequisite to better understand the molecular bases of their action, and also to design targeted structural/functional modifications. Peamaclein is a recently identified 7 kDa peach allergen that has been associated with severe allergic reactions in sensitive subjects. This protein represents the first component of a new allergen family, which has no 3D structure available yet. Here, we report the first experimental data on the 3D-structure of Peamaclein. Almost 75% of the backbone resonances, including two helical stretches in the N-terminal region, and four out of six cysteine pairs have been assigned by 2D-NMR using a natural protein sample. Simulated gastrointestinal digestion experiments have highlighted that Peamaclein is even more resistant to digestion than the peach major allergen Pru p 3. Only the heat-denatured protein becomes sensitive to intestinal proteases. Similar to Pru p 3, Peamaclein keeps its native 3D-structure up to 90°C, but it becomes unfolded at temperatures of 100-120°C. Heat denaturation affects the immunological properties of both peach allergens, which lose at least partially their IgE-binding epitopes. In conclusion, the data collected in this study provide a first set of information on the molecular properties of Peamaclein. Future studies could lead to the possible use of the denatured form of this protein as a vaccine, and of the inclusion of cooked peach in the diet of subjects allergic to Peamaclein. PMID:25130872

  12. Specific Fluorine Labeling of the HyHEL10 Antibody Affects Antigen Binding and Dynamics

    SciTech Connect

    Acchione, Mauro; Lee, Yi-Chien; DeSantis, Morgan E.; Lipschultz, Claudia A.; Wlodawer, Alexander; Li, Mi; Shanmuganathan, Aranganathan; Walter, Richard L.; Smith-Gill, Sandra; Barchi, Jr., Joseph J.

    2012-10-16

    To more fully understand the molecular mechanisms responsible for variations in binding affinity with antibody maturation, we explored the use of site specific fluorine labeling and {sup 19}F nuclear magnetic resonance (NMR). Several single-chain (scFv) antibodies, derived from an affinity-matured series of anti-hen egg white lysozyme (HEL) mouse IgG1, were constructed with either complete or individual replacement of tryptophan residues with 5-fluorotryptophan ({sup 5F}W). An array of biophysical techniques was used to gain insight into the impact of fluorine substitution on the overall protein structure and antigen binding. SPR measurements indicated that {sup 5F}W incorporation lowered binding affinity for the HEL antigen. The degree of analogue impact was residue-dependent, and the greatest decrease in affinity was observed when {sup 5F}W was substituted for residues near the binding interface. In contrast, corresponding crystal structures in complex with HEL were essentially indistinguishable from the unsubstituted antibody. {sup 19}F NMR analysis showed severe overlap of signals in the free fluorinated protein that was resolved upon binding to antigen, suggesting very distinct chemical environments for each {sup 5F}W in the complex. Preliminary relaxation analysis suggested the presence of chemical exchange in the antibody-antigen complex that could not be observed by X-ray crystallography. These data demonstrate that fluorine NMR can be an extremely useful tool for discerning structural changes in scFv antibody-antigen complexes with altered function that may not be discernible by other biophysical techniques.

  13. Experimental and molecular dynamics studies showed that CBP KIX mutation affects the stability of CBP:c-Myb complex.

    PubMed

    Odoux, Anne; Jindal, Darren; Tamas, Tamara C; Lim, Benjamin W H; Pollard, Drake; Xu, Wu

    2016-06-01

    The coactivators CBP (CREBBP) and its paralog p300 (EP300), two conserved multi-domain proteins in eukaryotic organisms, regulate gene expression in part by binding DNA-binding transcription factors. It was previously reported that the CBP/p300 KIX domain mutant (Y650A, A654Q, and Y658A) altered both c-Myb-dependent gene activation and repression, and that mice with these three point mutations had reduced numbers of platelets, B cells, T cells, and red blood cells. Here, our transient transfection assays demonstrated that mouse embryonic fibroblast cells containing the same mutations in the KIX domain and without a wild-type allele of either CBP or p300, showed decreased c-Myb-mediated transcription. Dr. Wright's group solved a 3-D structure of the mouse CBP:c-Myb complex using NMR. To take advantage of the experimental structure and function data and improved theoretical calculation methods, we performed MD simulations of CBP KIX, CBP KIX with the mutations, and c-Myb, as well as binding energy analysis for both the wild-type and mutant complexes. The binding between CBP and c-Myb is mainly mediated by a shallow hydrophobic groove in the center where the side-chain of Leu302 of c-Myb plays an essential role and two salt bridges at the two ends. We found that the KIX mutations slightly decreased stability of the CBP:c-Myb complex as demonstrated by higher binding energy calculated using either MM/PBSA or MM/GBSA methods. More specifically, the KIX mutations affected the two salt bridges between CBP and c-Myb (CBP-R646 and c-Myb-E306; CBP-E665 and c-Myb-R294). Our studies also revealed differing dynamics of the hydrogen bonds between CBP-R646 and c-Myb-E306 and between CBP-E665 and c-Myb-R294 caused by the CBP KIX mutations. In the wild-type CBP:c-Myb complex, both of the hydrogen bonds stayed relatively stable. In contrast, in the mutant CBP:c-Myb complex, hydrogen bonds between R646 and E306 showed an increasing trend followed by a decreasing trend, and hydrogen

  14. Stability and sequence-specific DNA binding of activation-labile mutants of the human glucocorticoid receptor

    SciTech Connect

    Elsasser, M.S.; Eisen, L.P.; Harmon, J.M. ); Riegel, A.T. )

    1991-11-19

    The stability and DNA-binding properties of activation-labile (act{sup 1}) human glucocorticoid receptors (hGRs) from the glucocorticoid-resistant mutant 3R7.6TG.4 were investigated. These receptors are able to bind reversible associating ligands with normal affinity and specificity, but become unstable during attempted activation to the DNA binding form. Affinity labeling and immunochemical analysis demonstrated that act{sup 1} receptors are not preferentially proteolyzed during attempted activation. In addition, analysis of binding to calf thymus DNA showed that after loss of ligand, act{sup 1} receptors retain the ability to bind to DNA nonspecifically. A 370 bp MMTV promoter fragment containing multiple GREs and an upstream 342 bp fragment lacking GRE sequences were used to assess the binding of act{sup 1} hGR to specific DNA sequences. Immunoadsorption of hGR-DNA complexes after incubation with {sup 32}P-end-labeled fragments showed that both normal and act{sup 1} both normal and act{sup 1} hGRs could be blocked with a synthetic oligonucleotide containing a perfect palindromic GRE, but not with an oligonucleotide in which the GRE was replaced by and ERE. Analogous results were obtained for normal and act{sup 1} hGR activated in the absence of ligand, or after incubation with the glucocorticoid antagonist RU 38486. These results suggest that sequence-specific binding of the hGR does not require the presence of bound ligand and suggest a role for the ligand in trans-activation of hormonally responsive genes.

  15. Spectrofluorimetric methods of stability-indicating assay of certain drugs affecting the cardiovascular system

    NASA Astrophysics Data System (ADS)

    Moussa, B. A.; Mohamed, M. F.; Youssef, N. F.

    2011-01-01

    Two stability-indicating spectrofluorimetric methods have been developed for the determination of ezetimibe and olmesartan medoxomil, drugs affecting the cardiovascular system, and validated in the presence of their degradation products. The first method, for ezetimibe, is based on an oxidative coupling reaction of ezetimibe with 3-methylbenzothiazolin-2-one hydrazone hydrochloride in the presence of cerium (IV) ammonium sulfate in an acidic medium. The quenching effect of ezetimibe on the fluorescence of excess cerous ions is measured at the emission wavelength, λem, of 345 nm with the excitation wavelength, λex, of 296 nm. Factors affecting the reaction were carefully studied and optimized. The second method, for olmesartan medoxomil, is based on measuring the native fluorescence intensity of olmesartan medoxomil in methanol at λem = 360 nm with λex = 286 nm. Regression plots revealed good linear relationships in the assay limits of 10-120 and 8-112 g/ml for ezetimibe and olmesartan medoxomil, respectively. The validity of the methods was assessed according to the United States Pharmacopeya guidelines. Statistical analysis of the results exposed good Student's t-test and F-ratio values. The introduced methods were successfully applied to the analysis of ezetimibe and olmesartan medoxomil in drug substances and drug products as well as in the presence of their degradation products.

  16. Cardiac Myosin Binding Protein C Phosphorylation Affects Cross-Bridge Cycle's Elementary Steps in a Site-Specific Manner

    PubMed Central

    Wang, Li; Sadayappan, Sakthivel; Kawai, Masakata

    2014-01-01

    Based on our recent finding that cardiac myosin binding protein C (cMyBP-C) phosphorylation affects muscle contractility in a site-specific manner, we further studied the force per cross-bridge and the kinetic constants of the elementary steps in the six-state cross-bridge model in cMyBP-C mutated transgenic mice for better understanding of the influence of cMyBP-C phosphorylation on contractile functions. Papillary muscle fibres were dissected from cMyBP-C mutated mice of ADA (Ala273-Asp282-Ala302), DAD (Asp273-Ala282-Asp302), SAS (Ser273-Ala282-Ser302), and t/t (cMyBP-C null) genotypes, and the results were compared to transgenic mice expressing wide-type (WT) cMyBP-C. Sinusoidal analyses were performed with serial concentrations of ATP, phosphate (Pi), and ADP. Both t/t and DAD mutants significantly reduced active tension, force per cross-bridge, apparent rate constant (2πc), and the rate constant of cross-bridge detachment. In contrast to the weakened ATP binding and enhanced Pi and ADP release steps in t/t mice, DAD mice showed a decreased ADP release without affecting the ATP binding and the Pi release. ADA showed decreased ADP release, and slightly increased ATP binding and cross-bridge detachment steps, whereas SAS diminished the ATP binding step and accelerated the ADP release step. t/t has the broadest effects with changes in most elementary steps of the cross-bridge cycle, DAD mimics t/t to a large extent, and ADA and SAS predominantly affect the nucleotide binding steps. We conclude that the reduced tension production in DAD and t/t is the result of reduced force per cross-bridge, instead of the less number of strongly attached cross-bridges. We further conclude that cMyBP-C is an allosteric activator of myosin to increase cross-bridge force, and its phosphorylation status modulates the force, which is regulated by variety of protein kinases. PMID:25420047

  17. Sequestration of maize crop straw C in different soils: role of oxyhydrates in chemical binding and stabilization as recalcitrance.

    PubMed

    Song, Xiangyun; Li, Lianqing; Zheng, Jufeng; Pan, Genxing; Zhang, Xuhui; Zheng, Jinwei; Hussain, Qaiser; Han, Xiaojun; Yu, Xinyan

    2012-05-01

    While biophysical controls on the sequestration capacity of soils have been well addressed with physical protection, chemical binding and stabilization processes as well as microbial community changes, the role of chemical binding and stabilization has not yet well characterized for soil organic carbon (SOC) sequestration in rice paddies. In this study, a 6-month laboratory incubation with and without maize straw amendment (MSA) was conducted using topsoil samples from soils with different clay mineralogy and free oxy-hydrate contents collected across Southern China. The increase in SOC under MSA was found coincident with that in Fe- and Al-bound OC (Fe/Al-OC) after incubation for 30 d (R(2)=0.90, P=0.05), and with sodium dithionate-citrate-bicarbonate (DCB) extractable Fe after incubation for 180 d (R(2)=0.99, P<0.01). The increase in SOC under MSA was found higher in soils rich in DCB extractable Fe than those poor in DCB extractable Fe. The greater SOC sequestration in soils rich in DCB extractable Fe was further supported by the higher abundance of (13)C which was a natural signature of MSA. Moreover, a weak positive correlation of the increased SOC under MSA with the increased humin (R(2)=0.87, P=0.06) observed after incubation for 180 d may indicate a chemical stabilization of sequestered SOC as humin in the long run. These results improved our understanding of SOC sequestration in China's rice paddies that involves an initial chemical binding of amended C and a final stabilization as recalcitrant C of humin. PMID:22341401

  18. The neurofibromin recruitment factor Spred1 binds to the GAP related domain without affecting Ras inactivation.

    PubMed

    Dunzendorfer-Matt, Theresia; Mercado, Ellen L; Maly, Karl; McCormick, Frank; Scheffzek, Klaus

    2016-07-01

    Neurofibromatosis type 1 (NF1) and Legius syndrome are related diseases with partially overlapping symptoms caused by alterations of the tumor suppressor genes NF1 (encoding the protein neurofibromin) and SPRED1 (encoding sprouty-related, EVH1 domain-containing protein 1, Spred1), respectively. Both proteins are negative regulators of Ras/MAPK signaling with neurofibromin functioning as a Ras-specific GTPase activating protein (GAP) and Spred1 acting on hitherto undefined components of the pathway. Importantly, neurofibromin has been identified as a key protein in the development of cancer, as it is genetically altered in a large number of sporadic human malignancies unrelated to NF1. Spred1 has previously been demonstrated to interact with neurofibromin via its N-terminal Ena/VASP Homology 1 (EVH1) domain and to mediate membrane translocation of its target dependent on its C-terminal Sprouty domain. However, the region of neurofibromin required for the interaction with Spred1 has remained unclear. Here we show that the EVH1 domain of Spred1 binds to the noncatalytic (GAPex) portion of the GAP-related domain (GRD) of neurofibromin. Binding is compatible with simultaneous binding of Ras and does not interfere with GAP activity. Our study points to a potential targeting function of the GAPex subdomain of neurofibromin that is present in all known canonical RasGAPs. PMID:27313208

  19. A single mutation in Escherichia coli ribonuclease II inactivates the enzyme without affecting RNA binding.

    PubMed

    Amblar, Mónica; Arraiano, Cecília M

    2005-01-01

    Exoribonuclease II (RNase II), encoded by the rnb gene, is a ubiquitous enzyme that is responsible for 90% of the hydrolytic activity in Escherichia coli crude extracts. The E. coli strain SK4803, carrying the mutant allele rnb296, has been widely used in the study of the role of RNase II. We determined the DNA sequence of rnb296 and cloned this mutant gene in an expression vector. Only a point mutation in the coding sequence of the gene was detected, which results in the single substitution of aspartate 209 for asparagine. The mutant and the wild-type RNase II enzymes were purified, and their 3' to 5' exoribonucleolytic activity, as well as their RNA binding capability, were characterized. We also studied the metal dependency of the exoribonuclease activity of RNase II. The results obtained demonstrated that aspartate 209 is absolutely essential for RNA hydrolysis, but is not required for substrate binding. This is the first evidence of an acidic residue that is essential for the activity of RNase II-like enzymes. The possible involvement of this residue in metal binding at the active site of the enzyme is discussed. These results are particularly relevant at this time given that no structural or mutational analysis has been performed for any protein of the RNR family of exoribonucleases. PMID:15654875

