[125I]2-(2-chloro-4-iodo-phenylamino)-5-methyl-pyrroline (LNP 911), a high-affinity radioligand selective for I1 imidazoline receptors.

PubMed

Greney, Hugues; Urosevic, Dragan; Schann, Stephan; Dupuy, Laurence; Bruban, Véronique; Ehrhardt, Jean-Daniel; Bousquet, Pascal; Dontenwill, Monique

2002-07-01

The I1 subtype of imidazoline receptors (I1R) is a plasma membrane protein that is involved in diverse physiological functions. Available radioligands used so far to characterize the I(1)R were able to bind with similar affinities to alpha2-adrenergic receptors (alpha2-ARs) and to I1R. This feature was a major drawback for an adequate characterization of this receptor subtype. New imidazoline analogs were therefore synthesized and the present study describes one of these compounds, 2-(2-chloro-4-iodo-phenylamino)-5-methyl-pyrroline (LNP 911), which was of high affinity and selectivity for the I1R. LNP 911 was radioiodinated and its binding properties characterized in different membrane preparations. Saturation experiments with [125I]LNP 911 revealed a single high affinity binding site in PC-12 cell membranes (K(D) = 1.4 nM; B(max) = 398 fmol/mg protein) with low nonspecific binding. [125I]LNP 911 specific binding was inhibited by various imidazolines and analogs but was insensitive to guanosine-5'-O-(3-thio)triphosphate. The rank order of potency of some competing ligands [LNP 911, PIC, rilmenidine, 4-chloro-2-(imidazolin-2-ylamino)-isoindoline (BDF 6143), lofexidine, and clonidine] was consistent with the definition of [125I]LNP 911 binding sites as I1R. However, other high-affinity I1R ligands (moxonidine, efaroxan, and benazoline) exhibited low affinities for these binding sites in standard binding assays. In contrast, when [125I]LNP 911 was preincubated at 4 degrees C, competition curves of moxonidine became biphasic. In this case, moxonidine exhibited similar high affinities on [125I]LNP 911 binding sites as on I1R defined with [125I]PIC. Moxonidine proved also able to accelerate the dissociation of [125I]LNP 911 from its binding sites. These results suggest the existence of an allosteric modulation at the level of the I1R, which seems to be corroborated by the dose-dependent enhancement by LNP 911 of the agonist effects on the adenylate cyclase pathway associated to I1R. Because [125I]LNP 911 was unable to bind to the I2 binding site and alpha2AR, our data indicate that [125I]LNP 911 is the first highly selective radioiodinated probe for I1R with a nanomolar affinity. This new tool should facilitate the molecular characterization of the I1 imidazoline receptor.

Engineered Escherichia coli Silver-Binding Periplasmic Protein That Promotes Silver Tolerance

PubMed Central

Hall Sedlak, Ruth; Hnilova, Marketa; Grosh, Carolynn; Fong, Hanson; Baneyx, Francois; Schwartz, Dan; Sarikaya, Mehmet; Tamerler, Candan

2012-01-01

Silver toxicity is a problem that microorganisms face in medical and environmental settings. Through exposure to silver compounds, some bacteria have adapted to growth in high concentrations of silver ions. Such adapted microbes may be dangerous as pathogens but, alternatively, could be potentially useful in nanomaterial-manufacturing applications. While naturally adapted isolates typically utilize efflux pumps to achieve metal resistance, we have engineered a silver-tolerant Escherichia coli strain by the use of a simple silver-binding peptide motif. A silver-binding peptide, AgBP2, was identified from a combinatorial display library and fused to the C terminus of the E. coli maltose-binding protein (MBP) to yield a silver-binding protein exhibiting nanomolar affinity for the metal. Growth experiments performed in the presence of silver nitrate showed that cells secreting MBP-AgBP2 into the periplasm exhibited silver tolerance in a batch culture, while those expressing a cytoplasmic version of the fusion protein or MBP alone did not. Transmission electron microscopy analysis of silver-tolerant cells revealed the presence of electron-dense silver nanoparticles. This is the first report of a specifically engineered metal-binding peptide exhibiting a strong in vivo phenotype, pointing toward a novel ability to manipulate bacterial interactions with heavy metals by the use of short and simple peptide motifs. Engineered metal-ion-tolerant microorganisms such as this E. coli strain could potentially be used in applications ranging from remediation to interrogation of biomolecule-metal interactions in vivo. PMID:22286990

Odorant-binding proteins display high affinities for behavioral attractants and repellents in the natural predator Chrysopa pallens.

PubMed

Li, Zhao-Qun; Zhang, Shuai; Luo, Jun-Yu; Wang, Si-Bao; Dong, Shuang-Lin; Cui, Jin-Jie

2015-07-01

Chrysopa pallens is an important natural predator of various pests in many different cropping systems. Understanding the sophisticated olfactory system of insect antennae is crucial for studying the physiological bases of olfaction and could also help enhance the effectiveness of C. pallens in biological control. However, functional studies of the olfactory genes in C. pallens are still lacking. In this study, we cloned five odorant-binding protein (OBP) genes from C. pallens (CpalOBPs). Quantitative RT-PCR results indicated that the five CpalOBPs had different tissue expression profiles. Ligand-binding assays showed that farnesol, farnesene, cis-3-hexenyl hexanoate, geranylacetone, beta-ionone, octyl aldehyde, decanal, nerolidol (Ki<20 μM), and especially 2-pentadecanone (Ki=1.19 μM) and 2-hexyl-1-decanol (Ki=0.37 μM) strongly bound to CpalOBP2. CpalOBP15 exhibited high binding affinities for beta-ionone, 2-tridecanone, trans-nerolidol, and dodecyl aldehyde. Behavioral trials using the 14 compounds exhibiting high binding affinities for the CpalOBPs revealed that nine were able to elicit significant behavioral responses from C. pallens. Among them, farnesene and its corresponding alcohol, farnesol, elicited remarkable repellent behavioral responses from C. pallens. Our study provides several compounds that could be selected to develop slow-release agents that attract/repel C. pallens and to improve the search for strategies to eliminate insect pests. Copyright © 2015 Elsevier Inc. All rights reserved.

The D-galactose specific lectin of field bean (Dolichos lablab) seed binds sugars with extreme negative cooperativity and half-of-the-sites binding.

PubMed

Rao, Devavratha H; Gowda, Lalitha R

2012-08-15

The field bean (Dolichos lablab) lectin designated as PPO-haemagglutinin (DLL-II) is bifunctional, exhibiting both polyphenol oxidase and haemagglutinating activity. The lectin is unusual in that it binds galactose (Gal), lactose (Lac) and N-acetylgalactosamine (GalNAc) only in the presence of (NH₄)₂SO₄ and exhibits negative cooperativity and half-of-the-sites binding. Circular dichroism, isothermal titration calorimetry and fluorescence quenching were used to assess the sugar binding in the presence of (NH₄)₂O₄. Comparison of the near-UV CD spectra with and without bound sugar revealed ligand induced conformational changes. The intrinsic fluorescence quenching data indicate that DLL-II exhibits weak binding to Gal in the presence of (NH₄)₂SO₄ with a stoichiometry of one bound ligand per dimer. ITC data fitted using a two sets of sites binding model presented a similar picture. The K(a)'s for Gal, Lac and GalNAc in the presence of (NH₄)₂SO₄ were 0.16±0.002, 0.21±0.004 and 8.45±0.78 (×10⁻³) M⁻¹ respectively. The Hill plot for the binding of these sugars to DLL-II was curvilinear with a tangent slope <1.0 indicating negative cooperativity. DLL-II thus exhibits half-of-the-site binding, an extreme form of negative cooperativity in which the second ligand does not bind at all. This is the first report of a legume lectin, exhibiting half-of-the-sites binding. Copyright © 2012 Elsevier Inc. All rights reserved.

Synthesis, hydrolysis rates, supercomputer modeling, and antibacterial activity of bicyclic tetrahydropyridazinones.

PubMed

Jungheim, L N; Boyd, D B; Indelicato, J M; Pasini, C E; Preston, D A; Alborn, W E

1991-05-01

Bicyclic tetrahydropyridazinones, such as 13, where X are strongly electron-withdrawing groups, were synthesized to investigate their antibacterial activity. These delta-lactams are homologues of bicyclic pyrazolidinones 15, which were the first non-beta-lactam containing compounds reported to bind to penicillin-binding proteins (PBPs). The delta-lactam compounds exhibit poor antibacterial activity despite having reactivity comparable to the gamma-lactams. Molecular modeling based on semiempirical molecular orbital calculations on a Cray X-MP supercomputer, predicted that the reason for the inactivity is steric bulk hindering high affinity of the compounds to PBPs, as well as high conformational flexibility of the tetrahydropyridazinone ring hampering effective alignment of the molecule in the active site. Subsequent PBP binding experiments confirmed that this class of compound does not bind to PBPs.