  20. The neurofibromin recruitment factor Spred1 binds to the GAP related domain without affecting Ras inactivation

    PubMed Central

    Dunzendorfer-Matt, Theresia; Mercado, Ellen L.; Maly, Karl; McCormick, Frank; Scheffzek, Klaus

    2016-01-01

    Neurofibromatosis type 1 (NF1) and Legius syndrome are related diseases with partially overlapping symptoms caused by alterations of the tumor suppressor genes NF1 (encoding the protein neurofibromin) and SPRED1 (encoding sprouty-related, EVH1 domain-containing protein 1, Spred1), respectively. Both proteins are negative regulators of Ras/MAPK signaling with neurofibromin functioning as a Ras-specific GTPase activating protein (GAP) and Spred1 acting on hitherto undefined components of the pathway. Importantly, neurofibromin has been identified as a key protein in the development of cancer, as it is genetically altered in a large number of sporadic human malignancies unrelated to NF1. Spred1 has previously been demonstrated to interact with neurofibromin via its N-terminal Ena/VASP Homology 1 (EVH1) domain and to mediate membrane translocation of its target dependent on its C-terminal Sprouty domain. However, the region of neurofibromin required for the interaction with Spred1 has remained unclear. Here we show that the EVH1 domain of Spred1 binds to the noncatalytic (GAPex) portion of the GAP-related domain (GRD) of neurofibromin. Binding is compatible with simultaneous binding of Ras and does not interfere with GAP activity. Our study points to a potential targeting function of the GAPex subdomain of neurofibromin that is present in all known canonical RasGAPs. PMID:27313208

  1. GTP-binding of ARL-3 is activated by ARL-13 as a GEF and stabilized by UNC-119

    PubMed Central

    Zhang, Qing; Li, Yan; Zhang, Yuxia; Torres, Vicente E.; Harris, Peter C.; Ling, Kun; Hu, Jinghua

    2016-01-01

    Primary cilia are sensory organelles indispensable for organogenesis and tissue pattern formation. Ciliopathy small GTPase ARLs are proposed as prominent ciliary switches, which when disrupted result in dysfunctional cilia, yet how ARLs are activated remain elusive. Here, we discover a novel small GTPase functional module, which contains ARL-3, ARL-13, and UNC-119, localizes near the poorly understood inversin (InV)-like compartment in C. elegans. ARL-13 acts synergistically with UNC-119, but antagonistically with ARL-3, in regulating ciliogenesis. We demonstrate that ARL-3 is a unique small GTPase with unusual high intrinsic GDP release but low intrinsic GTP binding rate. Importantly, ARL-13 acts as a nucleotide exchange factor (GEF) of ARL-3, while UNC-119 can stabilize the GTP binding of ARL-3. We further show that excess inactivated ARL-3 compromises ciliogenesis. The findings reveal a novel mechanism that one ciliopathy GTPase ARL-13, as a GEF, coordinates with UNC-119, which may act as a GTP-binding stabilizing factor, to properly activate another GTPase ARL-3 in cilia, a regulatory process indispensable for ciliogenesis. PMID:27102355

  2. The contribution of methionine to the stability of the Escherichia coli MetNIQ ABC transporter - substrate binding protein complex

    PubMed Central

    Nguyen, Phong T.; Li, Qi Wen; Kadaba, Neena S.; Lai, Jeffrey Y.; Yang, Janet G.; Rees, Douglas C.

    2015-01-01

    Despite the ubiquitous role of ATP Binding Cassette (ABC) importers in nutrient uptake, only the E. coli maltose and vitamin B12 ABC transporters have been structurally characterized in multiple conformations relevant to the alternating access transport mechanism. To complement our previous structure determination of the E. coli MetNI methionine importer in the inward facing conformation (Kadaba et al. (2008) Science 321, 250–253), we have explored conditions stabilizing the outward facing conformation. Using two variants, the Walker B E166Q mutation with ATP+EDTA to stabilize MetNI in the ATP-bound conformation and the N229A variant of the binding protein MetQ, shown in this work to disrupt methionine binding, a high affinity MetNIQ complex was formed with a dissociation constant measured to be 27 nM. Using wild type MetQ containing a co-purified methionine (for which the crystal structure is reported at 1.6 Å resolution), the dissociation constant for complex formation with MetNI is measured to be ~40-fold weaker, indicating that complex formation lowers the affinity of MetQ for methionine by this amount. Preparation of a stable MetNIQ complex is an essential step towards the crystallographic analysis of the outward facing conformation, a key intermediate in the uptake of methionine by this transport system. PMID:25803078

  3. PsbI affects the stability, function, and phosphorylation patterns of photosystem II assemblies in tobacco.

    PubMed

    Schwenkert, Serena; Umate, Pavan; Dal Bosco, Cristina; Volz, Stefanie; Mlçochová, Lada; Zoryan, Mikael; Eichacker, Lutz A; Ohad, Itzhak; Herrmann, Reinhold G; Meurer, Jörg

    2006-11-10

    Photosystem II (PSII) core complexes consist of CP47, CP43, D1, D2 proteins and of several low molecular weight integral membrane polypeptides, such as the chloroplast-encoded PsbE, PsbF, and PsbI proteins. To elucidate the function of PsbI in the photosynthetic process as well as in the biogenesis of PSII in higher plants, we generated homoplastomic knock-out plants by replacing most of the tobacco psbI gene with a spectinomycin resistance cartridge. Mutant plants are photoautotrophically viable under green house conditions but sensitive to high light irradiation. Antenna proteins of PSII accumulate to normal amounts, but levels of the PSII core complex are reduced by 50%. Bioenergetic and fluorescence studies uncovered that PsbI is required for the stability but not for the assembly of dimeric PSII and supercomplexes consisting of PSII and the outer antenna (PSII-LHCII). Thermoluminescence emission bands indicate that the presence of PsbI is required for assembly of a fully functional Q(A) binding site. We show that phosphorylation of the reaction center proteins D1 and D2 is light and redox-regulated in the wild type, but phosphorylation is abolished in the mutant, presumably due to structural alterations of PSII when PsbI is deficient. Unlike wild type, phosphorylation of LHCII is strongly increased in the dark due to accumulation of reduced plastoquinone, whereas even upon state II light phosphorylation is decreased in delta psbI. These data attest that phosphorylation of D1/D2, CP43, and LHCII is regulated differently. PMID:16920705

  4. Rice LGD1 containing RNA binding activity affects growth and development through alternative promoters.

    PubMed

    Thangasamy, Saminathan; Chen, Pei-Wei; Lai, Ming-Hsing; Chen, Jychian; Jauh, Guang-Yuh

    2012-07-01

    Tiller initiation and panicle development are important agronomical traits for grain production in Oryza sativa L. (rice), but their regulatory mechanisms are not yet fully understood. In this study, T-DNA mutant and RNAi transgenic approaches were used to functionally characterize a unique rice gene, LAGGING GROWTH AND DEVELOPMENT 1 (LGD1). The lgd1 mutant showed slow growth, reduced tiller number and plant height, altered panicle architecture and reduced grain yield. The fewer unelongated internodes and cells in lgd1 led to respective reductions in tiller number and to semi-dwarfism. Several independent LGD1-RNAi lines exhibited defective phenotypes similar to those observed in lgd1. Interestingly, LGD1 encodes multiple transcripts with different transcription start sites (TSSs), which were validated by RNA ligase-mediated rapid amplification of 5' and 3' cDNA ends (RLM-RACE). Additionally, GUS assays and a luciferase promoter assay confirmed the promoter activities of LGD1.1 and LGD1.5. LGD1 encoding a von Willebrand factor type A (vWA) domain containing protein is a single gene in rice that is seemingly specific to grasses. GFP-tagged LGD1 isoforms were predominantly detected in the nucleus, and weakly in the cytoplasm. In vitro northwestern analysis showed the RNA-binding activity of the recombinant C-terminal LGD1 protein. Our results demonstrated that LGD1 pleiotropically regulated rice vegetative growth and development through both the distinct spatiotemporal expression patterns of its multiple transcripts and RNA binding activity. Hence, the study of LGD1 will strengthen our understanding of the molecular basis of the multiple transcripts, and their corresponding polypeptides with RNA binding activity, that regulate pleiotropic effects in rice. PMID:22409537

  5. A novel ER–microtubule-binding protein, ERLIN2, stabilizes Cyclin B1 and regulates cell cycle progression

    PubMed Central

    Zhang, Xuebao; Cai, Juan; Zheng, Ze; Polin, Lisa; Lin, Zhenghong; Dandekar, Aditya; Li, Li; Sun, Fei; Finley, Russell L; Fang, Deyu; Yang, Zeng-Quan; Zhang, Kezhong

    2015-01-01

    The gene encoding endoplasmic reticulum (ER) lipid raft-associated protein 2 (ERLIN2) is amplified in human breast cancers. ERLIN2 gene mutations were also found to be associated with human childhood progressive motor neuron diseases. Yet, an understanding of the physiological function and mechanism for ERLIN2 remains elusive. In this study, we reveal that ERLIN2 is a spatially and temporally regulated ER–microtubule-binding protein that has an important role in cell cycle progression by interacting with and stabilizing the mitosis-promoting factors. Whereas ERLIN2 is highly expressed in aggressive human breast cancers, during normal development ERLIN2 is expressed at the postnatal stage and becomes undetectable in adulthood. ERLIN2 interacts with the microtubule component α-tubulin, and this interaction is maximal during the cell cycle G2/M phase where ERLIN2 simultaneously interacts with the mitosis-promoting complex Cyclin B1/Cdk1. ERLIN2 facilitates K63-linked ubiquitination and stabilization of Cyclin B1 protein in G2/M phase. Downregulation of ERLIN2 results in cell cycle arrest, represses breast cancer proliferation and malignancy and increases sensitivity of breast cancer cells to anticancer drugs. In summary, our study revealed a novel ER–microtubule-binding protein, ERLIN2, which interacts with and stabilizes mitosis-promoting factors to regulate cell cycle progression associated with human breast cancer malignancy.

  6. Restoration of structural stability and ligand binding after removal of the conserved disulfide bond in tear lipocalin

    PubMed Central

    Gasymov, Oktay K.; Abduragimov, Adil R.; Glasgow, Ben J.

    2014-01-01

    Disulfide bonds play diverse structural and functional roles in proteins. In tear lipocalin (TL), the conserved sole disulfide bond regulates stability and ligand binding. Probing protein structure often involves thiol selective labeling for which removal of the disulfide bonds may be necessary. Loss of the disulfide bond may destabilize the protein so strategies to retain the native state are needed. Several approaches were tested to regain the native conformational state in the disulfide-less protein. These included the addition of thrimethylamine N-oxide (TMAO) and the substitution of the Cys residues of disulfide bond with residues that can either form a potential salt bridge or others that can create a hydrophobic interaction. TMAO stabilized the protein relaxed by removal of the disulfide bond. In the disulfide-less mutants of TL, 1.0 M TMAO increased the free energy change (ΔG0) significantly from 2.1 to 3.8 kcal/mol. Moderate recovery was observed for the ligand binding tested with NBD-cholesterol. Because the disulfide bond of TL is solvent exposed, the substitution of the disulfide bond with a potential salt bridge or hydrophobic interaction did not stabilize the protein. This approach should work for buried disulfide bonds. However, for proteins with solvent exposed disulfide bonds, the use of TMAO may be an excellent strategy to restore the native conformational states in disulfide-less analogs of the proteins. PMID:25223802

  7. Restoration of structural stability and ligand binding after removal of the conserved disulfide bond in tear lipocalin.

    PubMed

    Gasymov, Oktay K; Abduragimov, Adil R; Glasgow, Ben J

    2014-10-01

    Disulfide bonds play diverse structural and functional roles in proteins. In tear lipocalin (TL), the conserved sole disulfide bond regulates stability and ligand binding. Probing protein structure often involves thiol selective labeling for which removal of the disulfide bonds may be necessary. Loss of the disulfide bond may destabilize the protein so strategies to retain the native state are needed. Several approaches were tested to regain the native conformational state in the disulfide-less protein. These included the addition of trimethylamine N-oxide (TMAO) and the substitution of the Cys residues of disulfide bond with residues that can either form a potential salt bridge or others that can create a hydrophobic interaction. TMAO stabilized the protein relaxed by removal of the disulfide bond. In the disulfide-less mutants of TL, 1.0M TMAO increased the free energy change (ΔG(0)) significantly from 2.1 to 3.8kcal/mol. Moderate recovery was observed for the ligand binding tested with NBD-cholesterol. Because the disulfide bond of TL is solvent exposed, the substitution of the disulfide bond with a potential salt bridge or hydrophobic interaction did not stabilize the protein. This approach should work for buried disulfide bonds. However, for proteins with solvent exposed disulfide bonds, the use of TMAO may be an excellent strategy to restore the native conformational states in disulfide-less analogs of the proteins. PMID:25223802

  8. Stabilizing a flexible interdomain hinge region harboring the SMB binding site drives uPAR into its closed conformation.

    PubMed

    Zhao, Baoyu; Gandhi, Sonu; Yuan, Cai; Luo, Zhipu; Li, Rui; Gårdsvoll, Henrik; de Lorenzi, Valentina; Sidenius, Nicolai; Huang, Mingdong; Ploug, Michael

    2015-03-27

    The urokinase-type plasminogen activator receptor (uPAR) is a multidomain glycolipid-anchored membrane protein, which facilitates extracellular matrix remodeling by focalizing plasminogen activation to cell surfaces via its high-affinity interaction with uPA. The modular assembly of its three LU (Ly6/uPAR-like) domains is inherently flexible and binding of uPA drives uPAR into its closed conformation, which presents the higher-affinity state for vitronectin thus providing an allosteric regulatory mechanism. Using a new class of epitope-mapped anti-uPAR monoclonal antibodies (mAbs), we now demonstrate that the reciprocal stabilization is indeed also possible. By surface plasmon resonance studies, we show that these mAbs and vitronectin have overlapping binding sites on uPAR and that they share Arg91 as hotspot residue in their binding interfaces. The crystal structure solved for one of these uPAR·mAb complexes at 3.0Å clearly shows that this mAb preselects the closed uPAR conformation with an empty but correctly assembled large hydrophobic binding cavity for uPA. Accordingly, these mAbs inhibit the uPAR-dependent lamellipodia formation and migration on vitronectin-coated matrices irrespective of the conformational status of uPAR and its occupancy with uPA. This is the first study to the best of our knowledge, showing that the dynamic assembly of the three LU domains in uPARwt can be driven toward the closed form by an external ligand, which is not engaging the hydrophobic uPA binding cavity. As this binding interface is also exploited by the somatomedin B domain of vitronectin, therefore, this relationship should be taken into consideration when exploring uPAR-dependent cell adhesion and migration in vitronectin-rich environments. PMID:25659907

  9. Calcium affects OX1 orexin (hypocretin) receptor responses by modifying both orexin binding and the signal transduction machinery

    PubMed Central

    Putula, Jaana; Pihlajamaa, Tero; Kukkonen, Jyrki P

    2014-01-01

    Background and Purpose One of the major responses upon orexin receptor activation is Ca2+ influx, and this influx seems to amplify the other responses mediated by orexin receptors. However, the reduction in Ca2+, often used to assess the importance of Ca2+ influx, might affect other properties, like ligand−receptor interactions, as suggested for some GPCR systems. Hence, we investigated the role of the ligand−receptor interaction and Ca2+ signal cascades in the apparent Ca2+ requirement of orexin-A signalling. Experimental Approach Receptor binding was assessed in CHO cells expressing human OX1 receptors with [125I]-orexin-A by conventional ligand binding as well as scintillation proximity assays. PLC activity was determined by chromatography. Key Results Both orexin receptor binding and PLC activation were strongly dependent on the extracellular Ca2+ concentration. The relationship between Ca2+ concentration and receptor binding was the same as that for PLC activation. However, when Ca2+ entry was reduced by depolarizing the cells or by inhibiting the receptor-operated Ca2+ channels, orexin-A-stimulated PLC activity was much more strongly inhibited than orexin-A binding. Conclusions and Implications Ca2+ plays a dual role in orexin signalling by being a prerequisite for both ligand−receptor interaction and amplifying orexin signals via Ca2+ influx. Some previous results obtained utilizing Ca2+ chelators have to be re-evaluated based on the results of the current study. From a drug discovery perspective, further experiments need to identify the target for Ca2+ in orexin-A−OX1 receptor interaction and its mechanism of action. PMID:25132134

  10. The importance of sulphide binding for leaching of heavy metals from contaminated Norwegian marine sediments treated by stabilization/solidification.

    PubMed

    Sparrevik, Magnus; Eek, Espen; Grini, Randi Skirstad

    2009-07-01

    Over time, Norwegian fjords and harbour areas have received contaminants from industrial activities and urban run-off, and measures to remediate contaminated marine sediments are therefore needed. Stabilization/solidification (S/S) technology, in which the contaminated marine sediments are mixed with cement and other binding agents, has been shown to be a promising remediation technology. This paper summarizes a study of the environmental effect of stabilization, highlighting the importance of sulphide binding governing the leaching of heavy metals from the S/S of contaminated marine sediments. The study is a part of a research project focusing on developing effective methods for S/S of contaminated seabed sediments for use in new construction areas. Four cementitious binders were tested on sediments from six different locations: Bergen, Gilhus, Grenland, Hammerfest, Sandvika and Trondheim. The sediments differed with respect to properties such as concentration of contaminants, water content, organic content and grain size distribution. Portland cement, Portland cement with fly ash, industry cement, and sulphate resistant cement, were tested as binders. The leaching from the S/S sediments after 28 days of curing was measured by using a standard leaching batch test (EN 12457-2: 2003), with seawater as leaching agent. The eluate was analysed for pH and redox, as well as content of heavy metals and organic contaminants. Available volatile sulphide (AVS) and simultaneously extractable metals (SEM) were also measured in the sediments. This paper focuses on the leaching of lead (Pb) and copper (Cu). A reduced leaching of Pb after stabilization was observed for the mixtures, whereas the leaching of Cu from Hammerfest sediments increased substantially after stabilization for all cementitious additions. Experiments show that Hammerfest samples had lower values of AVS than the other sediments. This was confirmed by the SEM/AVS analysis, highlighting the importance of

  11. Dihydrotanshinone-I interferes with the RNA-binding activity of HuR affecting its post-transcriptional function

    PubMed Central

    D’Agostino, Vito Giuseppe; Lal, Preet; Mantelli, Barbara; Tiedje, Christopher; Zucal, Chiara; Thongon, Natthakan; Gaestel, Matthias; Latorre, Elisa; Marinelli, Luciana; Seneci, Pierfausto; Amadio, Marialaura; Provenzani, Alessandro

    2015-01-01

    Post-transcriptional regulation is an essential determinant of gene expression programs in physiological and pathological conditions. HuR is a RNA-binding protein that orchestrates the stabilization and translation of mRNAs, critical in inflammation and tumor progression, including tumor necrosis factor-alpha (TNF). We identified the low molecular weight compound 15,16-dihydrotanshinone-I (DHTS), well known in traditional Chinese medicine practice, through a validated high throughput screening on a set of anti-inflammatory agents for its ability to prevent HuR:RNA complex formation. We found that DHTS interferes with the association step between HuR and the RNA with an equilibrium dissociation constant in the nanomolar range in vitro (Ki = 3.74 ± 1.63 nM). In breast cancer cell lines, short term exposure to DHTS influences mRNA stability and translational efficiency of TNF in a HuR-dependent manner and also other functional readouts of its post-transcriptional control, such as the stability of selected pre-mRNAs. Importantly, we show that migration and sensitivity of breast cancer cells to DHTS are modulated by HuR expression, indicating that HuR is among the preferential intracellular targets of DHTS. Here, we disclose a previously unrecognized molecular mechanism exerted by DHTS, opening new perspectives to therapeutically target the HuR mediated, post-transcriptional control in inflammation and cancer cells. PMID:26553968

  12. A DNA binding winged helix domain in CAF-1 functions with PCNA to stabilize CAF-1 at replication forks

    PubMed Central

    Zhang, Kuo; Gao, Yuan; Li, Jingjing; Burgess, Rebecca; Han, Junhong; Liang, Huanhuan; Zhang, Zhiguo; Liu, Yingfang

    2016-01-01

    Chromatin assembly factor 1 (CAF-1) is a histone H3–H4 chaperone that deposits newly synthesized histone (H3–H4)2 tetramers during replication-coupled nucleosome assembly. However, how CAF-1 functions in this process is not yet well understood. Here, we report the crystal structure of C terminus of Cac1 (Cac1C), a subunit of yeast CAF-1, and the function of this domain in stabilizing CAF-1 at replication forks. We show that Cac1C forms a winged helix domain (WHD) and binds DNA in a sequence-independent manner. Mutations in Cac1C that abolish DNA binding result in defects in transcriptional silencing and increased sensitivity to DNA damaging agents, and these defects are exacerbated when combined with Cac1 mutations deficient in PCNA binding. Similar phenotypes are observed for corresponding mutations in mouse CAF-1. These results reveal a mechanism conserved in eukaryotic cells whereby the ability of CAF-1 to bind DNA is important for its association with the DNA replication forks and subsequent nucleosome assembly. PMID:26908650

  13. A DNA binding winged helix domain in CAF-1 functions with PCNA to stabilize CAF-1 at replication forks.

    PubMed

    Zhang, Kuo; Gao, Yuan; Li, Jingjing; Burgess, Rebecca; Han, Junhong; Liang, Huanhuan; Zhang, Zhiguo; Liu, Yingfang

    2016-06-20

    Chromatin assembly factor 1 (CAF-1) is a histone H3-H4 chaperone that deposits newly synthesized histone (H3-H4)2 tetramers during replication-coupled nucleosome assembly. However, how CAF-1 functions in this process is not yet well understood. Here, we report the crystal structure of C terminus of Cac1 (Cac1C), a subunit of yeast CAF-1, and the function of this domain in stabilizing CAF-1 at replication forks. We show that Cac1C forms a winged helix domain (WHD) and binds DNA in a sequence-independent manner. Mutations in Cac1C that abolish DNA binding result in defects in transcriptional silencing and increased sensitivity to DNA damaging agents, and these defects are exacerbated when combined with Cac1 mutations deficient in PCNA binding. Similar phenotypes are observed for corresponding mutations in mouse CAF-1. These results reveal a mechanism conserved in eukaryotic cells whereby the ability of CAF-1 to bind DNA is important for its association with the DNA replication forks and subsequent nucleosome assembly. PMID:26908650

  14. Glycation of Ribonuclease A affects its enzymatic activity and DNA binding ability.

    PubMed

    Dinda, Amit Kumar; Tripathy, Debi Ranjan; Dasgupta, Swagata

    2015-11-01

    Prolonged non-enzymatic glycation of proteins results in the formation of advanced glycation end products (AGEs) that cause several diseases. The glycation of Ribonuclease A (RNase A) at pH 7.4 and 37 °C with ribose, glucose and fructose has been monitored by UV-vis, fluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix assisted laser desorption ionization spectroscopy-time of flight (MALDI-TOF) methods. The enzymatic activity and DNA binding ability of glycated RNase A was also investigated by an agarose gel-based assay. A precipitation assay examined the ribonucleolytic activity of the glycated enzyme. An increase in incubation time resulted in the formation of high molecular weight AGEs with a decrease in ribonucleolytic activity. Ribose exhibits the highest potency as a glycating agent and showed the greatest reduction in the ribonucleolytic activity of the enzyme. Interestingly, glycated RNase A was unable to bind with the ribonuclease inhibitor (RI) and DNA. The glycated form of the protein was also found to be ineffective in DNA melting unlike native RNase A. PMID:26365067

  15. Stability and Control Harmony in Approach and Landing. [analysis of factors affecting flight characteristics at low airspeeds

    NASA Technical Reports Server (NTRS)

    Anderson, S. B.

    1975-01-01

    A review of the factors which affect stability and control harmony in approach and landing is made to obtain a clearer understanding of the proper relationship, the trade-offs involved, and to show how limits in stability and control harmony are established for advanced aircraft. Factors which influence stability and control harmony include the longitudinal short period response of the aircraft and the level of several pitch control characteristics including control power, control sensitivity, and control feel. At low stability levels for advanced aircraft, less conventional control techniques such as DLC are needed to improve harmony and some form of stability augmentation must be provided to improve precession of flight path control and reduce pilot work load.

  16. Disulfide-Mediated Stabilization of the IκB Kinase Binding Domain of NF-κB Essential Modulator (NEMO)

    PubMed Central

    2015-01-01

    Human NEMO (NF-κB essential modulator) is a 419 residue scaffolding protein that, together with catalytic subunits IKKα and IKKβ, forms the IκB kinase (IKK) complex, a key regulator of NF-κB pathway signaling. NEMO is an elongated homodimer comprising mostly α-helix. It has been shown that a NEMO fragment spanning residues 44–111, which contains the IKKα/β binding site, is structurally disordered in the absence of bound IKKβ. Herein we show that enforcing dimerization of NEMO1–120 or NEMO44–111 constructs through introduction of one or two interchain disulfide bonds, through oxidation of the native Cys54 residue and/or at position 107 through a Leu107Cys mutation, induces a stable α-helical coiled-coil structure that is preorganized to bind IKKβ with high affinity. Chemical and thermal denaturation studies showed that, in the context of a covalent dimer, the ordered structure was stabilized relative to the denatured state by up to 3 kcal/mol. A full-length NEMO-L107C protein formed covalent dimers upon treatment of mammalian cells with H2O2. Furthermore, NEMO-L107C bound endogenous IKKβ in A293T cells, reconstituted TNF-induced NF-κB signaling in NEMO-deficient cells, and interacted with TRAF6. Our results indicate that the IKKβ binding domain of NEMO possesses an ordered structure in the unbound state, provided that it is constrained within a dimer as is the case in the constitutively dimeric full-length NEMO protein. The stability of the NEMO coiled coil is maintained by strong interhelix interactions in the region centered on residue 54. The disulfide-linked constructs we describe herein may be useful for crystallization of NEMO’s IKKβ binding domain in the absence of bound IKKβ, thereby facilitating the structural characterization of small-molecule inhibitors. PMID:25400026

  17. Disulfide-mediated stabilization of the IκB kinase binding domain of NF-κB essential modulator (NEMO).

    PubMed

    Zhou, Li; Yeo, Alan T; Ballarano, Carmine; Weber, Urs; Allen, Karen N; Gilmore, Thomas D; Whitty, Adrian

    2014-12-23

    Human NEMO (NF-κB essential modulator) is a 419 residue scaffolding protein that, together with catalytic subunits IKKα and IKKβ, forms the IκB kinase (IKK) complex, a key regulator of NF-κB pathway signaling. NEMO is an elongated homodimer comprising mostly α-helix. It has been shown that a NEMO fragment spanning residues 44-111, which contains the IKKα/β binding site, is structurally disordered in the absence of bound IKKβ. Herein we show that enforcing dimerization of NEMO1-120 or NEMO44-111 constructs through introduction of one or two interchain disulfide bonds, through oxidation of the native Cys54 residue and/or at position 107 through a Leu107Cys mutation, induces a stable α-helical coiled-coil structure that is preorganized to bind IKKβ with high affinity. Chemical and thermal denaturation studies showed that, in the context of a covalent dimer, the ordered structure was stabilized relative to the denatured state by up to 3 kcal/mol. A full-length NEMO-L107C protein formed covalent dimers upon treatment of mammalian cells with H2O2. Furthermore, NEMO-L107C bound endogenous IKKβ in A293T cells, reconstituted TNF-induced NF-κB signaling in NEMO-deficient cells, and interacted with TRAF6. Our results indicate that the IKKβ binding domain of NEMO possesses an ordered structure in the unbound state, provided that it is constrained within a dimer as is the case in the constitutively dimeric full-length NEMO protein. The stability of the NEMO coiled coil is maintained by strong interhelix interactions in the region centered on residue 54. The disulfide-linked constructs we describe herein may be useful for crystallization of NEMO's IKKβ binding domain in the absence of bound IKKβ, thereby facilitating the structural characterization of small-molecule inhibitors. PMID:25400026

  18. The Study of Stability of Compression-loaded Multispan Composite Panel Upon Failure of elements Binding it to Panel Supports

    NASA Technical Reports Server (NTRS)

    Zamula, G. N.; Ierusalimsky, K. M.; Fomin, V. P.; Grishin, V. I.; Kalmykova, G. S.