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

Stachel, Shawn J.; Sanders, John M.; Henze, Darrell A.

We have identified several series of small molecule inhibitors of TrkA with unique binding modes. The starting leads were chosen to maximize the structural and binding mode diversity derived from a high throughput screen of our internal compound collection. These leads were optimized for potency and selectivity employing a structure based drug design approach adhering to the principles of ligand efficiency to maximize binding affinity without overly relying on lipophilic interactions. This endeavor resulted in the identification of several small molecule pan-Trk inhibitor series that exhibit high selectivity for TrkA/B/C versus a diverse panel of kinases. We have also demonstratedmore » efficacy in both inflammatory and neuropathic pain models upon oral dosing. Herein we describe the identification process, hit-to-lead progression, and binding profiles of these selective pan-Trk kinase inhibitors.« less

Novel interactions of CAPS (Ca2+-dependent activator protein for secretion) with the three neuronal SNARE proteins required for vesicle fusion.

PubMed

Daily, Neil J; Boswell, Kristin L; James, Declan J; Martin, Thomas F J

2010-11-12

CAPS (aka CADPS) is required for optimal vesicle exocytosis in neurons and endocrine cells where it functions to prime the exocytic machinery for Ca(2+)-triggered fusion. Fusion is mediated by trans complexes of the SNARE proteins VAMP-2, syntaxin-1, and SNAP-25 that bridge vesicle and plasma membrane. CAPS promotes SNARE complex formation on liposomes, but the SNARE binding properties of CAPS are unknown. The current work revealed that CAPS exhibits high affinity binding to syntaxin-1 and SNAP-25 and moderate affinity binding to VAMP-2. CAPS binding is specific for a subset of exocytic SNARE protein isoforms and requires membrane integration of the SNARE proteins. SNARE protein binding by CAPS is novel and mediated by interactions with the SNARE motifs in the three proteins. The C-terminal site for CAPS binding on syntaxin-1 does not overlap the Munc18-1 binding site and both proteins can co-reside on membrane-integrated syntaxin-1. As expected for a C-terminal binding site on syntaxin-1, CAPS stimulates SNARE-dependent liposome fusion with N-terminal truncated syntaxin-1 but exhibits impaired activity with C-terminal syntaxin-1 mutants. Overall the results suggest that SNARE complex formation promoted by CAPS may be mediated by direct interactions of CAPS with each of the three SNARE proteins required for vesicle exocytosis.

Novel Interactions of CAPS (Ca2+-dependent Activator Protein for Secretion) with the Three Neuronal SNARE Proteins Required for Vesicle Fusion*

PubMed Central

Daily, Neil J.; Boswell, Kristin L.; James, Declan J.; Martin, Thomas F. J.

2010-01-01

CAPS (aka CADPS) is required for optimal vesicle exocytosis in neurons and endocrine cells where it functions to prime the exocytic machinery for Ca2+-triggered fusion. Fusion is mediated by trans complexes of the SNARE proteins VAMP-2, syntaxin-1, and SNAP-25 that bridge vesicle and plasma membrane. CAPS promotes SNARE complex formation on liposomes, but the SNARE binding properties of CAPS are unknown. The current work revealed that CAPS exhibits high affinity binding to syntaxin-1 and SNAP-25 and moderate affinity binding to VAMP-2. CAPS binding is specific for a subset of exocytic SNARE protein isoforms and requires membrane integration of the SNARE proteins. SNARE protein binding by CAPS is novel and mediated by interactions with the SNARE motifs in the three proteins. The C-terminal site for CAPS binding on syntaxin-1 does not overlap the Munc18-1 binding site and both proteins can co-reside on membrane-integrated syntaxin-1. As expected for a C-terminal binding site on syntaxin-1, CAPS stimulates SNARE-dependent liposome fusion with N-terminal truncated syntaxin-1 but exhibits impaired activity with C-terminal syntaxin-1 mutants. Overall the results suggest that SNARE complex formation promoted by CAPS may be mediated by direct interactions of CAPS with each of the three SNARE proteins required for vesicle exocytosis. PMID:20826818

Rapid and Reliable Binding Affinity Prediction of Bromodomain Inhibitors: A Computational Study

PubMed Central

2016-01-01

Binding free energies of bromodomain inhibitors are calculated with recently formulated approaches, namely ESMACS (enhanced sampling of molecular dynamics with approximation of continuum solvent) and TIES (thermodynamic integration with enhanced sampling). A set of compounds is provided by GlaxoSmithKline, which represents a range of chemical functionality and binding affinities. The predicted binding free energies exhibit a good Spearman correlation of 0.78 with the experimental data from the 3-trajectory ESMACS, and an excellent correlation of 0.92 from the TIES approach where applicable. Given access to suitable high end computing resources and a high degree of automation, we can compute individual binding affinities in a few hours with precisions no greater than 0.2 kcal/mol for TIES, and no larger than 0.34 and 1.71 kcal/mol for the 1- and 3-trajectory ESMACS approaches. PMID:28005370

The 1.3 A resolution structure of the RNA tridecamer r(GCGUUUGAAACGC): metal ion binding correlates with base unstacking and groove contraction.

PubMed

Timsit, Youri; Bombard, Sophie

2007-12-01

Metal ions play a key role in RNA folding and activity. Elucidating the rules that govern the binding of metal ions is therefore an essential step for better understanding the RNA functions. High-resolution data are a prerequisite for a detailed structural analysis of ion binding on RNA and, in particular, the observation of monovalent cations. Here, the high-resolution crystal structures of the tridecamer duplex r(GCGUUUGAAACGC) crystallized under different conditions provides new structural insights on ion binding on GAAA/UUU sequences that exhibit both unusual structural and functional properties in RNA. The present study extends the repertory of RNA ion binding sites in showing that the two first bases of UUU triplets constitute a specific site for sodium ions. A striking asymmetric pattern of metal ion binding in the two equivalent halves of the palindromic sequence demonstrates that sequence and its environment act together to bind metal ions. A highly ionophilic half that binds six metal ions allows, for the first time, the observation of a disodium cluster in RNA. The comparison of the equivalent halves of the duplex provides experimental evidences that ion binding correlates with structural alterations and groove contraction.

The influence of surface integrin binding patterns on specific biomaterial-cell interactions

NASA Astrophysics Data System (ADS)

Beranek, Maggi Marie

As the future of biomaterials progresses toward bioactivity, the biomaterial surface must control non-specific protein adsorption and encourage selective protein and cell adsorption. Integrins alphavbeta3, alpha 1beta1, alpha5beta1 and alpha Mbeta2 are expressed on cells involved in endothelialization, inflammation, and intimal hyperplasia. These cellular events play a vital role in biomaterial biocompatibility, especially in the vascular environment. The overall hypothesis of these studies is that biomaterial surfaces exhibit selective integrin binding, which then specifies differential cell binding. To test this hypothesis, four specific aims were developed. The first aim was designed to determine whether metal and polymeric biomaterials exhibit selective integrin binding. The tested materials included 316L stainless steel, nitinol, gold, Elgiloy RTM, poly(D, L-lactide-co-glycolide), polycarbonate urethane and expanded polytetrafluoroethylene. Discrete integrin binding patterns were detected microscopically using integrin specific fluorescent antibodies. Stainless steel exhibited high level integrin alpha1beta 1 and low level integrin alphaMbeta2 binding pattern. This suggests that this metal surface should selectively encourage endothelial cell to inflammatory cell binding. In contrast, gold bound ten times the amount of integrin alphaMbeta2 compared to integrin alpha1beta1, which should encourage inflammatory cell adhesion. The 65/35 poly(D, L-lactide-co-glycolide) was the only polymeric biomaterial tested that had integrin binding levels comparable to metal biomaterials. Based on these observations, a combinational biomaterial with a surface pattern of 65/35 poly(D, L-lactide-co-glycolide) dots on a 316L stainless steel background was created. A pattern of high level integrin alpha1beta1 binding and low level integrin alpha Mbeta2 binding on this combinational surface indicates that this surface should selectively favor endothelial cell binding. In the second aim, the response of surface-bound integrins to flow-related shear stress was examined. Based on fluorescent analysis, total alphavbeta 3, alpha1beta1, and alpha5beta 1 appeared to increase on stainless steel after 90-minute low shear stress exposure, whereas only alpha5beta1 appeared to increase when exposed to high shear. 65/35 poly(D, L-lactide-co-glycolide) exhibited increased total binding of alpha5beta1 and alphaMbeta2, when exposed to either shear stress level. Exposure to either shear stress regimen appeared to increase binding of all integrins on the combinational surface. These responses to shear stress suggest differential integrin binding affinity compared to stainless steel. Using antibodies specific to the integrin subunits, the apparent increase in surface-bound integrins was found to be related to a surface disassociation of alpha and beta subunits. The third aim evaluated human aortic endothelial cells and acute monocytic leukemia cells (THP-1) cell binding to the tested biomaterial surfaces under both static and flow conditions. Both stainless steel and the combinational surface had increased endothelial cell binding compared to monocyte attachment. Pre-incubation of the surface with the specific integrins significantly inhibited human aortic endothelial cell binding. Aim four was designed to investigate the influence of surface bound integrins on human aortic endothelial cell migration under shear stress. If biomaterial surface integrin binding patterns are specific, then pre-bound surface integrins should competitively inhibit binding of cellular integrins to the surface. Cell migration distance on to alphavbeta3, alpha 1beta1, and alpha5beta1 pre-incubated stainless steel was decreased ten-fold, and decreased by three-fold on both 65/35 poly(D, L-lactide-coglycolide) and combinational surfaces compared to the respective bare surfaces. In contrast, migration distance on to alphaMbeta2 pre-coated stainless steel and combinational surface was decreased by only sixty percent and only fifty percent on alphaMbeta2 precoated 65/35 poly(D, L -lactide-co-glycolide). These results suggested that surface binding sites are selective and critical in governing endothelial cell migration. In conclusion, these results support the hypothesis that a surface that encourages specific integrin binding would promote differential cell binding. The novel integrin binding model used in this investigation may be a methodology that can be employed to evaluate potential vascular biomaterials.