    1999-01-01

    The present document is a final technical report under the NCC-1-233 research program (dated September 15, 1998; see Appendix 5) carried out within co-operation between United States'NASA Langley RC and Russia's Goskomoboronprom in aeronautics, and continues similar programs, NCCW-73, NCC-1-233 and NCCW 1-233 accomplished in 1996, 1997, and 1998, respectively. The report provides results of "The study of stability of compression-loaded multispan composite panels upon failure of elements binding it to panel supports"; these comply with requirements established at TsAGI on 24 March 1998 and at NASA on 15 September 1998.

  19. Recombinant human nerve growth factor for clinical trials: protein expression, purification, stability and characterisation of binding to infusion pumps.

    PubMed

    Allen, S J; Robertson, A G; Tyler, S J; Wilcock, G K; Dawbarn, D

    2001-02-26

    Nerve growth factor (NGF) has been suggested to be of therapeutic benefit to patients with Alzheimer's disease. One of the early changes in this disease is a loss of cholinergic function within the brain, and NGF is able to rescue cholinergic neurons both in vitro and in vivo. We describe the production of recombinant human beta-NGF (rhNGF), using baculovirus infection of insect cells; its purification, formulation and subsequent stability for use in clinical trials. Tests were also carried out to monitor release of protein from infusion pumps and catheters for intracerebroventricular administration (icv). Initial problems with non-specific binding were overcome using a blocking formula. PMID:11245895

  20. Factors affecting the stability and performance of ipratropium bromide; fenoterol hydrobromide pressurized-metered dose inhalers.

    PubMed

    Ninbovorl, Jenjira; Sawatdee, Somchai; Srichana, Teerapol

    2013-12-01

    The aim of the study was to investigate the factors affecting the stability and performance of ipratropium bromide and fenoterol hydrobromide in a pressurized-metered dose inhaler (pMDI). A factorial design was applied to investigate the effects of three parameters (propellant, water, and ethanol) on the performance of 27 designed formulations of a solution-based pMDI. The formulations that contained a hydrofluoroalkane (HFA) propellant lower than 72% v/v and an ethanol concentration higher than 27% v/v remained as clear solutions. Nine formulations that contained the HFA propellant higher than 74% v/v precipitated. The results indicated that it was not only the HFA propellant content of the formulations that was related to the formulation instability but also ethanol content. Only six formulations from the 18 formulations, that did not precipitate, produced drug contents that were within the acceptable range (80-120%). These six formulations generated aerosols with mass median aerodynamic diameters (MMAD) of approximately 2 μm with a fine particle fraction (FPF; particle size, <6.4 μm) between 45% and 52%. The MMAD and FPF did not change significantly after 6 months of storage (P > 0.05). PMID:23975571

  1. Arch coordination does not affect the stability in class III orthognathic surgery patients.

    PubMed

    Hong, Sung Ok; Ryu, Dong-Mok; Lee, Deok-Won; Jung, Jae Hoon

    2013-11-01

    The purpose of this study was to evaluate if the arch coordination manifested by preorthodontics had an effect on the short-term stability after orthognathic surgery by evaluating the B point, menton, overjet, and overbite. The subjects were 10 healthy adult female and male Koreans (mean age, 24.9 years) with insufficient arch coordination and 10 healthy adult female and male Koreans (mean age, 22 years) with sufficient arch coordination. All subjects had sagittal split ramus osteotomy with 1-piece maxillary Le Fort I surgery with/without genioplasty done from the same practitioner at Kyung Hee University Hospital at Gangdong from 2009 to 2011. All arch widths of the maxilla and mandible were measured. Cephalometric tracings of the subjects were made of the presurgical and postsurgical period with a follow-up at 3 months. Relapse was measured according to cephalometric tracing changes using the V-ceph Cephalometric Analysis Software version 5.5(Osstem, Seoul, South Korea). Insufficient arch coordination did not definitively affect the overall treatment outcome. There was significant difference in the horizontal dimensions of the mandible (vertical plane to point B, overjet) in the study group. The study group showed instability in orthodontic factors, whereas skeletal factors were stable. Vertical dimensions (horizontal plane to point B, horizontal plane to menton, overbite) were not statistically significant. PMID:24220471

  2. Characterization of How DNA Modifications Affect DNA Binding by C2H2 Zinc Finger Proteins

    PubMed Central

    Patel, A.; Hashimoto, H.; Zhang, X.; Cheng, X.

    2016-01-01

    Much is known about vertebrate DNA methylation and oxidation; however, much less is known about how modified cytosine residues within particular sequences are recognized. Among the known methylated DNA-binding domains, the Cys2-His2 zinc finger (ZnF) protein superfamily is the largest with hundreds of members, each containing tandem ZnFs ranging from 3 to >30 fingers. We have begun to biochemically and structurally characterize these ZnFs not only on their sequence specificity but also on their sensitivity to various DNA modifications. Rather than following published methods of refolding insoluble ZnF arrays, we have expressed and purified soluble forms of ZnFs, ranging in size from a tandem array of two to six ZnFs, from seven different proteins. We also describe a fluorescence polarization assay to measure ZnFs affinity with oligonucleotides containing various modifications and our approaches for cocrystallization of ZnFs with oligonucleotides. PMID:27372763

  3. TANK-binding kinase-1 broadly affects oyster immune response to bacteria and viruses.

    PubMed

    Tang, Xueying; Huang, Baoyu; Zhang, Linlin; Li, Li; Zhang, Guofan

    2016-09-01

    As a benthic filter feeder of estuaries, the immune system of oysters provides one of the best models for studying the genetic and molecular basis of the innate immune pathway in marine invertebrates and examining the influence of environmental factors on the immune system. Here, the molecular function of molluscan TANK-binding kinase-1 (TBK1) (which we named CgTBK1) was studied in the Pacific oyster, Crassostrea gigas. Compared with known TBK1 proteins in other model organisms, CgTBK1 contains a conserved S-TKc domain and a coiled coil domain at the N- and C-terminals but lacks an important ubiquitin domain. Quantitative real-time PCR analysis revealed that the expression level of CgTBK1 was ubiquitous in all selected tissues, with highest expression in the gills. CgTBK1 expression was significantly upregulated in response to infections with Vibrio alginolyticus, ostreid herpesvirus 1 (OsHV-1 reference strain and μvar), and polyinosinic:polycytidylic acid sodium salt, suggesting its broad function in immune response. Subcellular localization showed the presence of CgTBK1 in the cytoplasm of HeLa cells, suggesting its potential function as the signal transducer between the receptor and transcription factor. We further demonstrated that CgTBK1 interacted with CgSTING in HEK293T cells, providing evidence that CgTBK1 could be activated by direct binding to CgSTING. In summary, we characterized the TBK1 gene in C. gigas and demonstrated its role in the innate immune response to pathogen infections. PMID:27422757

  4. A Phytophthora sojae effector suppresses endoplasmic reticulum stress-mediated immunity by stabilizing plant Binding immunoglobulin Proteins.

    PubMed

    Jing, Maofeng; Guo, Baodian; Li, Haiyang; Yang, Bo; Wang, Haonan; Kong, Guanghui; Zhao, Yao; Xu, Huawei; Wang, Yan; Ye, Wenwu; Dong, Suomeng; Qiao, Yongli; Tyler, Brett M; Ma, Wenbo; Wang, Yuanchao

    2016-01-01

    Phytophthora pathogens secrete an array of specific effector proteins to manipulate host innate immunity to promote pathogen colonization. However, little is known about the host targets of effectors and the specific mechanisms by which effectors increase susceptibility. Here we report that the soybean pathogen Phytophthora sojae uses an essential effector PsAvh262 to stabilize endoplasmic reticulum (ER)-luminal binding immunoglobulin proteins (BiPs), which act as negative regulators of plant resistance to Phytophthora. By stabilizing BiPs, PsAvh262 suppresses ER stress-triggered cell death and facilitates Phytophthora infection. The direct targeting of ER stress regulators may represent a common mechanism of host manipulation by microbes. PMID:27256489

  5. A Phytophthora sojae effector suppresses endoplasmic reticulum stress-mediated immunity by stabilizing plant Binding immunoglobulin Proteins

    PubMed Central

    Jing, Maofeng; Guo, Baodian; Li, Haiyang; Yang, Bo; Wang, Haonan; Kong, Guanghui; Zhao, Yao; Xu, Huawei; Wang, Yan; Ye, Wenwu; Dong, Suomeng; Qiao, Yongli; Tyler, Brett M.; Ma, Wenbo; Wang, Yuanchao

    2016-01-01

    Phytophthora pathogens secrete an array of specific effector proteins to manipulate host innate immunity to promote pathogen colonization. However, little is known about the host targets of effectors and the specific mechanisms by which effectors increase susceptibility. Here we report that the soybean pathogen Phytophthora sojae uses an essential effector PsAvh262 to stabilize endoplasmic reticulum (ER)-luminal binding immunoglobulin proteins (BiPs), which act as negative regulators of plant resistance to Phytophthora. By stabilizing BiPs, PsAvh262 suppresses ER stress-triggered cell death and facilitates Phytophthora infection. The direct targeting of ER stress regulators may represent a common mechanism of host manipulation by microbes. PMID:27256489

  6. Whole Genome Sequencing Identifies a Deletion in Protein Phosphatase 2A That Affects Its Stability and Localization in Chlamydomonas reinhardtii

    PubMed Central

    Lin, Huawen; Miller, Michelle L.; Granas, David M.; Dutcher, Susan K.

    2013-01-01

    Whole genome sequencing is a powerful tool in the discovery of single nucleotide polymorphisms (SNPs) and small insertions/deletions (indels) among mutant strains, which simplifies forward genetics approaches. However, identification of the causative mutation among a large number of non-causative SNPs in a mutant strain remains a big challenge. In the unicellular biflagellate green alga Chlamydomonas reinhardtii, we generated a SNP/indel library that contains over 2 million polymorphisms from four wild-type strains, one highly polymorphic strain that is frequently used in meiotic mapping, ten mutant strains that have flagellar assembly or motility defects, and one mutant strain, imp3, which has a mating defect. A comparison of polymorphisms in the imp3 strain and the other 15 strains allowed us to identify a deletion of the last three amino acids, Y313F314L315, in a protein phosphatase 2A catalytic subunit (PP2A3) in the imp3 strain. Introduction of a wild-type HA-tagged PP2A3 rescues the mutant phenotype, but mutant HA-PP2A3 at Y313 or L315 fail to rescue. Our immunoprecipitation results indicate that the Y313, L315, or YFLΔ mutations do not affect the binding of PP2A3 to the scaffold subunit, PP2A-2r. In contrast, the Y313, L315, or YFLΔ mutations affect both the stability and the localization of PP2A3. The PP2A3 protein is less abundant in these mutants and fails to accumulate in the basal body area as observed in transformants with either wild-type HA-PP2A3 or a HA-PP2A3 with a V310T change. The accumulation of HA-PP2A3 in the basal body region disappears in mated dikaryons, which suggests that the localization of PP2A3 may be essential to the mating process. Overall, our results demonstrate that the terminal YFL tail of PP2A3 is important in the regulation on Chlamydomonas mating. PMID:24086163

  7. The effects of buffers and pH on the thermal stability, unfolding and substrate binding of RecA.

    PubMed

    Metrick, Michael A; Temple, Joshua E; MacDonald, Gina

    2013-12-31

    The Escherichia coli protein RecA is responsible for catalysis of the strand transfer reaction used in DNA repair and recombination. Previous studies in our lab have shown that high concentrations of salts stabilize RecA in a reverse-anionic Hofmeister series. Here we investigate how changes in pH and buffer alter the thermal unfolding and cofactor binding. RecA in 20mM HEPES, MES, Tris and phosphate buffers was studied in the pH range from 6.5 to 8.5 using circular dichroism (CD), infrared (IR) and fluorescence spectroscopies. The results show all of the buffers studied stabilize RecA up to 50°C above the Tris melting temperature and influence RecA's ability to nucleate on double-stranded DNA. Infrared and CD spectra of RecA in the different buffers do not show that secondary structural changes are associated with increased stability or decreased ability to nucleate on dsDNA. These results suggest the differences in stability arise from decreasing positive charge and/or buffer interactions. PMID:24036048

  8. Binding stability of peptides derived from 1ALA residue and 7GLY residues to sites near active center of fluctuating papain

    NASA Astrophysics Data System (ADS)

    Nishiyama, Katsuhiko

    2012-05-01

    We investigated the binding stability of peptides derived from 1ALA residue and 7GLY residues to sites near active center of fluctuating papain via molecular dynamics and docking simulations. Replacing GLY residue in 8GLY with ALA residue had a positive effect on binding stability to the sites in some cases although the replacing had a negative effect on it in other cases. Furthermore the replacing had a negative effect on the chance of binding to the sites. Residue in peptide should be replaced on the basis of systematic exploration of its position.

  9. Ca2+-stabilized adhesin helps an Antarctic bacterium reach out and bind ice

    PubMed Central

    Vance, Tyler D. R.; Olijve, Luuk L. C.; Campbell, Robert L.; Voets, Ilja K.; Davies, Peter L.; Guo, Shuaiqi

    2014-01-01

    The large size of a 1.5-MDa ice-binding adhesin [MpAFP (Marinomonas primoryensis antifreeze protein)] from an Antarctic Gram-negative bacterium, M. primoryensis, is mainly due to its highly repetitive RII (Region II). MpAFP_RII contains roughly 120 tandem copies of an identical 104-residue repeat. We have previously determined that a single RII repeat folds as a Ca2+-dependent immunoglobulin-like domain. Here, we solved the crystal structure of RII tetra-tandemer (four tandem RII repeats) to a resolution of 1.8 Å. The RII tetra-tandemer reveals an extended (~190-Å × ~25-Å), rod-like structure with four RII-repeats aligned in series with each other. The inter-repeat regions of the RII tetra-tandemer are strengthened by Ca2+ bound to acidic residues. SAXS (small-angle X-ray scattering) profiles indicate the RII tetra-tandemer is significantly rigidified upon Ca2+ binding, and that the protein's solution structure is in excellent agreement with its crystal structure. We hypothesize that >600 Ca2+ help rigidify the chain of ~120 104-residue repeats to form a ~0.6 μm rod-like structure in order to project the ice-binding domain of MpAFP away from the bacterial cell surface. The proposed extender role of RII can help the strictly aerobic, motile bacterium bind ice in the upper reaches of the Antarctic lake where oxygen and nutrients are most abundant. Ca2+-induced rigidity of tandem Ig-like repeats in large adhesins might be a general mechanism used by bacteria to bind to their substrates and help colonize specific niches. PMID:24892750

  10. Variation in Biofilm Stability with Decreasing pH Affects Porous Medium Hydraulic Properties

    NASA Astrophysics Data System (ADS)

    Kirk, M. F.; Santillan, E. F.; McGrath, L. K.; Altman, S. J.

    2010-12-01

    Changes to microbial communities caused by subsurface CO2 injection may have many consequences, including possible impacts to CO2 transport. We used column experiments to examine how decreasing pH, a geochemical change associated with CO2 injection, will affect biofilm stability and ultimately the hydraulic properties of porous media. Columns consisted of 1 mm2 square capillary tubes filled with 105-150 µm diameter glass beads. Artificial groundwater medium containing 1 mM glucose was pumped through the columns at a rate of 0.01 mL/min (q = 14.4 m/day; Re = 0.03). Columns were inoculated with 3 × 10^8 CFU (avg.) of Pseudomonas fluorescens, a model biofilm former, transformed with a green fluorescent protein. Biomass distribution and transport was examined using scanning laser confocal microscopy and effluent plating. Variation in the bulk hydraulic properties of the columns was measured using manometers. In an initial experiment, biofilm growth was allowed to occur for seven days in medium with pH 7.3. Within this period, cells uniformly coated bead surfaces, effluent cell numbers stabilized at 1 × 10^9 CFU/mL, and hydraulic conductivity (K) decreased 77%. Next, medium with pH 4 was introduced. As a result, biomass within the reactor redistributed from bead surfaces to pores, effluent cell numbers decreased to 3 × 10^5 CFU/mL, and K decreased even further (>94% reduction). This decreased K was maintained until the experiment was terminated, seven days after introducing low pH medium. These results suggest that changes in biomass distribution as a result of decreased pH may initially limit transport of solubility-trapped CO2 following CO2 injection. Experiments in progress and planned will test this result in more detail and over longer periods of time. This material is based upon work supported as part of the Center for Frontiers of Subsurface Energy Security, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office

  11. Ca2+-dependent Calmodulin Binding to FcRn Affects Immunoglobulin G Transport in the Transcytotic Pathway

    PubMed Central

    Dickinson, Bonny L.; Claypool, Steven M.; D'Angelo, June A.; Aiken, Martha L.; Venu, Nanda; Yen, Elizabeth H.; Wagner, Jessica S.; Borawski, Jason A.; Pierce, Amy T.; Hershberg, Robert; Blumberg, Richard S.

    2008-01-01

    The Fcγ receptor FcRn transports immunoglobulin G (IgG) so as to avoid lysosomal degradation and to carry it bidirectionally across epithelial barriers to affect mucosal immunity. Here, we identify a calmodulin-binding site within the FcRn cytoplasmic tail that affects FcRn trafficking. Calmodulin binding to the FcRn tail is direct, calcium-dependent, reversible, and specific to residues comprising a putative short amphipathic α-helix immediately adjacent to the membrane. FcRn mutants with single residue substitutions in this motif, or FcRn mutants lacking the cytoplasmic tail completely, exhibit a shorter half-life and attenuated transcytosis. Chemical inhibitors of calmodulin phenocopy the mutant FcRn defect in transcytosis. These results suggest a novel mechanism for regulation of IgG transport by calmodulin-dependent sorting of FcRn and its cargo away from a degradative pathway and into a bidirectional transcytotic route. PMID:18003977

  12. A functional MiR-124 binding-site polymorphism in IQGAP1 affects human cognitive performance.

    PubMed

    Yang, Lixin; Zhang, Rui; Li, Ming; Wu, Xujun; Wang, Jianhong; Huang, Lin; Shi, Xiaodong; Li, Qingwei; Su, Bing

    2014-01-01

    As a product of the unique evolution of the human brain, human cognitive performance is largely a collection of heritable traits. Rather surprisingly, to date there have been no reported cases to highlight genes that underwent adaptive evolution in humans and which carry polymorphisms that have a marked effect on cognitive performance. IQ motif containing GTPase activating protein 1 (IQGAP1), a scaffold protein, affects learning and memory in a dose-dependent manner. Its expression is regulated by miR-124 through the binding sites in the 3'UTR, where a SNP (rs1042538) exists in the core-binding motif. Here we showed that this SNP can influence the miR-target interaction both in vitro and in vivo. Individuals carrying the derived T alleles have higher IQGAP1 expression in the brain as compared to the ancestral A allele carriers. We observed a significant and male-specific association between rs1042538 and tactile performances in two independent cohorts. Males with the derived allele displayed higher tactual performances as compared to those with the ancestral allele. Furthermore, we found a highly diverged allele-frequency distribution of rs1042538 among world human populations, likely caused by natural selection and/or recent population expansion. These results suggest that current human populations still carry sequence variations that affect cognitive performances and that these genetic variants may likely have been subject to comparatively recent natural selection. PMID:25222038

  13. Starch-binding domain affects catalysis in two Lactobacillus alpha-amylases.

    PubMed

    Rodríguez-Sanoja, R; Ruiz, B; Guyot, J P; Sanchez, S

    2005-01-01

    A new starch-binding domain (SBD) was recently described in alpha-amylases from three lactobacilli (Lactobacillus amylovorus, Lactobacillus plantarum, and Lactobacillus manihotivorans). Usually, the SBD is formed by 100 amino acids, but the SBD sequences of the mentioned lactobacillus alpha-amylases consist of almost 500 amino acids that are organized in tandem repeats. The three lactobacillus amylase genes share more than 98% sequence identity. In spite of this identity, the SBD structures seem to be quite different. To investigate whether the observed differences in the SBDs have an effect on the hydrolytic capability of the enzymes, a kinetic study of L. amylovorus and L. plantarum amylases was developed, with both enzymes acting on several starch sources in granular and gelatinized forms. Results showed that the amylolytic capacities of these enzymes are quite different; the L. amylovorus alpha-amylase is, on average, 10 times more efficient than the L. plantarum enzyme in hydrolyzing all the tested polymeric starches, with only a minor difference in the adsorption capacities. PMID:15640201

  14. Starch-Binding Domain Affects Catalysis in Two Lactobacillus α-Amylases

    PubMed Central

    Rodríguez-Sanoja, R.; Ruiz, B.; Guyot, J. P.; Sanchez, S.

    2005-01-01

    A new starch-binding domain (SBD) was recently described in α-amylases from three lactobacilli (Lactobacillus amylovorus, Lactobacillus plantarum, and Lactobacillus manihotivorans). Usually, the SBD is formed by 100 amino acids, but the SBD sequences of the mentioned lactobacillus α-amylases consist of almost 500 amino acids that are organized in tandem repeats. The three lactobacillus amylase genes share more than 98% sequence identity. In spite of this identity, the SBD structures seem to be quite different. To investigate whether the observed differences in the SBDs have an effect on the hydrolytic capability of the enzymes, a kinetic study of L. amylovorus and L. plantarum amylases was developed, with both enzymes acting on several starch sources in granular and gelatinized forms. Results showed that the amylolytic capacities of these enzymes are quite different; the L. amylovorus α-amylase is, on average, 10 times more efficient than the L. plantarum enzyme in hydrolyzing all the tested polymeric starches, with only a minor difference in the adsorption capacities. PMID:15640201

  15. Proteinase 3 Is a Phosphatidylserine-binding Protein That Affects the Production and Function of Microvesicles.

    PubMed

    Martin, Katherine R; Kantari-Mimoun, Chahrazade; Yin, Min; Pederzoli-Ribeil, Magali; Angelot-Delettre, Fanny; Ceroi, Adam; Grauffel, Cédric; Benhamou, Marc; Reuter, Nathalie; Saas, Philippe; Frachet, Philippe; Boulanger, Chantal M; Witko-Sarsat, Véronique

    2016-05-13

    Proteinase 3 (PR3), the autoantigen in granulomatosis with polyangiitis, is expressed at the plasma membrane of resting neutrophils, and this membrane expression increases during both activation and apoptosis. Using surface plasmon resonance and protein-lipid overlay assays, this study demonstrates that PR3 is a phosphatidylserine-binding protein and this interaction is dependent on the hydrophobic patch responsible for membrane anchorage. Molecular simulations suggest that PR3 interacts with phosphatidylserine via a small number of amino acids, which engage in long lasting interactions with the lipid heads. As phosphatidylserine is a major component of microvesicles (MVs), this study also examined the consequences of this interaction on MV production and function. PR3-expressing cells produced significantly fewer MVs during both activation and apoptosis, and this reduction was dependent on the ability of PR3 to associate with the membrane as mutating the hydrophobic patch restored MV production. Functionally, activation-evoked MVs from PR3-expressing cells induced a significantly larger respiratory burst in human neutrophils compared with control MVs. Conversely, MVs generated during apoptosis inhibited the basal respiratory burst in human neutrophils, and those generated from PR3-expressing cells hampered this inhibition. Given that membrane expression of PR3 is increased in patients with granulomatosis with polyangiitis, MVs generated from neutrophils expressing membrane PR3 may potentiate oxidative damage of endothelial cells and promote the systemic inflammation observed in this disease. PMID:26961880

  16. A Varp-Binding Protein, RACK1, Regulates Dendrite Outgrowth through Stabilization of Varp Protein in Mouse Melanocytes.

    PubMed

    Marubashi, Soujiro; Ohbayashi, Norihiko; Fukuda, Mitsunori

    2016-08-01

    Varp (VPS9-ankyrin repeat protein) in melanocytes is thought to function as a key player in the pigmentation of mammals. Varp regulates two different melanocyte functions: (i) transport of melanogenic enzymes to melanosomes by functioning as a Rab32/38 effector and (ii) promotion of dendrite outgrowth by functioning as a Rab21-guanine nucleotide exchange factor. The Varp protein level has recently been shown to be negatively regulated by proteasomal degradation through interaction of the ankyrin repeat 2 (ANKR2) domain of Varp with Rab40C. However, the molecular mechanisms by which Varp escapes from Rab40C and retains its own expression level remain completely unknown. Here, we identified RACK1 (receptor of activated protein kinase C 1) as a Varp-ANKR2 binding partner and investigated its involvement in Varp stabilization in mouse melanocytes. The results showed that knockdown of endogenous RACK1 in melanocytes caused dramatic reduction of the Varp protein level and inhibition of dendrite outgrowth, and intriguingly, overexpression of RACK1 inhibited the interaction between Varp and Rab40C and counteracted the negative effect of Rab40C on dendrite outgrowth. These findings indicated that RACK1 competes with Rab40C for binding to the ANKR2 domain of Varp and regulates dendrite outgrowth through stabilization of Varp in mouse melanocytes. PMID:27066885

  17. A theoretical study of the stability of DNA binding with cis/trans platin.

    PubMed

    Srivastava, Seema; Khan, Irfan Ali; Srivastava, Shinoo; Gupta, Vishwambhar Dayal

    2004-12-01

    Both cis- and trans-platins are known to form intra- and interstrand cross-linking with DNA. Since the nature and strength of binding is different, it makes their efficacy as anti-tumour drug different. In the present communication, we report theoretical analysis by using an amended Zimm and Bragg theory, to explain the melting behaviour and heat capacity of DNA with and without platin binding. The sharpness of transition has been examined in terms of half width and sensitivity parameter (deltaH/sigma). The experimental measurements of Pilch et al (J Mol Biol 2000, 296, 803) and Ctirad and Brabec (J Biol Chem 2001, 276, 9655) have been used. PMID:22900359

  18. Metabolic rate, latitude and thermal stability of roosts, but not phylogeny, affect rewarming rates of bats.

    PubMed

    Menzies, Allyson K; Webber, Quinn M R; Baloun, Dylan E; McGuire, Liam P; Muise, Kristina A; Coté, Damien; Tinkler, Samantha; Willis, Craig K R

    2016-10-01

    Torpor is an adaptation that allows many endotherms to save energy by abandoning the energetic cost of maintaining elevated body temperatures. Although torpor reduces energy consumption, the metabolic heat production required to arouse from torpor is energetically expensive and can impact the overall cost of torpor. The rate at which rewarming occurs can impact the cost of arousal, therefore, factors influencing rewarming rates of heterothermic endotherms could have influenced the evolution of rewarming rates and overall energetic costs of arousal from torpor. Bats are a useful taxon for studies of ecological and behavioral correlates of rewarming rate because of the widespread expression of heterothermy and ecological diversity across the >1200 known species. We used a comparative analysis of 45 bat species to test the hypothesis that ecological, behavioral, and physiological factors affect rewarming rates. We used basal metabolic rate (BMR) as an index of thermogenic capacity, and local climate (i.e., latitude of geographic range), roost stability and maximum colony size as ecological and behavioral predictors of rewarming rate. After controlling for phylogeny, high BMR was associated with rapid rewarming while species that live at higher absolute latitudes and in less thermally stable roosts also rewarmed most rapidly. These patterns suggests that some bat species rely on passive rewarming and social thermoregulation to reduce costs of rewarming, while others might rely on thermogenic capacity to maintain rapid rewarming rates in order to reduce energetic costs of arousal. Our results highlight species-specific traits associated with maintaining positive energy balance in a wide range of climates, while also providing insight into possible mechanisms underlying the evolution of heterothermy in endotherms. PMID:27317837

  19. A cyst nematode effector binds to diverse plant proteins, increases nematode susceptibility and affects root morphology.

    PubMed

    Pogorelko, Gennady; Juvale, Parijat S; Rutter, William B; Hewezi, Tarek; Hussey, Richard; Davis, Eric L; Mitchum, Melissa G; Baum, Thomas J

    2016-08-01

    Cyst nematodes are plant-parasitic roundworms that are of significance in many cropping systems around the world. Cyst nematode infection is facilitated by effector proteins secreted from the nematode into the plant host. The cDNAs of the 25A01-like effector family are novel sequences that were isolated from the oesophageal gland cells of the soybean cyst nematode (Heterodera glycines). To aid functional characterization, we identified an orthologous member of this protein family (Hs25A01) from the closely related sugar beet cyst nematode H. schachtii, which infects Arabidopsis. Constitutive expression of the Hs25A01 CDS in Arabidopsis plants caused a small increase in root length, accompanied by up to a 22% increase in susceptibility to H. schachtii. A plant-expressed RNA interference (RNAi) construct targeting Hs25A01 transcripts in invading nematodes significantly reduced host susceptibility to H. schachtii. These data document that Hs25A01 has physiological functions in planta and a role in cyst nematode parasitism. In vivo and in vitro binding assays confirmed the specific interactions of Hs25A01 with an Arabidopsis F-box-containing protein, a chalcone synthase and the translation initiation factor eIF-2 β subunit (eIF-2bs), making these proteins probable candidates for involvement in the observed changes in plant growth and parasitism. A role of eIF-2bs in the mediation of Hs25A01 virulence function is further supported by the observation that two independent eIF-2bs Arabidopsis knock-out lines were significantly more susceptible to H. schachtii. PMID:26575318

  20. Mod5 protein binds to tRNA gene complexes and affects local transcriptional silencing

    PubMed Central

    Pratt-Hyatt, Matthew; Pai, Dave A.; Haeusler, Rebecca A.; Wozniak, Glenn G.; Good, Paul D.; Miller, Erin L.; McLeod, Ian X.; Yates, John R.; Hopper, Anita K.; Engelke, David R.