In Vitro Binding Capacity of Bile Acids by Defatted Corn Protein Hydrolysate

PubMed Central

Kongo-Dia-Moukala, Jauricque Ursulla; Zhang, Hui; Irakoze, Pierre Claver

2011-01-01

Defatted corn protein was digested using five different proteases, Alcalase, Trypsin, Neutrase, Protamex and Flavourzyme, in order to produce bile acid binding peptides. Bile acid binding capacity was analyzed in vitro using peptides from different proteases of defatted corn hydrolysate. Some crystalline bile acids like sodium glycocholate, sodium cholate and sodium deoxycholate were individually tested using HPLC to see which enzymes can release more peptides with high bile acid binding capacity. Peptides from Flavourzyme defatted corn hydrolysate exhibited significantly (p < 0.05) stronger bile acid binding capacity than all others hydrolysates tested and all crystalline bile acids tested were highly bound by cholestyramine, a positive control well known as a cholesterol-reducing agent. The bile acid binding capacity of Flavourzyme hydrolysate was almost preserved after gastrointestinal proteases digestion. The molecular weight of Flavourzyme hydrolysate was determined and most of the peptides were found between 500–180 Da. The results showed that Flavourzyme hydrolysate may be used as a potential cholesterol-reducing agent. PMID:21541043

Synthesis and pharmacological characterization of novel xanthine carboxylate amides as A2A adenosine receptor ligands exhibiting bronchospasmolytic activity.

PubMed

Yadav, Rakesh; Bansal, Ranju; Rohilla, Suman; Kachler, Sonja; Klotz, Karl-Norbert

2016-04-01

The carboxylate amides of 8-phenyl-1,3-dimethylxanthine described herein represent a new series of selective ligands of the adenosine A2A receptors exhibiting bronchospasmolytic activity. The effects of location of 8-phenyl substitutions on the adenosine receptor (AR) binding affinities of the newly synthesized xanthines have also been studied. The compounds displayed moderate to potent binding affinities toward various adenosine receptor subtypes when evaluated through radioligand binding studies. However, most of the compounds showed the maximum affinity for the A2A subtype, some with high selectivity versus all other subtypes. Xanthine carboxylate amide 13b with a diethylaminoethylamino moiety at the para-position of the 8-phenylxanthine scaffold was identified as the most potent A2A adenosine receptor ligand with Ki=0.06μM. Similarly potent and highly A2A-selective are the isovanillin derivatives 16a and 16d. In addition, the newly synthesized xanthine derivatives showed good in vivo bronchospasmolytic activity when tested in guinea pigs. Copyright © 2016 Elsevier Inc. All rights reserved.

Modulation of ouabain binding and potassium pump fluxes by cellular sodium and potassium in human and sheep erythrocytes.

PubMed Central

Joiner, C H; Lauf, P K

1978-01-01

1. Erythrocytes were treated with nystatin to alter internal Na (Nai) and K (Ki) composition. Although the rates of K pumping and [3H]ouabain binding were altered dramatically, the relationship between glycoside binding and K pump inhibition was unaffected. 2. Human cells with high Nai and low Ki exhibited an increased rate of ouabain binding as compared to high Ki, low Nai cells; this paralleled the stimulated K pump activity of high Nai cells. 3. At constant Ki, increasing internal Na stimulated K pump and ouabain binding rates concomitantly. 4. At low Nai, increasing Ki inhibited both K pumping and ouabain binding. However, at high Nai, increasing Ki from 4 to 44 mM stimulated the rate of glycoside binding, parallel to its effect of increasing the rate of active K influx. 5. Anti-L, an isoantibody to low K (LK) sheep red cells, increased the rate of ouabain binding via its stimulation of K pump turnover. Since the latter effect is the result of affinity changes at the internal cation activation site(s) of the pump (Lauf, Rasmusen, Hoffman, Dunham, Cook, Parmelee & Tosteson, 1970), the antibody's effect on ouabain binding reflected the positive correlation between the rates of K pump turnover and glycoside binding. 6. These data provide the first evidence in intact cells for the occurrence of a Nai-induced conformational change in the Na/K pump during its normal operational cycle. PMID:722574

Photocontrol of the expression of genes encoding chlorophyll a/b binding proteins and small subunit of ribulose-1,5-bisphosphate carboxylase in etiolated seedlings of Lycopersicon esculentum (L. ) and Nicotiana tabacum (L. )

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

Wehmeyer, B.; Cashmore, A.R.; Schaefer, E.

Phytochrome and the blue ultraviolet-A photoreceptor control light-induced expression of genes encoding the chlorophyll a/b binding protein of photosystem II and photosystem I and the genes for the small subunit of the ribulose-1,5-bisphosphate carboxylase in etiolated seedlings of Lycopersicon esculentum (tomato) and Nicotiana tabacum (tobacco). A high irradiance response also controls the induction of these genes. Genes encoding photosystem II- and I-associated chlorophyll a/b binding proteins both exhibit a transient rapid increase in expression in response to light pulse or to continuous irradiation. In contrast, genes encoding the small subunit exhibit a continuous increase in expression in response to light.more » These distinct expression characteristics are shown to reflect differences at the level of transcription.« less

Agonist and antagonist actions of antipsychotic agents at 5-HT1A receptors: a [35S]GTPgammaS binding study.

PubMed

Newman-Tancredi, A; Gavaudan, S; Conte, C; Chaput, C; Touzard, M; Verrièle, L; Audinot, V; Millan, M J

1998-08-21

Recombinant human (h) 5-HT1A receptor-mediated G-protein activation was characterised in membranes of transfected Chinese hamster ovary (CHO) cells by use of guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS binding). The potency and efficacy of 21 5-HT receptor agonists and antagonists was determined. The agonists, 5-CT (carboxamidotryptamine) and flesinoxan displayed high affinity (subnanomolar Ki values) and high efficacy (Emax > 90%, relative to 5-HT = 100%). In contrast, ipsapirone, zalospirone and buspirone displayed partial agonist activity. EC50s for agonist stimulation of [35S]GTPgammaS binding correlated well with Ki values from competition binding (r = +0.99). Among the compounds tested for antagonist activity, methiothepin and (+)butaclamol exhibited 'inverse agonist' behaviour, inhibiting basal [35S]GTPgammaS binding. The actions of 17 antipsychotic agents were investigated. Clozapine and several putatively 'atypical' antipsychotic agents, including ziprasidone, quetiapine and tiospirone, exhibited partial agonist activity and marked affinity at h5-HT1A receptors, similar to their affinity at hD2 dopamine receptors. In contrast, risperidone and sertindole displayed low affinity at h5-HT1A receptors and behaved as 'neutral' antagonists, inhibiting 5-HT-stimulated [35S]GTPgammaS binding. Likewise the 'typical' neuroleptics, haloperidol, pimozide, raclopride and chlorpromazine exhibited relatively low affinity and 'neutral' antagonist activity at h5-HT1A receptors with Ki values which correlated with their respective Kb values. The present data show that (i) [35S]GTPgammaS binding is an effective method to evaluate the efficacy and potency of agonists and antagonists at recombinant human 5-HT1A receptors. (ii) Like clozapine, several putatively 'atypical' antipsychotic drugs display balanced serotonin h5-HT1A/dopamine hD2 receptor affinity and partial agonist activity at h5-HT1A receptors. (iii) Several 'typical' and some putatively 'atypical' antipsychotic agents displayed antagonist properties at h5-HT1A sites with generally much lower affinity than at hD2 dopamine receptors. It is suggested that agonist activity at 5-HT1A receptors may be of utility for certain antipsychotic agents.