    2013-01-01

    The tRNA gene-mediated (tgm) silencing of RNA polymerase II promoters is dependent on subnuclear clustering of the tRNA genes, but genetic analysis shows that the silencing requires additional mechanisms. We have identified proteins that bind tRNA gene transcription complexes and are required for tgm silencing but not required for gene clustering. One of the proteins, Mod5, is a tRNA modifying enzyme that adds an N6-isopentenyl adenosine modification at position 37 on a small number of tRNAs in the cytoplasm, although a subpopulation of Mod5 is also found in the nucleus. Recent publications have also shown that Mod5 has tumor suppressor characteristics in humans as well as confers drug resistance through prion-like misfolding in yeast. Here, we show that a subpopulation of Mod5 associates with tRNA gene complexes in the nucleolus. This association occurs and is required for tgm silencing regardless of whether the pre-tRNA transcripts are substrates for Mod5 modification. In addition, Mod5 is bound to nuclear pre-tRNA transcripts, although they are not substrates for the A37 modification. Lastly, we show that truncation of the tRNA transcript to remove the normal tRNA structure also alleviates silencing, suggesting that synthesis of intact pre-tRNAs is required for the silencing mechanism. These results are discussed in light of recent results showing that silencing near tRNA genes also requires chromatin modification. PMID:23898186

  1. Testing the Coulomb/Accessible Surface Area solvent model for protein stability, ligand binding, and protein design

    PubMed Central

    am Busch, Marcel Schmidt; Lopes, Anne; Amara, Najette; Bathelt, Christine; Simonson, Thomas

    2008-01-01

    Background Protein structure prediction and computational protein design require efficient yet sufficiently accurate descriptions of aqueous solvent. We continue to evaluate the performance of the Coulomb/Accessible Surface Area (CASA) implicit solvent model, in combination with the Charmm19 molecular mechanics force field. We test a set of model parameters optimized earlier, and we also carry out a new optimization in this work, using as a target a set of experimental stability changes for single point mutations of various proteins and peptides. The optimization procedure is general, and could be used with other force fields. The computation of stability changes requires a model for the unfolded state of the protein. In our approach, this state is represented by tripeptide structures of the sequence Ala-X-Ala for each amino acid type X. We followed an iterative optimization scheme which, at each cycle, optimizes the solvation parameters and a set of tripeptide structures for the unfolded state. This protocol uses a set of 140 experimental stability mutations and a large set of tripeptide conformations to find the best tripeptide structures and solvation parameters. Results Using the optimized parameters, we obtain a mean unsigned error of 2.28 kcal/mol for the stability mutations. The performance of the CASA model is assessed by two further applications: (i) calculation of protein-ligand binding affinities and (ii) computational protein design. For these two applications, the previous parameters and the ones optimized here give a similar performance. For ligand binding, we obtain reasonable agreement with a set of 55 experimental mutation data, with a mean unsigned error of 1.76 kcal/mol with the new parameters and 1.47 kcal/mol with the earlier ones. We show that the optimized CASA model is not inferior to the Generalized Born/Surface Area (GB/SA) model for the prediction of these binding affinities. Likewise, the new parameters perform well for the design of 8

  2. Four novel cystic fibrosis mutations in splice junction sequences affecting the CFTR nucleotide binding folds

    SciTech Connect

    Doerk, T.; Wulbrand, U.; Tuemmler, B. )

    1993-03-01

    Single cases of the four novel splice site mutations 1525[minus]1 G [r arrow] A (intron 9), 3601[minus]2 A [r arrow] G (intron 18), 3850[minus]3 T [r arrow] G (intron 19), and 4374+1 G [r arrow] T (intron 23) were detected in the CFTR gene of cystic fibrosis patients of Indo-Iranian, Turkish, Polish, and Germany descent. The nucleotide substitutions at the +1, [minus]1, and [minus]2 positions all destroy splice sites and lead to severe disease alleles associated with features typical of gastrointestinal and pulmonary cystic fibrosis disease. The 3850[minus]3 T-to-G change was discovered in a very mildly affected 33-year-old [Delta]F508 compound heterozygote, suggesting that the T-to-G transversion at the less conserved [minus]3 position of the acceptor splice site may retain some wildtype function. 13 refs., 1 fig., 2 tabs.

  3. A novel RNA binding protein affects rbcL gene expression and is specific to bundle sheath chloroplasts in C4 plants

    PubMed Central

    2013-01-01

    Background Plants that utilize the highly efficient C4 pathway of photosynthesis typically possess kranz-type leaf anatomy that consists of two morphologically and functionally distinct photosynthetic cell types, the bundle sheath (BS) and mesophyll (M) cells. These two cell types differentially express many genes that are required for C4 capability and function. In mature C4 leaves, the plastidic rbcL gene, encoding the large subunit of the primary CO2 fixation enzyme Rubisco, is expressed specifically within BS cells. Numerous studies have demonstrated that BS-specific rbcL gene expression is regulated predominantly at post-transcriptional levels, through the control of translation and mRNA stability. The identification of regulatory factors associated with C4 patterns of rbcL gene expression has been an elusive goal for many years. Results RLSB, encoded by the nuclear RLSB gene, is an S1-domain RNA binding protein purified from C4 chloroplasts based on its specific binding to plastid-encoded rbcL mRNA in vitro. Co-localized with LSU to chloroplasts, RLSB is highly conserved across many plant species. Most significantly, RLSB localizes specifically to leaf bundle sheath (BS) cells in C4 plants. Comparative analysis using maize (C4) and Arabidopsis (C3) reveals its tight association with rbcL gene expression in both plants. Reduced RLSB expression (through insertion mutation or RNA silencing, respectively) led to reductions in rbcL mRNA accumulation and LSU production. Additional developmental effects, such as virescent/yellow leaves, were likely associated with decreased photosynthetic function and disruption of associated signaling networks. Conclusions Reductions in RLSB expression, due to insertion mutation or gene silencing, are strictly correlated with reductions in rbcL gene expression in both maize and Arabidopsis. In both plants, accumulation of rbcL mRNA as well as synthesis of LSU protein were affected. These findings suggest that specific accumulation

  4. Soil aggregate stability as affected by clay mineralogy and polyacrylamide addition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The addition of polyacrylamide (PAM) to soil leads to stabilization of existing aggregates and improved bonding between, and aggregation of adjacent soil particles However, the dependence of PAM efficacy as an aggregate stabilizing agent on soil-clay mineralogy has not been studied. Sixteen soil sam...

  5. Soil-Structural Stability as Affected by Clay Mineralogy, Soil Texture and Polyacrylamide Application

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soil-structural stability (expressed in terms of aggregate stability and pore size distribution) depends on (i) soil inherent properties, (ii) extrinsic condition prevailing in the soil that may vary temporally and spatially, and (iii) addition of soil amendments. Different soil management practices...

  6. SOIL AGGREGATE STABILITY AS AFFECTED BY LONG-TERM TILLAGE AND CLAY TYPE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soil aggregate stability and dispersivity depend on clay mineralogy. However, little is known about the effect of soil mineralogy on soil crustability for long-term cultivated soil. The effect of long-term tillage on aggregate stability was the objective of our study. More than 20 soil samples chara...

  7. Are herbage yield and yield stability affected by plant species diversity in sown pasture mixtures?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A tenet of plant biodiversity theory in grasslands is that increased diversity contributes to the stability of ecosystems. In managed grasslands, such as pastures, greater stability of herbage production as a result of increased plant species diversity would be beneficial. In this study, I combined ...

  8. New index for the stability of a type I collagen affected by hydrophobic environment.

    PubMed

    Nezu, Takashi; Morikawa, Tomohiro; Sasaki, Kaori; Saitoh, Setsuo; Taira, Masayuki; Terada, Yoshihiro; Araki, Yoshima

    2007-05-01

    Effects of hydrophobic environment adjusted by various alcohols on the structural stability of calfskin collagen (CSC) were studied to elucidate the nature of collagen-monomer interaction in adhesion. The stability of CSC in aqueous alcohol solutions was represented by its denaturation temperature, Td, measured by DSC. The hydrophobicity of the alcohol solutions was quantified with their specific dielectric constants, epsilon(r), calculated from their concentrations. The effect of each alcohol to stabilize or destabilize CSC was evaluated by the initial slope of each Td vs. epsilon(r) plot, denoted as -(dTd/d epsilon(r))ini and termed as stabilization power. Results showed that a hydrophobic environment with a smaller epsilon(r) lowered the stabilization power. Stabilization power ranged from -3 (strong destabilization) for phenol (epsilon(r) =12) to +0.3 (weak stabilization) for glycerol (epsilon(r)=47). In view of the encouraging results obtained in this study, the new index was therefore helpful in predicting the effects of new dental materials of known epsilon(r) values on the stability of dentinal collagen. PMID:17694747

  9. Structural and Dynamics Studies of Pax5 Reveal Asymmetry in Stability and DNA Binding by the Paired Domain.

    PubMed

    Perez-Borrajero, Cecilia; Okon, Mark; McIntosh, Lawrence P

    2016-06-01

    The eukaryotic transcription factor Pax5 or B-cell specific activator protein (BSAP) is central to B-cell development and has been implicated in a large number of cellular malignancies resulting from loss- or gain-of-function mutations. In this study, we characterized the DNA-binding Paired domain (PD) of Pax5 in its free and DNA-bound forms using NMR spectroscopy. In isolation, the PD folds as two independent helical bundle subdomains separated by a conformationally disordered linker. The two subdomains differ in stability, with the C-terminal subdomain (CTD) being ~10-fold more protected from amide hydrogen exchange (HX) than the N-terminal subdomain (NTD). Upon binding DNA, the linker and an induced N-terminal β-hairpin become ordered with significantly dampened motions and increased HX protection. Both subdomains of the PD contribute to specific DNA binding, resulting in an equilibrium dissociation constant more than three orders of magnitude lower than exhibited by the separate subdomains for their respective half-sites (nM versus μM). The isolated CTD binds non-specific DNA sequences with only ~10-fold weaker affinity than cognate sequences. In contrast, the NTD associates very poorly with non-specific DNA. We propose that the more stable CTD has evolved to provide relatively low affinity non-specific contacts with DNA. In contrast, the more dynamic NTD discriminates between cognate and non-specific sites. The distinct roles of the PD subdomains may enable efficient searching of genomic DNA by Pax5 while retaining specificity for functional regulatory sites. PMID:27067111

  10. Characterization and small-molecule stabilization of the multisite tandem binding between 14-3-3 and the R domain of CFTR.

    PubMed

    Stevers, Loes M; Lam, Chan V; Leysen, Seppe F R; Meijer, Femke A; van Scheppingen, Daphne S; de Vries, Rens M J M; Carlile, Graeme W; Milroy, Lech G; Thomas, David Y; Brunsveld, Luc; Ottmann, Christian

    2016-03-01

    Cystic fibrosis is a fatal genetic disease, most frequently caused by the retention of the CFTR (cystic fibrosis transmembrane conductance regulator) mutant protein in the endoplasmic reticulum (ER). The binding of the 14-3-3 protein to the CFTR regulatory (R) domain has been found to enhance CFTR trafficking to the plasma membrane. To define the mechanism of action of this protein-protein interaction, we have examined the interaction in vitro. The disordered multiphosphorylated R domain contains nine different 14-3-3 binding motifs. Furthermore, the 14-3-3 protein forms a dimer containing two amphipathic grooves that can potentially bind these phosphorylated motifs. This results in a number of possible binding mechanisms between these two proteins. Using multiple biochemical assays and crystal structures, we show that the interaction between them is governed by two binding sites: The key binding site of CFTR (pS768) occupies one groove of the 14-3-3 dimer, and a weaker, secondary binding site occupies the other binding groove. We show that fusicoccin-A, a natural-product tool compound used in studies of 14-3-3 biology, can stabilize the interaction between 14-3-3 and CFTR by selectively interacting with a secondary binding motif of CFTR (pS753). The stabilization of this interaction stimulates the trafficking of mutant CFTR to the plasma membrane. This definition of the druggability of the 14-3-3-CFTR interface might offer an approach for cystic fibrosis therapeutics. PMID:26888287

  11. Size-dependent stability toward dissociation and ligand binding energies of phosphine-ligated gold cluster ions

    SciTech Connect

    Johnson, Grant E.; Priest, Thomas A.; Laskin, Julia

    2014-01-01

    The stability of sub-nanometer size gold clusters ligated with organic molecules is of paramount importance to the scalable synthesis of monodisperse size-selected metal clusters with highly tunable chemical and physical properties. For the first time, a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR-MS) equipped with surface induced dissociation (SID) has been employed to investigate the time and collision energy resolved fragmentation behavior of cationic doubly charged gold clusters containing 7-9 gold atoms and 6-7 triphenylphosphine (TPP) ligands prepared by reduction synthesis in solution. The TPP ligated gold clusters are demonstrated to fragment through three primary dissociation pathways: (1) Loss of a neutral TPP ligand from the precursor gold cluster, (2) asymmetric fission and (3) symmetric fission and charge separation of the gold core resulting in formation of complementary pairs of singly charged fragment ions. Threshold energies and activation entropies of these fragmentation pathways have been determined employing Rice-Ramsperger-Kassel-Marcus (RRKM) modeling of the experimental SID data. It is demonstrated that the doubly charged cluster ion containing eight gold atoms and six TPP ligands, (8,6)2+, exhibits exceptional stability compared to the other cationic gold clusters examined in this study due to its large ligand binding energy of 1.76 eV. Our findings demonstrate the dramatic effect of the size and extent of ligation on the gas-phase stability and preferred fragmentation pathways of small TPP-ligated gold clusters.