Autoradiographic analysis of tritiated imipramine binding in the human brain post mortem: effects of suicide

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

Gross-Isseroff, R.; Israeli, M.; Biegon, A.

In vitro quantitative autoradiography of high-affinity tritiated imipramine binding sites was performed on brains of 12 suicide victims and 12 matched controls. Region-specific differences in imipramine binding were found between the two groups. Thus, the pyramidal and molecular layers of the cornu ammoni hippocampal fields and the hilus of the dentate gyrus exhibited 80%, 60%, and 90% increases in binding in the suicide group, respectively. The postcentral cortical gyrus, insular cortex, and claustrum had 45%, 28%, and 75% decreases in binding in the suicide group, respectively. No difference in imipramine binding was observed in prefrontal cortical regions, in the basalmore » ganglia, and in mesencephalic nuclei. No sex and postmortem delay effects on imipramine binding were found. Imipramine binding was positively correlated with age, the effect of age being most pronounced in portions of the basal ganglia and temporal cortex.« less

High-oleic canola oil consumption enriches LDL particle cholesteryl oleate content and reduces LDL proteoglycan binding in humans

PubMed Central

Jones, Peter J. H.; MacKay, Dylan. S.; Senanayake, Vijitha K.; Pu, Shuaihua; Jenkins, David J. A.; Connelly, Philip W.; Lamarche, Benoît; Couture, Patrick; Kris-Etherton, Penny M.; West, Sheila G.; Liu, Xiaoran; Fleming, Jennifer A.; Hantgan, Roy R.; Rudel, Lawrence L.

2015-01-01

Oleic acid consumption is considered cardio-protective according to studies conducted examining effects of the Mediterranean diet. However, animal models have shown that oleic acid consumption increases LDL particle cholesteryl oleate content which is associated with increased LDL-proteoglycan binding and atherosclerosis. The objective was to examine effects of varying oleic, linoleic and docosahexaenoic acid consumption on human LDL-proteoglycan binding in a non-random subset of the Canola Oil Multi-center Intervention Trial (COMIT) participants. COMIT employed a randomized, double-blind, five-period, cross-over trial design. Three of the treatment oil diets; 1) a blend of corn/safflower oil (25:75); 2) high oleic canola oil; and 3) DHA-enriched high oleic canola oil were selected for analysis of LDL-proteoglycan binding in 50 participants exhibiting good compliance. LDL particles were isolated from frozen plasma by gel filtration chromatography and LDL cholesteryl esters quantified by mass-spectrometry. LDL-proteoglycan binding was assessed using surface plasmon resonance. LDL particle cholesterol ester fatty acid composition was sensitive to the treatment fatty acid compositions, with the main fatty acids in the treatments increasing in the LDL cholesterol esters. The corn/safflower oil and high-oleic canola oil diets lowered LDL-proteoglycan binding relative to their baseline values (p=0.0005 and p=0.0012, respectively). At endpoint, high-oleic canola oil feeding resulted in lower LDL-proteoglycan binding than corn/safflower oil (p=0.0243) and DHA-enriched high oleic canola oil (p=0.0249), although high-oleic canola oil had the lowest binding at baseline (p=0.0344). Our findings suggest that high-oleic canola oil consumption in humans increases cholesteryl oleate percentage in LDL, but in a manner not associated with a rise in LDL-proteoglycan binding. PMID:25528432

Functional and Selective Bacterial Interfaces Using Cross-Scaffold Gold Binding Peptides

NASA Astrophysics Data System (ADS)

Adams, Bryn L.; Hurley, Margaret M.; Jahnke, Justin P.; Stratis-Cullum, Dimitra N.

2015-11-01

We investigated the functional and selective activity of three phage-derived gold-binding peptides on the Escherichia coli ( E. coli) bacterial cell surface display scaffold (eCPX) for the first time. Gold-binding peptides, p3-Au12 (LKAHLPPSRLPS), p8#9 (VSGSSPDS), and Midas-2 (TGTSVLIATPYV), were compared side-by-side through experiment and simulation. All exhibited strong binding to an evaporated gold film, with approximately a 4-log difference in binding between each peptide and the control sample. The increased affinity for gold was also confirmed by direct visualization of samples using Scanning Electron Microscopy (SEM). Peptide dynamics in solution were performed to analyze innate structure, and all three were found to have a high degree of flexibility. Preferential binding to gold over silicon for all three peptides was demonstrated, with up to four orders of magnitude selectivity exhibited by p3-Au12. The selectivity was also clearly evident through SEM analysis of the boundary between the gold film and silicon substrate. Functional activity of bound E. coli cells was further demonstrated by stimulating filamentation and all three peptides were characterized as prolific relative to control samples. This work shows great promise towards functional and active bacterial-hybrid gold surfaces and the potential to enable the next generation living material interfaces.

Near-infrared fluorophores as biomolecular probes

NASA Astrophysics Data System (ADS)

Patonay, Gabor; Beckford, Garfield; Strekowski, Lucjan; Henary, Maged; Merid, Yonathan

2010-02-01

Near-Infrared (NIR) fluorescence has been valuable in analytical and bioanalytical chemistry. NIR probes and labels have been used for several applications, including hydrophobicity of protein binding sites, DNA sequencing, immunoassays, CE separations, etc. The NIR region (700-1100 nm) has advantages for the spectroscopist due to the inherently lower background interference from the biological matrix and the high molar absorptivities of NIR chromophores. During the studies we report here several NIR dyes were prepared to determine the role of the hydrophobicity of NIR dyes and their charge in binding to amino acids and proteins, e.g., serum albumins. We synthesized NIR dye homologs containing the same chromophore but substituents of varying hydrophobicity. Hydrophobic moieties were represented by alkyl and aryl groups. These NIR dyes of varying hydrophobicity exhibited varying degrees of H-aggregation in aqueous solution indicating that the degree of H-aggregation could be used as an indicator to predict binding characteristics to serum albumins. In order to understand what factors may be important in the binding process, spectral behavior of these varying hydrophobicity dyes were examined in the presence of amino acids. Typical dye structures that exhibit large binding constants to biomolecules were compared in order to optimize applications utilizing non-covalent interactions.

Enzymes in Commercial Cellulase Preparations Bind Differently to Dioxane Extracted Lignins

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

Yarbrough, John M.; Mittal, Ashutosh; Katahira, Rui

Commercial fungal cellulases used in biomass-to-biofuels processes can be grouped into three general classes: native, augmented, and engineered. To evaluate lignin binding affinities of different enzyme activities in various commercial cellulase formulations in order to determine if enzyme losses due to lignin binding can be modulated by using different enzymes of the same activity We used water:dioxane (1:9) to extract lignin from pretreated corn stover. Commercial cellulases were incubated with lignin and the unbound supernatants were evaluated for individual enzyme loss by SDS=PAGE and these were correlated with activity loss using various pNP-sugar substrates. Colorimetric assays for general glycosyl hydrolasemore » activities showed distinct differences in enzyme binding to lignin for each enzyme activity. Native systems demonstrated low binding of endo- and exo-cellulases, high binding of xylanase, and moderate ..beta..-glucosidase binding. Engineered cellulase mixtures exhibited low binding of exo-cellulases, very strong binding of endocellulases and ..beta..- glucosidase, and mixed binding of xylanase activity. The augmented cellulase had low binding of exocellulase, high binding of endocellulase and xylanase, and moderate binding of ..beta..-glucosidase activities. Bound and unbound activities were correlated with general molecular weight ranges of proteins as measured by loss of proteins bands in bound fractions on SDS-PAGE gels. Lignin-bound high molecular weight bands correlated with binding of ..beta..-glucosidase activity. While ..beta..-glucosidases demonstrated high binding in many cases, they have been shown to remain active. Bound low molecular weight bands correlated with xylanase activity binding. Contrary to other literature, exocellulase activity did not show strong lignin binding. The variation in enzyme activity binding between the three classes of cellulases preparations indicate that it is certainly possible to alter the binding of specific glycosyl hydrolase activities. It remains unclear whether loss of endocellulase activity to lignin binding is problematic for biomass conversion.« less

Comparison of Genome-Wide Binding of MyoD in Normal Human Myogenic Cells and Rhabdomyosarcomas Identifies Regional and Local Suppression of Promyogenic Transcription Factors

PubMed Central

MacQuarrie, Kyle L.; Yao, Zizhen; Fong, Abraham P.; Diede, Scott J.; Rudzinski, Erin R.; Hawkins, Douglas S.

2013-01-01

Rhabdomyosarcoma is a pediatric tumor of skeletal muscle that expresses the myogenic basic helix-loop-helix protein MyoD but fails to undergo terminal differentiation. Prior work has determined that DNA binding by MyoD occurs in the tumor cells, but myogenic targets fail to activate. Using MyoD chromatin immunoprecipitation coupled to high-throughput sequencing and gene expression analysis in both primary human muscle cells and RD rhabdomyosarcoma cells, we demonstrate that MyoD binds in a similar genome-wide pattern in both tumor and normal cells but binds poorly at a subset of myogenic genes that fail to activate in the tumor cells. Binding differences are found both across genomic regions and locally at specific sites that are associated with binding motifs for RUNX1, MEF2C, JDP2, and NFIC. These factors are expressed at lower levels in RD cells than muscle cells and rescue myogenesis when expressed in RD cells. MEF2C is located in a genomic region that exhibits poor MyoD binding in RD cells, whereas JDP2 exhibits local DNA hypermethylation in its promoter in both RD cells and primary tumor samples. These results demonstrate that regional and local silencing of differentiation factors contributes to the differentiation defect in rhabdomyosarcomas. PMID:23230269

Engineering high-potency R-spondin adult stem cell growth factors.

PubMed

Warner, Margaret L; Bell, Tufica; Pioszak, Augen A

2015-01-01

Secreted R-spondin proteins (RSPOs1-4) function as adult stem cell growth factors by potentiating Wnt signaling. Simultaneous binding of distinct regions of the RSPO Fu1-Fu2 domain module to the extracellular domains (ECDs) of the LGR4 G protein-coupled receptor and the ZNRF3 transmembrane E3 ubiquitin ligase regulates Wnt receptor availability. Here, we examine the molecular basis for the differing signaling strengths of RSPOs1-4 using purified RSPO Fu1-Fu2, LGR4 ECD, and ZNRF3 ECD proteins in Wnt signaling and receptor binding assays, and we engineer novel high-potency RSPOs. RSPO2/3/4 had similar signaling potencies that were stronger than that of RSPO1, whereas RSPO1/2/3 had similar efficacies that were greater than that of RSPO4. The RSPOs bound LGR4 with affinity rank order RSPO4 > RSPO2/3 > RSPO1 and ZNRF3 with affinity rank order RSPO2/3 > > RSPO1 > RSPO4. An RSPO2-4 chimera combining RSPO2 ZNRF3 binding with RSPO4 LGR4 binding was a "Superspondin" that exhibited enhanced ternary complex formation and 10-fold stronger signaling potency than RSPO2 and efficacy equivalent to RSPO2. An RSPO4-1 chimera combining RSPO4 ZNRF3 binding with RSPO1 LGR4 binding was a "Poorspondin" that exhibited signaling potency similar to RSPO1 and efficacy equivalent to RSPO4. Conferring increased ZNRF3 binding upon RSPO4 with amino acid substitutions L56F, I58L, and I63M enhanced its signaling potency and efficacy. Our results reveal the molecular basis for RSPOs1-4 activity differences and suggest that signaling potency is determined by ternary complex formation ability, whereas efficacy depends on ZNRF3 recruitment. High-potency RSPOs may be of value for regenerative medicine and/or therapeutic applications. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Characterization of 18F-FPyKYNE-Losartan for Imaging AT1 Receptors.

PubMed

Hachem, Maryam; Tiberi, Mario; Ismail, Basma; Hunter, Chad R; Arksey, Natasha; Hadizad, Tayebeh; Beanlands, Rob S; deKemp, Robert A; DaSilva, Jean N

2016-10-01

Most physiologic effects of the renin angiotensin system (RAS) are mediated via the angiotensin (Ang) type 1 receptor (AT 1 R). The 18 F-FPyKYNE derivative of the clinically used AT 1 R blocker losartan exhibits high binding selectivity for kidney AT 1 R and rapid metabolism in rats. The aim of this study was to further assess the binding profile of this novel PET agent for imaging AT 1 R in rats and pigs. In vitro binding assays were performed with 18 F-FPyKYNE-losartan in rat kidneys. Male Sprague-Dawley rats were used to assess dosimetry, antagonistic efficacy via blood pressure measurements, and presence of labeled metabolites in kidneys. Test-retest PET imaging, blocking with AT 1 R antagonist candesartan (10 mg/kg), and plasma metabolism analysis were performed in female Yorkshire pigs. 18 F-FPyKYNE-losartan bound with high affinity (dissociation constant of 49.4 ± 18.0 nM and maximal binding of 348 ± 112 fmol/mm 2 ) to rat kidney AT 1 R. It bound strongly to plasma proteins in rats (97%), and its labeled metabolites displayed minimal interference on renal AT 1 R binding. FPyKYNE-losartan fully antagonized the Ang II pressor effect, albeit with 4-fold potency reduction (the effective dose inhibiting 50% of the Ang II-induced maximal pressor response of 25.5 mg/kg) relative to losartan. PET imaging exhibited high kidney-to-blood contrast and slow renal clearance, with an SUV of 14.1 ± 6.2. Excellent reproducibility was observed in the calculated test-retest variability (7.2% ± 0.75%). Only hydrophilic-labeled metabolites were present in plasma samples, and renal retention was reduced (-60%) at 10-15 min after blockade with candesartan. 18 F-FPyKYNE-losartan has a favorable binding profile and displays high potential for translational work in humans as an AT 1 R PET imaging agent. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Surface properties of adipocyte lipid-binding protein: Response to lipid binding, and comparison with homologous proteins.

PubMed

LiCata, V J; Bernlohr, D A

1998-12-01

Adipocyte lipid-binding protein (ALBP) is one of a family of intracellular lipid-binding proteins (iLBPs) that bind fatty acids, retinoids, and other hydrophobic ligands. The different members of this family exhibit a highly conserved three-dimensional structure; and where structures have been determined both with (holo) and without (apo) bound lipid, observed conformational changes are extremely small (Banaszak, et al., 1994, Adv. Prot. Chem. 45, 89; Bernlohr, et al., 1997, Annu. Rev. Nutr. 17, 277). We have examined the electrostatic, hydrophobic, and water accessible surfaces of ALBP in the apo form and of holo forms with a variety of bound ligands. These calculations reveal a number of previously unrecognized changes between apo and holo ALBP, including: 1) an increase in the overall protein surface area when ligand binds, 2) expansion of the binding cavity when ligand is bound, 3) clustering of individual residue exposure increases in the area surrounding the proposed ligand entry portal, and 4) ligand-binding dependent variation in the topology of the electrostatic potential in the area surrounding the ligand entry portal. These focused analyses of the crystallographic structures thus reveal a number of subtle but consistent conformational and surface changes that might serve as markers for differential targeting of protein-lipid complexes within the cell. Most changes are consistent from ligand to ligand, however there are some ligand-specific changes. Comparable calculations with intestinal fatty-acid-binding protein and other vertebrate iLBPs show differences in the electrostatic topology, hydrophobic topology, and in localized changes in solvent exposure near the ligand entry portal. These results provide a basis toward understanding the functional and mechanistic differences among these highly structurally homologous proteins. Further, they suggest that iLBPs from different tissues exhibit one of two predominant end-state structural distributions of the ligand entry portal.

Characteristics of low-and high-fat beef patties: effect of high hydrostatic pressure.

PubMed

Carballo, J; Fernandez, P; Carrascosa, A V; Solas, M T; Colmenero, F J

1997-01-01

The purpose of this study was to analyze the consequences of applying high pressures (100 and 300 MPa for 5 or 20 min) on characteristics such as water- and fat-binding properties, texture, color, microstructure, and microbiology of low-fat (9.2%) and high-fat (20.3%) beef patties. In nonpressurized patties, the low-fat product exhibited significantly poorer (P < 0.05) binding properties and higher (P < 0.05) Kramer shear force and Kramer energy than did high-fat patties. Although high pressure did not clearly influence the binding properties of low- and high-fat beef patties, it did produce a rise in the Kramer shear force and energy which were more pronounced at 300 MPa. High pressures altered patty color, the extent of alteration depending on fat content, pressure, and pressurizing time. Pressurizing high- and low-fat beef patties at 300 MPa not only produced a lethal effect (P < 0.05) on microorganisms, but caused sublethal damage as well.

Studies on the chitin/chitosan binding properties of six cuticular proteins analogous to peritrophin 3 from Bombyx mori.