  12. Interaction Signatures Stabilizing the NAD(P)-Binding Rossmann Fold: A Structure Network Approach

    PubMed Central

    Bhattacharyya, Moitrayee; Upadhyay, Roopali; Vishveshwara, Saraswathi

    2012-01-01

    The fidelity of the folding pathways being encoded in the amino acid sequence is met with challenge in instances where proteins with no sequence homology, performing different functions and no apparent evolutionary linkage, adopt a similar fold. The problem stated otherwise is that a limited fold space is available to a repertoire of diverse sequences. The key question is what factors lead to the formation of a fold from diverse sequences. Here, with the NAD(P)-binding Rossmann fold domains as a case study and using the concepts of network theory, we have unveiled the consensus structural features that drive the formation of this fold. We have proposed a graph theoretic formalism to capture the structural details in terms of the conserved atomic interactions in global milieu, and hence extract the essential topological features from diverse sequences. A unified mathematical representation of the different structures together with a judicious concoction of several network parameters enabled us to probe into the structural features driving the adoption of the NAD(P)-binding Rossmann fold. The atomic interactions at key positions seem to be better conserved in proteins, as compared to the residues participating in these interactions. We propose a “spatial motif” and several “fold specific hot spots” that form the signature structural blueprints of the NAD(P)-binding Rossmann fold domain. Excellent agreement of our data with previous experimental and theoretical studies validates the robustness and validity of the approach. Additionally, comparison of our results with statistical coupling analysis (SCA) provides further support. The methodology proposed here is general and can be applied to similar problems of interest. PMID:23284738

  13. A JUMONJI Protein with E3 Ligase and Histone H3 Binding Activities Affects Transposon Silencing in Arabidopsis.

    PubMed

    Kabelitz, Tina; Brzezinka, Krzysztof; Friedrich, Thomas; Górka, Michał; Graf, Alexander; Kappel, Christian; Bäurle, Isabel

    2016-05-01

    Transposable elements (TEs) make up a large proportion of eukaryotic genomes. As their mobilization creates genetic variation that threatens genome integrity, TEs are epigenetically silenced through several pathways, and this may spread to neighboring sequences. JUMONJI (JMJ) proteins can function as antisilencing factors and prevent silencing of genes next to TEs Whether TE silencing is counterbalanced by the activity of antisilencing factors is still unclear. Here, we characterize JMJ24 as a regulator of TE silencing. We show that loss of JMJ24 results in increased silencing of the DNA transposon AtMu1c, while overexpression of JMJ24 reduces silencing. JMJ24 has a JumonjiC (JmjC) domain and two RING domains. JMJ24 autoubiquitinates in vitro, demonstrating E3 ligase activity of the RING domain(s). JMJ24-JmjC binds the N-terminal tail of histone H3, and full-length JMJ24 binds histone H3 in vivo. JMJ24 activity is anticorrelated with histone H3 Lys 9 dimethylation (H3K9me2) levels at AtMu1c Double mutant analyses with epigenetic silencing mutants suggest that JMJ24 antagonizes histone H3K9me2 and requires H3K9 methyltransferases for its activity on AtMu1c Genome-wide transcriptome analysis indicates that JMJ24 affects silencing at additional TEs Our results suggest that the JmjC domain of JMJ24 has lost demethylase activity but has been retained as a binding domain for histone H3. This is in line with phylogenetic analyses indicating that JMJ24 (with the mutated JmjC domain) is widely conserved in angiosperms. Taken together, this study assigns a role in TE silencing to a conserved JmjC-domain protein with E3 ligase activity, but no demethylase activity. PMID:26979329

  14. Binding of Alkyl Polyglucoside Surfactants to Bacteriorhodopsin and its Relation to Protein Stability

    PubMed Central

    Santonicola, M. Gabriella; Lenhoff, Abraham M.; Kaler, Eric W.

    2008-01-01

    The binding of alkyl polyglucoside surfactants to the integral membrane protein bacteriorhodopsin (BR) and the formation of protein-surfactant complexes are investigated by sedimentation equilibrium via analytical ultracentrifugation and by small-angle neutron scattering (SANS). Contrast variation techniques in SANS enable measurement of the composition of the protein-surfactant complexes and determination of the thickness of the surfactant shell bound to the protein. The results indicate that alkyl polyglucosides can bind to BR as single surfactant layers or as a thicker shell. The thickness of the surfactant shell increases with increasing surfactant tail length, and it is generally unrelated to the aggregation number of the micelles even for a small and predominantly hydrophobic membrane protein such as BR. The aggregation numbers determined by sedimentation equilibrium methods match those measured by SANS, which also allows reconstruction of the shape of the protein-detergent complex. When the surfactant is present as a single layer, the BR loses activity, as measured by absorption spectroscopy, more quickly than it does when the surfactant forms a thicker shell. PMID:18234822

  15. Effect of Lysine Modification on the Stability and Cellular Binding of Human Amyloidogenic Light Chains

    SciTech Connect

    O'Neill, Hugh Michael; Davern, Sandra M.; Murphy, Charles L.; Wall, Jonathan; Deborah, Weiss T.; Solomon, Alan

    2011-01-01

    AL amyloidosis is characterized by the pathologic deposition as fibrils of monoclonal light chains (i.e., Bence Jones proteins [BJPs]) in particular organs and tissues. This phenomenon has been attributed to the presence in amyloidogenic proteins of particular amino acids that cause these molecules to become unstable, as well as post-translational modifications and, in regard to the latter, we have investigated the effect of biotinylation of lysyl residues on cell binding. We utilized an experimental system designed to test if BJPs obtained from patients with AL amyloidosis or, as a control, multiple myeloma (MM), bound human fibroblasts and renal epithelial cells. As documented by fluorescent microscopy and ELISA, the amyloidogenic BJPs, as compared with MM components, bound preferentially and this reactivity increased significantly after chemical modification of their lysyl residues with sulfo-NHS-biotin. Further, based on tryptophan fluorescence and circular dichorism data, it was apparent that their conformation was altered, which we hypothesize exposed a binding site not accessible on the native protein. The results of our studies indicate that post-translational structural modifications of pathologic light chains can enhance their capacity for cellular interaction and thus may contribute to the pathogenesis of AL amyloidosis and multiple myeloma.

  16. DNA Binding by Sgf11 Protein Affects Histone H2B Deubiquitination by Spt-Ada-Gcn5-Acetyltransferase (SAGA)*

    PubMed Central

    Koehler, Christian; Bonnet, Jacques; Stierle, Matthieu; Romier, Christophe; Devys, Didier; Kieffer, Bruno

    2014-01-01

    The yeast Spt-Ada-Gcn5-acetyltransferase (SAGA) complex is a transcription coactivator that contains a histone H2B deubiquitination activity mediated by its Ubp8 subunit. Full enzymatic activity requires the formation of a quaternary complex, the deubiquitination module (DUBm) of SAGA, which is composed of Ubp8, Sus1, Sgf11, and Sgf73. The crystal structures of the DUBm have shed light on the structure/function relationship of this complex. Specifically, both Sgf11 and Sgf73 contain zinc finger domains (ZnF) that appear essential for the DUBm activity. Whereas Sgf73 N-terminal ZnF is important for DUBm stability, Sgf11 C-terminal ZnF appears to be involved in DUBm function. To further characterize the role of these two zinc fingers, we have solved their structure by NMR. We show that, contrary to the previously reported structures, Sgf73 ZnF adopts a C2H2 coordination with unusual tautomeric forms for the coordinating histidines. We further report that the Sgf11 ZnF, but not the Sgf73 ZnF, binds to nucleosomal DNA with a binding interface composed of arginine residues located within the ZnF α-helix. Mutational analyses both in vitro and in vivo provide evidence for the functional relevance of our structural observations. The combined interpretation of our results leads to an uncommon ZnF-DNA interaction between the SAGA DUBm and nucleosomes, thus providing further functional insights into SAGA's epigenetic modulation of the chromatin structure. PMID:24509845

  17. Phenylalanine-Rich Peptides Potently Bind ESAT6, a Virulence Determinant of Mycobacterium tuberculosis, and Concurrently Affect the Pathogen's Growth

    PubMed Central

    Kumar, Krishan; Tharad, Megha; Ganapathy, Swetha; Ram, Geeta; Narayan, Azeet; Khan, Jameel Ahmad; Pratap, Rana; Ghosh, Anamika; Samuchiwal, Sachin Kumar; Kumar, Sushil; Bhalla, Kuhulika; Gupta, Deepti; Natarajan, Krishnamurthy; Singh, Yogendra; Ranganathan, Anand

    2009-01-01

    Background The secretory proteins of Mycobacterium tuberculosis (M. tuberculosis) have been known to be involved in the virulence, pathogenesis as well as proliferation of the pathogen. Among this set, many proteins have been hypothesized to play a critical role at the genesis of the onset of infection, the primary site of which is invariably the human lung. Methodology/Principal Findings During our efforts to isolate potential binding partners of key secretory proteins of M. tuberculosis from a human lung protein library, we isolated peptides that strongly bound the virulence determinant protein Esat6. All peptides were less than fifty amino acids in length and the binding was confirmed by in vivo as well as in vitro studies. Curiously, we found all three binders to be unusually rich in phenylalanine, with one of the three peptides a short fragment of the human cytochrome c oxidase-3 (Cox-3). The most accessible of the three binders, named Hcl1, was shown also to bind to the Mycobacterium smegmatis (M. smegmatis) Esat6 homologue. Expression of hcl1 in M. tuberculosis H37Rv led to considerable reduction in growth. Microarray analysis showed that Hcl1 affects a host of key cellular pathways in M. tuberculosis. In a macrophage infection model, the sets expressing hcl1 were shown to clear off M. tuberculosis in much greater numbers than those infected macrophages wherein the M. tuberculosis was not expressing the peptide. Transmission electron microscopy studies of hcl1 expressing M. tuberculosis showed prominent expulsion of cellular material into the matrix, hinting at cell wall damage. Conclusions/Significance While the debilitating effects of Hcl1 on M. tuberculosis are unrelated and not because of the peptide's binding to Esat6–as the latter is not an essential protein of M. tuberculosis–nonetheless, further studies with this peptide, as well as a closer inspection of the microarray data may shed important light on the suitability of such small phenylalanine

  18. Emotional modulation of control dilemmas: the role of positive affect, reward, and dopamine in cognitive stability and flexibility.

    PubMed

    Goschke, Thomas; Bolte, Annette

    2014-09-01

    Goal-directed action in changing environments requires a dynamic balance between complementary control modes, which serve antagonistic adaptive functions (e.g., to shield goals from competing responses and distracting information vs. to flexibly switch between goals and behavioral dispositions in response to significant changes). Too rigid goal shielding promotes stability but incurs a cost in terms of perseveration and reduced flexibility, whereas too weak goal shielding promotes flexibility but incurs a cost in terms of increased distractibility. While research on cognitive control has long been conducted relatively independently from the study of emotion and motivation, it is becoming increasingly clear that positive affect and reward play a central role in modulating cognitive control. In particular, evidence from the past decade suggests that positive affect not only influences the contents of cognitive processes, but also modulates the balance between complementary modes of cognitive control. In this article we review studies from the past decade that examined effects of induced positive affect on the balance between cognitive stability and flexibility with a focus on set switching and working memory maintenance and updating. Moreover, we review recent evidence indicating that task-irrelevant positive affect and performance-contingent rewards exert different and sometimes opposite effects on cognitive control modes, suggesting dissociations between emotional and motivational effects of positive affect. Finally, we critically review evidence for the popular hypothesis that effects of positive affect may be mediated by dopaminergic modulations of neural processing in prefrontal and striatal brain circuits, and we refine this "dopamine hypothesis of positive affect" by specifying distinct mechanisms by which dopamine may mediate effects of positive affect and reward on cognitive control. We conclude with a discussion of limitations of current research, point to

  19. Co-solvents as stabilizing agents during heterologous overexpression in Escherichia coli - application to chlamydial penicillin-binding protein 6.

    PubMed

    Otten, Christian; De Benedetti, Stefania; Gaballah, Ahmed; Bühl, Henrike; Klöckner, Anna; Brauner, Jarryd; Sahl, Hans-Georg; Henrichfreise, Beate

    2015-01-01

    Heterologous overexpression of foreign proteins in Escherichia coli often leads to insoluble aggregates of misfolded inactive proteins, so-called inclusion bodies. To solve this problem use of chaperones or in vitro refolding procedures are the means of choice. These methods are time consuming and cost intensive, due to additional purification steps to get rid of the chaperons or the process of refolding itself. We describe an easy to use lab-scale method to avoid formation of inclusion bodies. The method systematically combines use of co-solvents, usually applied for in vitro stabilization of biologicals in biopharmaceutical formulation, and periplasmic expression and can be completed in one week using standard equipment in any life science laboratory. Demonstrating the unique power of our method, we overproduced and purified for the first time an active chlamydial penicillin-binding protein, demonstrated its function as penicillin sensitive DD-carboxypeptidase and took a major leap towards understanding the "chlamydial anomaly." PMID:25849314

  20. Denaturation and Oxidative Stability of Hemp Seed (Cannabis sativa L.) Protein Isolate as Affected by Heat Treatment.

    PubMed

    Raikos, Vassilios; Duthie, Garry; Ranawana, Viren

    2015-09-01

    The present study investigated the impact of heat treatments on the denaturation and oxidative stability of hemp seed protein during simulated gastrointestinal digestion (GID). Heat-denatured hemp protein isolate (HPI) solutions were prepared by heating HPI (2 mg/ml, pH 6.8) to 40, 60, 80 and 100 °C for 10 min. Heat-induced denaturation of the protein isolates was monitored by polyacrylamide gel electrophoresis. Heating HPI at temperatures above 80 °C significantly reduced solubility and led to the formation of large protein aggregates. The isolates were then subjected to in vitro GID and the oxidative stability of the generated peptides was investigated. Heating did not significantly affect the formation of oxidation products during GID. The results suggest that heat treatments should ideally remain below 80 °C if heat stability and solubility of HPI are to be preserved. PMID:26142888

  1. Thermal stability and unfolding pathways of hyperthermophilic and mesophilic periplasmic binding proteins studied by molecular dynamics simulation.