PubMed

Qu, M; Ren, Y; Liu, Y; Yang, Q

2017-08-01

Chitin deacetylation is required to make the cuticle rigid and compact through chitin chain crosslinking. Thus it is presumed that specialized proteins are required to bind deacetylated chitin chains together. However, deacetylated-chitin binding proteins have not ever been reported. In a previous work, six cuticular proteins analogous to peritrophin 3 (CPAP3s) were found to be abundant in the moulting fluid of Bombyx mori. In this study, these BmCPAP3s (BmCPAP3-A1, BmCPAP3-A2, BmCPAP3-B, BmCPAP3-C, BmCPAP3-D1 and BmCPAP3-D2) were cloned and expressed in Escherichia coli and purified using metal-chelating affinity chromatography. Their binding activities demonstrated that although all of the BmCPAP3s showed similar binding abilities toward crystalline chitin and colloidal chitin, they differed in their affinities toward partially and fully deacetylated chitin. Amongst them, BmCPAP3-D1 exhibited the highest binding activity toward deacetylated chitin. The gene expression pattern of BmCPAP3-D1 was similar to BmCPAP3-A1 and BmCPAP3-C at most stages except that it was dramatically upregulated at the beginning of the pupa to adult transition stage. This work is the first report of a chitin-binding protein, BmCPAP3-D1, which exhibits high binding affinity to deacetylated chitin. © 2017 The Royal Entomological Society.

Functional characterization of mongoose nicotinic acetylcholine receptor alpha-subunit: resistance to alpha-bungarotoxin and high sensitivity to acetylcholine.

PubMed

Asher, O; Lupu-Meiri, M; Jensen, B S; Paperna, T; Fuchs, S; Oron, Y

1998-07-24

The mongoose is resistant to snake neurotoxins. The mongoose muscle nicotinic acetylcholine receptor (AChR) alpha-subunit contains a number of mutations in the ligand-binding domain and exhibits poor binding of alpha-bungarotoxin (alpha-BTX). We characterized the functional properties of a hybrid (alpha-mongoose/beta gamma delta-rat) AChR. Hybrid AChRs, expressed in Xenopus oocytes, respond to acetylcholine with depolarizing current, the mean maximal amplitude of which was greater than that mediated by the rat AChR. The IC50 of alpha-BTX to the hybrid AChR was 200-fold greater than that of the rat, suggesting much lower affinity for the toxin. Hybrid AChRs exhibited an apparent higher rate of desensitization and higher affinity for ACh (EC50 1.3 vs. 23.3 microM for the rat AChR). Hence, changes in the ligand-binding domain of AChR not only affect the binding properties of the receptor, but also result in marked changes in the characteristics of the current.

Atomically precise organomimetic cluster nanomolecules assembled via perfluoroaryl-thiol SNAr chemistry

NASA Astrophysics Data System (ADS)

Qian, Elaine A.; Wixtrom, Alex I.; Axtell, Jonathan C.; Saebi, Azin; Jung, Dahee; Rehak, Pavel; Han, Yanxiao; Moully, Elamar Hakim; Mosallaei, Daniel; Chow, Sylvia; Messina, Marco S.; Wang, Jing Yang; Royappa, A. Timothy; Rheingold, Arnold L.; Maynard, Heather D.; Král, Petr; Spokoyny, Alexander M.

2017-04-01

The majority of biomolecules are intrinsically atomically precise, an important characteristic that enables rational engineering of their recognition and binding properties. However, imparting a similar precision to hybrid nanoparticles has been challenging because of the inherent limitations of existing chemical methods and building blocks. Here we report a new approach to form atomically precise and highly tunable hybrid nanomolecules with well-defined three-dimensionality. Perfunctionalization of atomically precise clusters with pentafluoroaryl-terminated linkers produces size-tunable rigid cluster nanomolecules. These species are amenable to facile modification with a variety of thiol-containing molecules and macromolecules. Assembly proceeds at room temperature within hours under mild conditions, and the resulting nanomolecules exhibit high stabilities because of their full covalency. We further demonstrate how these nanomolecules grafted with saccharides can exhibit dramatically improved binding affinity towards a protein. Ultimately, the developed strategy allows the rapid generation of precise molecular assemblies to investigate multivalent interactions.

Identification of a Novel Penicillin-Binding Protein from Helicobacter pylori

PubMed Central

Krishnamurthy, Partha; Parlow, Mary H.; Schneider, John; Burroughs, Stephanie; Wickland, Catherine; Vakil, Nimish B.; Dunn, Bruce E.; Phadnis, Suhas H.

1999-01-01

The Helicobacter pylori genome encodes four penicillin-binding proteins (PBPs). PBPs 1, 2, and 3 exhibit similarities to known PBPs. The sequence of PBP 4 is unique in that it displays a novel combination of two highly conserved PBP motifs and an absence of a third motif. Expression of PBP 4, but not PBP 1, 2, or 3, is significantly increased during mid- to late-log-phase growth. PMID:10438788

Reconstitution of high affinity. cap alpha. /sub 2/ adrenergic agonist binding by fusion with a pertussis toxin substrate

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

Kim, M.H.; Neubig, R.R.

1986-03-05

High affinity ..cap alpha../sub 2/ adrenergic agonist binding is thought to occur via a coupling of the ..cap alpha../sub 2/ receptor with N/sub i/, the inhibitory guanyl nucleotide binding protein. Human platelet membranes pretreated at pH 11.5 exhibit a selective inactivation of agonist binding and N/sub i/. To further study the mechanism of agonist binding, alkali treated membranes (ATM) were mixed with membranes pretreated with 10 ..mu..M phenoxybenzamine to block ..cap alpha../sub 2/ receptors (POB-M). The combined membrane pellet was incubated in 50% polyethylene glycol (PEG) to promote membrane-membrane fusion and assayed for binding to the ..cap alpha../sub 2/ agonistmore » (/sup 3/H)UK 14,304 (UK) and the antagonist (/sup 3/H) yohimbine. PEG treatment resulted in a 2-4 fold enhancement of UK binding whereas yohimbine binding was unchanged. No enhancement of UK binding was observed in the absence of PEG treatment. The reconstitution was dependent on the addition of POB-M. They found that a 1:1 ratio of POB-M:ATM was optimal. Reconstituted binding was inhibited by GppNHp. Fusion of rat C6 glioma cell membranes, which do not contain ..cap alpha../sub 2/ receptors, also enhanced agonist binding to ATM. Fusion of C6 membranes from cells treated with pertussis toxin did not enhance (/sup 3/H) UK binding. These data show that a pertussis toxin sensitive membrane component, possibly N/sub i/, can reconstitute high affinity ..cap alpha../sub 2/ agonist binding.« less

Nanohole Arrays of Mixed Designs and Microwriting for Simultaneous and Multiple Protein Binding Studies

PubMed Central

Ji, Jin; Yang, Jiun-Chan; Larson, Dale N.

2009-01-01

We demonstrate using nanohole arrays of mixed designs and a microwriting process based on dip-pen nanolithography to monitor multiple, different protein binding events simultaneously in real time based on the intensity of Extraordinary Optical Transmission of nanohole arrays. The microwriting process and small footprint of the individual nanohole arrays enabled us to observe different binding events located only 16μm apart, achieving high spatial resolution. We also present a novel concept that incorporates nanohole arrays of different designs to improve confidence and accuracy of binding studies. For proof of concept, two types of nanohole arrays, designed to exhibit opposite responses to protein bindings, were fabricated on one transducer. Initial studies indicate that the mixed designs could help to screen out artifacts such as protein intrinsic signals, providing improved accuracy of binding interpretation. PMID:19297143

Switch control pocket inhibitors of p38-MAP kinase. Durable type II inhibitors that do not require binding into the canonical ATP hinge region

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

Ahn, Yu Mi; Clare, Michael; Ensinger, Carol L.

Switch control pocket inhibitors of p38-alpha kinase are described. Durable type II inhibitors were designed which bind to arginines (Arg67 or Arg70) that function as key residues for mediating phospho-threonine 180 dependant conformational fluxing of p38-alpha from an inactive type II state to an active type I state. Binding to Arg70 in particular led to potent inhibitors, exemplified by DP-802, which also exhibited high kinase selectivity. Binding to Arg70 obviated the requirement for binding into the ATP Hinge region. X-ray crystallography revealed that DP-802 and analogs induce an enhanced type II conformation upon binding to either the unphosphorylated or themore » doubly phosphorylated form of p38-alpha kinase.« less

Modification of agonist binding moiety in hybrid derivative 5/7-{[2-(4-aryl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro-naphthalen-1-ol/-2-amino versions: Impact on functional activity and selectivity for dopamine D2/D3 receptors

PubMed Central

Gopishetty, Bhaskar; Zhang, Suhong; Kharkar, Prashant S.; Antonio, Tamara; Reith, Maarten; Dutta, Aloke K.