    PubMed

    Chen, Lin; Li, Xue; Wang, Ruige; Fang, Fengqin; Yang, Wanli; Kan, Wei

    2016-07-01

    The ribose binding protein (RBP), a sugar-binding periplasmic protein, is involved in the transport and signaling processes in both prokaryotes and eukaryotes. Although several cellular and structural studies have been reported, a description of the thermostability of RBP at the molecular level remains elusive. Focused on the hyperthermophilic Thermoytoga maritima RBP (tmRBP) and mesophilic Escherichia coli homolog (ecRBP), we applied molecular dynamics simulations at four different temperatures (300, 380, 450, and 500 K) to obtain a deeper insight into the structural features responsible for the reduced thermostability of the ecRBP. The simulations results indicate that there are distinct structural differences in the unfolding pathway between the two homologs and the ecRBP unfolds faster than the hyperthermophilic homologs at certain temperatures in accordance with the lower thermal stability found experimentally. Essential dynamics analysis uncovers that the essential subspaces of ecRBP and tmRBP are non-overlapping and these two proteins show different directions of motion within the simulations trajectories. Such an understanding is required for designing efficient proteins with characteristics for a particular application. PMID:26292713

  2. Plakophilins 1 and 3 Bind to FXR1 and Thereby Influence the mRNA Stability of Desmosomal Proteins

    PubMed Central

    Fischer-Kešo, Regina; Breuninger, Sonja; Hofmann, Sarah; Henn, Manuela; Röhrig, Theresa; Ströbel, Philipp; Stoecklin, Georg

    2014-01-01

    Plakophilins 1 and 3 (PKP1/3) are members of the arm repeat family of catenin proteins and serve as structural components of desmosomes, which are important for cell-cell-adhesion. In addition, PKP1/3 occur as soluble proteins outside desmosomes, yet their role in the cytoplasm is not known. We found that cytoplasmic PKP1/3 coprecipitated with the RNA-binding proteins FXR1, G3BP, PABPC1, and UPF1, and these PKP1/3 complexes also comprised desmoplakin and PKP2 mRNAs. Moreover, we showed that the interaction of PKP1/3 with G3BP, PABPC1, and UPF1 but not with FXR1 was RNase sensitive. To address the cytoplasmic function of PKP1/3, we performed gain-and-loss-of-function studies. Both PKP1 and PKP3 knockdown cell lines showed reduced protein and mRNA levels for desmoplakin and PKP2. Whereas global rates of translation were unaffected, desmoplakin and PKP2 mRNA were destabilized. Furthermore, binding of PKP1/3 to FXR1 was RNA independent, and both PKP3 and FXR1 stabilized PKP2 mRNA. Our results demonstrate that cytoplasmic PKP1/3 are components of mRNA ribonucleoprotein particles and act as posttranscriptional regulators of gene expression. PMID:25225333

  3. Covalent binding of sulfamethazine to natural and synthetic humic acids: assessing laccase catalysis and covalent bond stability.

    PubMed

    Gulkowska, Anna; Sander, Michael; Hollender, Juliane; Krauss, Martin

    2013-07-01

    Sulfonamide antibiotics form stable covalent bonds with quinone moieties in organic matter via nucleophilic addition reactions. In this work, we combined analytical electrochemistry with trace analytics to assess the catalytic role of the oxidoreductase laccase in the binding of sulfamethazine (SMZ) to Leonardite humic acid (LHA) and to four synthetic humic acids (SHAs) polymerized from low molecular weight precursors and to determine the stability of the formed bonds. In the absence of laccase, a significant portion of the added SMZ formed covalent bonds with LHA, but only a very small fraction (<0.4%) of the total quinone moieties in LHA reacted. Increasing absolute, but decreasing relative concentrations of SMZ-LHA covalent bonds with increasing initial SMZ concentration suggested that the quinone moieties in LHA covered a wide distribution in reactivity for the nucleophilic addition of SMZ. Laccase catalyzed the formation of covalent bonds by oxidizing unreactive hydroquinone moieties in LHA to reactive, electrophilic quinone moieties, of which a large fraction (5%) reacted with SMZ. Compared to LHA, the SHA showed enhanced covalent bond formation in the absence of laccase, suggesting a higher reactivity of their quinone moieties toward nucleophilic addition. This work supports that binding to soil organic matter (SOM) is an important process governing the fate, bioactivity, and extractability of sulfonamides in soils. PMID:23384282

  4. Monoclonal antibodies that bind the renal Na/sup +//glucose symport system. 2. Stabilization of an active conformation

    SciTech Connect

    Wu, J.S.R.; Lever, J.E.

    1987-09-08

    Conformation-dependent fluorescein isothiocyanate (FITC) labeling of the pig renal Na/sup +//glucose symporter was investigated with specific monoclonal antibodies (MAb's). When renal brush border membranes were pretreated with phenyl isothiocyanate (PITC), washed, and then treated at neutral pH with FITC in the presence of transporter substrates Na/sup +/ and glucose, most of the incorporated fluorescence was associated with a single peak after resolution by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular mass of the FITC-labeled species ranged from 79 to 92 kDa. Labeling of this peak was specifically reduced by 70% if Na/sup +/ and glucose were omitted. Na/sup +/ could not be replaced by K/sup +/, Rb/sup +/, or Li/sup +/. FITC labeling of this peak was also stimulated after incubation of membranes with MAb's known to influence high-affinity phlorizin binding, and stimulation was synergistically increased when MAb's were added in the presence of Na/sup +/ and glucose. Substrate-induced or MAb-induced labeling correlated with inactivation of Na/sup +/-dependent phlorizin binding. MAb's recognized an antigen of 75 kDa in the native membranes whereas substrate-induced FITC labeling was accompanied by loss of antigen recognition and protection from proteolysis. These findings are consistent with a model in which MAb's stabilize a Na/sup +/-induced active conformer of the Na/sup +//glucose symport system.

  5. Comparison of Temperature and Additives Affecting the Stability of the Probiotic Weissella cibaria.

    PubMed

    Kang, Mi-Sun; Kim, Youn-Shin; Lee, Hyun-Chul; Lim, Hoi-Soon; Oh, Jong-Suk

    2012-12-01

    Daily use of probiotic chewing gum might have a beneficial effect on oral health, and it is important that the viability of the probiotics be maintained in this food product. In this study, we examined the stability of probiotic chewing gum containing Weissella cibaria. We evaluated the effects of various factors, including temperature and additives, on the survival of freeze-dried probiotic W. cibaria powder. No changes in viability were detected during storage at 4℃ for 5 months, whereas the viability of bacteria stored at 20℃ decreased. The stability of probiotic chewing gum decreased steadily during storage at 20℃ for 4 weeks. The viability of the freeze-dried W. cibaria mixed with various additives, such as xylitol, sorbitol, menthol, sugar ester, magnesium stearate, and vitamin C, was determined over a 4-week storage period at 20℃. Most of the freeze-dried bacteria except for those mixed with menthol and vitamin C were generally stable during a 3-week storage period. Overall, our study showed that W. cibaria was more stable at 4℃ than that at 20℃. In addition, menthol and vitamin C had a detrimental effect on the storage stability of W. cibaria. This is the first study to examine the effects of various chewing gum additives on the stability of W. cibaria. Further studies will be needed to improve the stability of probiotic bacteria for developing a novel probiotic W. cibaria gum. PMID:23323221

  6. Comparison of Temperature and Additives Affecting the Stability of the Probiotic Weissella cibaria

    PubMed Central

    Kang, Mi-Sun; Kim, Youn-Shin; Lee, Hyun-Chul; Lim, Hoi-Soon

    2012-01-01

    Daily use of probiotic chewing gum might have a beneficial effect on oral health, and it is important that the viability of the probiotics be maintained in this food product. In this study, we examined the stability of probiotic chewing gum containing Weissella cibaria. We evaluated the effects of various factors, including temperature and additives, on the survival of freeze-dried probiotic W. cibaria powder. No changes in viability were detected during storage at 4℃ for 5 months, whereas the viability of bacteria stored at 20℃ decreased. The stability of probiotic chewing gum decreased steadily during storage at 20℃ for 4 weeks. The viability of the freeze-dried W. cibaria mixed with various additives, such as xylitol, sorbitol, menthol, sugar ester, magnesium stearate, and vitamin C, was determined over a 4-week storage period at 20℃. Most of the freeze-dried bacteria except for those mixed with menthol and vitamin C were generally stable during a 3-week storage period. Overall, our study showed that W. cibaria was more stable at 4℃ than that at 20℃. In addition, menthol and vitamin C had a detrimental effect on the storage stability of W. cibaria. This is the first study to examine the effects of various chewing gum additives on the stability of W. cibaria. Further studies will be needed to improve the stability of probiotic bacteria for developing a novel probiotic W. cibaria gum. PMID:23323221

  7. Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124

    SciTech Connect

    Auld, Douglas S.; Lovell, Scott; Thorne, Natasha; Lea, Wendy A.; Maloney, David J.; Shen, Min; Rai, Ganesha; Battaile, Kevin P.; Thomas, Craig J.; Simeonov, Anton; Hanzlik, Robert P.; Inglese, James

    2010-04-07

    Firefly luciferase (FLuc), an ATP-dependent bioluminescent reporter enzyme, is broadly used in chemical biology and drug discovery assays. PTC124 Ataluren; (3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid) discovered in an FLuc-based assay targeting nonsense codon suppression, is an unusually potent FLuc-inhibitor. Paradoxically, PTC124 and related analogs increase cellular FLuc activity levels by posttranslational stabilization. In this study, we show that FLuc inhibition and stabilization is the result of an inhibitory product formed during the FLuc-catalyzed reaction between its natural substrate, ATP, and PTC124. A 2.0 {angstrom} cocrystal structure revealed the inhibitor to be the acyl-AMP mixed-anhydride adduct PTC124-AMP, which was subsequently synthesized and shown to be a high-affinity multisubstrate adduct inhibitor (MAI; KD = 120 pM) of FLuc. Biochemical assays, liquid chromatography/mass spectrometry, and near-attack conformer modeling demonstrate that formation of this novel MAI is absolutely dependent upon the precise positioning and reactivity of a key meta-carboxylate of PTC124 within the FLuc active site. We also demonstrate that the inhibitory activity of PTC124-AMP is relieved by free coenzyme A, a component present at high concentrations in luciferase detection reagents used for cell-based assays. This explains why PTC124 can appear to increase, instead of inhibit, FLuc activity in cell-based reporter gene assays. To our knowledge, this is an unusual example in which the 'off-target' effect of a small molecule is mediated by an MAI mechanism.

  8. Effect of metal binding on the structural stability of pigeon liver malic enzyme.

    PubMed

    Chang, Hui-Chuan; Chou, Wei-Yuan; Chang, Gu-Gang

    2002-02-15

    The cytosolic malic enzyme from the pigeon liver is sensitive to chemical denaturant urea. When monitored by protein intrinsic fluorescence or circular dichroism spectral changes, an unfolding of the enzyme in urea at 25 degrees C and pH 7.4 revealed a biphasic phenomenon with an intermediate state detected at 4-5 m urea. The enzyme activity was activated by urea up to 1 m but was completely lost before the intermediate state was detected. This suggests that the active site region of the enzyme was more sensitive to chemical denaturant than other structural scaffolds. In the presence of 4 mm Mn(2+), the urea denaturation pattern of malic enzyme changed to monophasic. Mn(2+) helped the enzyme to resist phase I urea denaturation. The [urea](0.5) for the enzyme inactivation shifted from 2.2 to 3.8 m. Molecular weight determined by the analytical ultracentrifuge indicated that the tetrameric enzyme was dissociated to dimers in the early stage of phase I denaturation. In the intermediate state at 4-5 m urea, the enzyme showed polymerization. However, the polymer forms were dissociated to unfolded monomers at a urea concentration greater than 6 m. Mn(2+) retarded the polymerization of the malic enzyme. Three mutants of the enzyme with a defective metal ligand (E234Q, D235N, E234Q/D235N) were cloned and purified to homogeneity. These mutant malic enzymes showed a biphasic urea denaturation pattern in the absence or presence of Mn(2+). These results indicate that the Mn(2+) has dual roles in the malic enzyme. The metal ion not only plays a catalytic role in stabilization of the reaction intermediate, enol-pyruvate, but also stabilizes the overall tetrameric protein architecture. PMID:11739398

  9. Molecular basis for the high-affinity binding and stabilization of firefly luciferase by PTC124

    PubMed Central

    Auld, Douglas S.; Lovell, Scott; Thorne, Natasha; Lea, Wendy A.; Maloney, David J.; Shen, Min; Rai, Ganesha; Battaile, Kevin P.; Thomas, Craig J.; Simeonov, Anton; Hanzlik, Robert P.; Inglese, James

    2010-01-01

    Firefly luciferase (FLuc), an ATP-dependent bioluminescent reporter enzyme, is broadly used in chemical biology and drug discovery assays. PTC124 (Ataluren; (3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid) discovered in an FLuc-based assay targeting nonsense codon suppression, is an unusually potent FLuc-inhibitor. Paradoxically, PTC124 and related analogs increase cellular FLuc activity levels by posttranslational stabilization. In this study, we show that FLuc inhibition and stabilization is the result of an inhibitory product formed during the FLuc-catalyzed reaction between its natural substrate, ATP, and PTC124. A 2.0 Å cocrystal structure revealed the inhibitor to be the acyl-AMP mixed-anhydride adduct PTC124-AMP, which was subsequently synthesized and shown to be a high-affinity multisubstrate adduct inhibitor (MAI; KD = 120 pM) of FLuc. Biochemical assays, liquid chromatography/mass spectrometry, and near-attack conformer modeling demonstrate that formation of this novel MAI is absolutely dependent upon the precise positioning and reactivity of a key meta-carboxylate of PTC124 within the FLuc active site. We also demonstrate that the inhibitory activity of PTC124-AMP is relieved by free coenzyme A, a component present at high concentrations in luciferase detection reagents used for cell-based assays. This explains why PTC124 can appear to increase, instead of inhibit, FLuc activity in cell-based reporter gene assays. To our knowledge, this is an unusual example in which the “off-target” effect of a small molecule is mediated by an MAI mechanism. PMID:20194791

  10. Type of packaging affects the colour stability of vitamin E enriched beef.

    PubMed

    Nassu, Renata T; Uttaro, Bethany; Aalhus, Jennifer L; Zawadski, Sophie; Juárez, Manuel; Dugan, Michael E R

    2012-12-01

    Colour stability is a very important parameter for meat retail display, as appearance of the product is the deciding factor for consumers at time of purchase. This study investigated the possibility of extending appearance shelf-life through the combined use of packaging method (overwrapping - OVER, modified atmosphere - MAP, vacuum skin packaging - VSP and a combination of modified atmosphere and vacuum skin packaging - MAPVSP) and antioxidants (vitamin E enriched beef). Retail attributes (appearance, lean colour, % surface discolouration), as well as colour space analysis of images for red, green and blue parameters were measured over 18days. MAPVSP provided the most desirable retail appearance during the first 4days of retail display, while VSP-HB had the best colour stability. Overall, packaging type was more influential than α-tocopherol levels on meat colour stability, although α-tocopherol levels (>4μgg(-1) meat) had a protective effect when using high oxygen packaging methods. PMID:22953936