2013-01-01

The goal of the present study was to explore, in our previously developed hybrid template, the effect of introduction of additional heterocyclic rings (mimicking catechol hydroxyl groups as bioisosteric replacement) on selectivity and affinity for the D3 versus D2 receptor. In addition, we wanted to explore the effect of derivatization of functional groups of the agonist binding moiety in compounds developed by us earlier from the hybrid template. Binding affinity (Ki) of the new compounds was measured with tritiated spiperone as the radioligand and HEK-293 cells expressing either D2 or D3 receptors. Functional activity of selected compounds was assessed in the GTPγS binding assay. In the imidazole series, compound 10a exhibited the highest D3 affinity whereas the indole derivative 13 exhibited similar high D3 affinity. Functionalization of the amino group in agonist (+)-9d with different sulfonamides derivatives improved the D3 affinity significantly with (+)-14f exhibiting the highest affinity. However, functionalization of the hydroxyl and amino groups of 15 and (+)-9d, known agonist and partial agonist, to sulfonate ester and amide in general modulated the affinity. In both cases loss of agonist potency resulted from such derivatization. PMID:23623679

Follitropin receptors in rat testis. Characterization with enzymatically 125I-labeled human follitropin.

PubMed

Ketelslegers, J M; Catt, K J

1978-07-03

The interaction between enzymatically radioiodinated human follitropin and the follitropin receptors in testis homogenate was investigated in immature and adult rats. The 125I-labeled human follitropin exhibited high binding activity with specific binding of up to 17% in the presence of an excess of testis homogenate. Approx. 50% of the bound hormone could be eluted at pH 5, and the receptor purified tracer exhibited a 3.6-fold increase in binding activity when compared with the original tracer preparation. Quantitative analysis of equilibrium binding data was performed with corrections for the measured specific activity and maximum binding activity of the tracer hormone. The equilibrium association constants (Ka) determined 24 degrees C were not significantly different in immature and adult rat testis, and the mean value for Ka was 3.9 . 10(9) M-1. At 37 degrees C, the Ka value obtained using immature rat testis was 1.3 . 10(10) M-1. The association of 125I-labeled human follitropin with immature rat testis homogenate was time and temperature dependent. In the presence of an excess of unlabeled hormone, 30--60% of the preformed hormone . receptor complex was dissociated after 24 h incubation. A specific and sensitive radioligand-receptor assay for follitropin was developed using immature rat testis homogenate. The minimum detectable dose of purified human follitropin was 0.6 ng, and human urinary and pituitary follitropin, ovine follitropin and pregnant mare serum gonadotropin reacted in the assay with equivalent slopes. The potencies of highly purified pregnent mare serum gonadotropin and highly purified human follitropin were similar in the radioligand-receptor assay, consistent with the follitropin bioactivity of the equine gonadotropin.

The opioid receptor pharmacology of GSK1521498 compared to other ligands with differential effects on compulsive reward-related behaviours.

PubMed

Kelly, Eamonn; Mundell, Stuart J; Sava, Anna; Roth, Adelheid L; Felici, Antonio; Maltby, Kay; Nathan, Pradeep J; Bullmore, Edward T; Henderson, Graeme

2015-01-01

The novel opioid receptor antagonist, GSK1421498, has been shown to attenuate reward-driven compulsive behaviours, such as stimulant drug seeking or binge eating, in animals and humans. Here, we report new data on the receptor pharmacology of GSK121498, in comparison to naltrexone, naloxone, 6-β-naltrexol and nalmefene. To determine whether the novel opioid antagonist, GSK1521498, is an orthosteric or allosteric antagonist at the μ opioid receptor (MOPr) and whether it has neutral antagonist or inverse agonist properties. A combination of radioligand binding assays and [(35)S]GTPγS binding assays was employed. GSK1521498 completely displaced [(3)H]naloxone binding to MOPr and did not alter the rate of [(3)H]naloxone dissociation from MOPr observations compatible with it binding to the orthosteric site on MOPr. GSK1521498 exhibited inverse agonism when MOPr was overexpressed but not when the level of MOPr expression was low. In parallel studies under conditions of high receptor expression density, naloxone, naltrexone, 6-β-naltrexol and nalmefene exhibited partial agonism, not inverse agonism as has been reported previously for naloxone and naltrexone. In brain tissue from mice receiving a prolonged morphine pre-treatment, GSK1521498 exhibited slight inverse agonism. Differences between GSK1521498 and naltrexone in their effects on compulsive reward seeking are arguably linked to the more selective and complete MOPr antagonism of GSK1521498 versus the partial MOPr agonism of naltrexone. GSK1521498 is also pharmacologically differentiated by its inverse agonist efficacy at high levels of MOPr expression, but this may be less likely to contribute to behavioural differentiation at patho-physiological levels of expression.

“Turn-On” Protein Fluorescence: In Situ Formation of Cyanine Dyes

PubMed Central

2015-01-01

Protein reengineering of cellular retinoic acid binding protein II (CRABPII) has yielded a genetically addressable system, capable of binding a profluorophoric chromophore that results in fluorescent protein/chromophore complexes. These complexes exhibit far-red emission, with high quantum efficiencies and brightness and also exhibit excellent pH stability spanning the range of 2–11. In the course of this study, it became evident that single mutations of L121E and R59W were most effective in improving the fluorescent characteristics of CRABPII mutants as well as the kinetics of complex formation. The readily crystallizable nature of these proteins was invaluable to provide clues for the observed spectroscopic behavior that results from single mutation of key residues. PMID:25534273

"Turn-on" protein fluorescence: in situ formation of cyanine dyes.

PubMed

Yapici, Ipek; Lee, Kin Sing Stephen; Berbasova, Tetyana; Nosrati, Meisam; Jia, Xiaofei; Vasileiou, Chrysoula; Wang, Wenjing; Santos, Elizabeth M; Geiger, James H; Borhan, Babak

2015-01-28

Protein reengineering of cellular retinoic acid binding protein II (CRABPII) has yielded a genetically addressable system, capable of binding a profluorophoric chromophore that results in fluorescent protein/chromophore complexes. These complexes exhibit far-red emission, with high quantum efficiencies and brightness and also exhibit excellent pH stability spanning the range of 2-11. In the course of this study, it became evident that single mutations of L121E and R59W were most effective in improving the fluorescent characteristics of CRABPII mutants as well as the kinetics of complex formation. The readily crystallizable nature of these proteins was invaluable to provide clues for the observed spectroscopic behavior that results from single mutation of key residues.

“Turn-On” Protein Fluorescence: In Situ Formation of Cyanine Dyes

DOE PAGES

Yapici, Ipek; Lee, Kin Sing Stephen; Berbasova, Tetyana; ...

2014-12-22

Protein reengineering of cellular retinoic acid binding protein II (CRABPII) has yielded a genetically addressable system, capable of binding a profluorophoric chromophore that results in fluorescent protein/chromophore complexes. These complexes exhibit far-red emission, with high quantum efficiencies and brightness and also exhibit excellent pH stability spanning the range of 2$-$11. In the course of this study, it became evident that single mutations of L121E and R59W were most effective in improving the fluorescent characteristics of CRABPII mutants as well as the kinetics of complex formation. The readily crystallizable nature of these proteins was invaluable to provide clues for the observedmore » spectroscopic behavior that results from single mutation of key residues.« less

Effect of thiol pendant conjugates on plasmid DNA binding, release, and stability of polymeric delivery vectors.

PubMed

Bacalocostantis, Irene; Mane, Viraj P; Kang, Michael S; Goodley, Addison S; Muro, Silvia; Kofinas, Peter

2012-05-14

Polymers have attracted much attention as potential gene delivery vectors due to their chemical and structural versatility. However, several challenges associated with polymeric carriers, including low transfection efficiencies, insufficient cargo release, and high cytotoxicity levels have prevented clinical implementation. Strong electrostatic interactions between polymeric carriers and DNA cargo can prohibit complete cargo release within the cell. As a result, cargo DNA never reaches the cell's nucleus where gene expression takes place. In addition, highly charged cationic polymers have been correlated with high cytotoxicity levels, making them unsuitable carriers in vivo. Using poly(allylamine) (PAA) as a model, we investigated how pH-sensitive disulfide cross-linked polymer networks can improve the delivery potential of cationic polymer carriers. To accomplish this, we conjugated thiol-terminated pendant chains onto the primary amines of PAA using 2-iminothiolane, developing three new polymer vectors with 5, 13, or 20% thiol modification. Unmodified PAA and thiol-conjugated polymers were tested for their ability to bind and release plasmid DNA, their capacity to protect genetic cargo from enzymatic degradation, and their potential for endolysosomal escape. Our results demonstrate that polymer-plasmid complexes (polyplexes) formed by the 13% thiolated polymer demonstrate the greatest delivery potential. At high N/P ratios, all thiolated polymers (but not unmodified counterparts) were able to resist decomplexation in the presence of heparin, a negatively charged polysaccharide used to mimic in vivo polyplex-protein interactions. Further, all thiolated polymers exhibited higher buffering capacities than unmodified PAA and, therefore, have a greater potential for endolysosomal escape. However, 5 and 20% thiolated polymers exhibited poor DNA binding-release kinetics, making them unsuitable carriers for gene delivery. The 13% thiolated polymers, on the other hand, displayed high DNA binding efficiency and pH-sensitive release.

Autoradiographic analysis of the in vivo distribution of 3H-imipramine and 3H-desipramine in brain: Comparison to in vitro binding patterns

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

Duncan, G.E.; Paul, I.A.; Fassberg, J.B.

1991-03-01

Using high resolution autoradiographic techniques, the distribution of radioactivity in forebrain and brainstem was assessed after 4 injection of 3H-impramine or 3H-desipramine. Results were compared with regional binding of the drugs to brain sections in vitro. Similar topographic binding of 3H-imipramine and 3H-desipramine was observed in vitro among brain regions, except in the paraventricular nucleus of the hypothalamus and locus coeruleus, where binding was greater for 3H-desipramine. For both 3H-desipramine and 3H-imipramine, some brain regions that exhibited high binding in vitro also showed high accumulation after in vivo injection. However, certain regions that contained high densities of binding sites formore » the antidepressant drugs as measured by in vitro binding showed very low accumulation of radioactivity after in vivo treatment. Such regions included the dentate gyrus of the hippocampus, layer 1 of piriform cortex, caudate-putamen, pontine and midbrain central gray, and cerebellar granular layer. Compared to in vitro binding of the drugs, the distribution of imipramine and desipramine in vivo appears more anatomically selective. For imipramine, primary sites of action in vivo, as indicated by the topographic distribution in brain, appear to be the locus coeruleus, hippocampus, lateral septal nucleus, and amygdala. For desipramine, the greatest accumulation in vivo was found in the locus coeruleus, paraventricular nucleus of the hypothalamus, and anterior thalamic nuclei.« less

Interaction between phloretin and the red blood cell membrane

PubMed Central

1976-01-01

Phloretin binding to red blood cell components has been characterized at pH6, where binding and inhibitory potency are maximal. Binding to intact red cells and to purified hemoglobin are nonsaturated processes approximately equal in magnitude, which strongly suggests that most of the red cell binding may be ascribed to hemoglobin. This conclusion is supported by the fact that homoglobin-free red cell ghosts can bind only 10% as much phloretin as an equivalent number of red cells. The permeability of the red cell membrane to phloretin has been determined by a direct measurement at the time-course of the phloretin uptake. At a 2% hematocrit, the half time for phloretin uptake is 8.7s, corresponding to a permeability coefficient of 2 x 10(-4) cm/s. The concentration dependence of the binding to ghosts reveals two saturable components. Phloretin binds with high affinity (K diss = 1.5 muM) to about 2.5 x 10(6) sites per cell; it also binds with lower affinity (Kdiss = 54 muM) to a second (5.5 x 10(7) per cell) set of sites. In sonicated total lipid extracts of red cell ghosts, phloretin binding consists of a single, saturable component. Its affinity and total number of sites are not significantly different from those of the low affinity binding process in ghosts. No high affinity binding of phloretin is exhibited by the red cell lipid extracts. Therefore, the high affinity phloretin binding sites are related to membrane proteins, and the low affinity sites result from phloretin binding to lipid. The identification of these two types of binding sites allows phloretin effects on protein-mediated transport processes to be distinguished from effects on the lipid region of the membrane. PMID:5575

Exploring the DNA binding/cleavage, cellular accumulation and topoisomerase inhibition of 2-hydroxy-3-(aminomethyl)-1,4-naphthoquinone Mannich bases and their platinum(II) complexes.

PubMed

Neves, Amanda P; Pereira, Michelle X G; Peterson, Erica J; Kipping, Ralph; Vargas, Maria D; Silva, Floriano P; Carneiro, J Walkimar M; Farrell, Nicholas P

2013-02-01

Several chlorido and amino Pt(2+) complexes of 2-hydroxy-3-(aminomethyl)-1,4-naphthoquinone Mannich bases HL exhibiting moderate to high cytotoxicity against cancer cell lines were studied in order to investigate their modes of DNA binding, in vitro DNA strand breaks, mechanism of topoisomerase (Topo I) inhibition and cellular accumulation. DNA model base studies have shown that complex 1a [Pt(HL1)Cl(2)] was capable of binding covalently to 9-ethylguanine (9-EtG) and 5'-GMP. (1)H NMR and mass spectrometry studies have shown that both chlorides were substituted by 9-EtG ligands, whereas 5'-GMP was able to replace only one chlorido ligand, due to steric hindrance. The chlorido Pt(2+) complexes [Pt(HL)Cl(2)] highly accumulate in prostate (PC-3) and melanoma (MDA-MB-435) cell lines, being able to induce DNA strand breaks in vitro and inhibit Topo I by a catalytic mode. On the other hand, the free 2-hydroxy-3-(aminomethyl)-1,4-naphthoquinones HL and the amino Pt(2+) complexes [Pt(L(-))(NH(3))(2)]NO(3) neither cause DNA strand breakage nor exhibit strong DNA interaction, nevertheless the latter were also found to be catalytic inhibitors of Topo I at 100μM. Thus, coordination of the Mannich bases HL to the "PtCl(2)" fragment substantially affects the chemical and biophysical properties of the pro-ligands, leading to an improvement of their DNA binding properties and generating compounds that cleave DNA and catalytically inhibit Topo I. Finally, the high cytotoxicity exhibited by the free (uncomplexed) 2-hydroxy-3-(aminomethyl)-1,4-naphthoquinones might be associated with their decomposition in solution, which is not observed for the Pt(2+) complexes. Copyright © 2012 Elsevier Inc. All rights reserved.

Developing a Novel Hydrogen Sponge with Ideal Binding Energy and High Surface Area for Practical Hydrogen Storage

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

Chung, T. C. Mike

This Phase I (5 quarters) research project was to examine the validity of a new class of boron-containing polymer (B-polymer) frameworks, serving as the adsorbents for the practical onboard H2 storage applications. Three B-polymer frameworks were synthesized and investigated, which include B-poly(butyenylstyrene) (B-PBS) framework (A), B-poly(phenyldiacetyene) (B-PPDA) framework (B), and B-poly(phenyltriacetylene) (B-PPTA) framework (C). They are 2-D polymer structures with the repeating cyclic units that spontaneously form open morphology and the B-doped (p-type) π-electrons delocalized surfaces. The ideal B-polymer framework shall exhibit open micropores (pore size in the range of 1-1.5nm) with high surface area (>3000 m 2/g), and themore » B-dopants in the conjugated framework shall provide high surface energy for interacting with H 2 molecules (an ideal H 2 binding energy in the range of 15-25 kJ/mol). The pore size distribution and H2 binding energy were investigated at both Penn State and NREL laboratories. So far, the experimental results show the successful synthesis of B-polymer frameworks with the relatively well-defined planar (2-D) structures. The intrinsically formed porous morphology exhibits a broad pore size distribution (in the range of 0.5-10 nm) with specific surface area (~1000 m 2/g). The miss-alignment between 2-D layers may block some micropore channels and limit gas diffusion throughout the entire matrix. In addition, the 2-D planar conjugated structure may also allow free π-electrons delocalization throughout the framework, which significantly reduces the acidity of B-moieties (electron-deficiency).The resulting 2-D B-polymer frameworks only exhibit a small increase of H 2 binding energy in the range of 8-9 KJ/mole (quite constant over the whole sorption range).« less

Temperature and doping dependence of the high-energy kink in cuprates.

PubMed

Zemljic, M M; Prelovsek, P; Tohyama, T

2008-01-25

It is shown that spectral functions within the extended t-J model, evaluated using the finite-temperature diagonalization of small clusters, exhibit the high-energy kink in single-particle dispersion consistent with recent angle-resolved photoemission results on hole-doped cuprates. The kink and waterfall-like features persist up to large doping and to temperatures beyond J; hence, the origin can be generally attributed to strong correlations and incoherent hole propagation at large binding energies. In contrast, our analysis predicts that electron-doped cuprates do not exhibit these phenomena in photoemission.

Supramolecular approach to enantioselective DNA recognition using enantiomerically resolved cationic 4-amino-1,8-naphthalimide-based Tröger's bases.

PubMed

Banerjee, Swagata; Bright, Sandra A; Smith, Jayden A; Burgeat, Jeremy; Martinez-Calvo, Miguel; Williams, D Clive; Kelly, John M; Gunnlaugsson, Thorfinnur

2014-10-03

The synthesis and photophysical studies of two cationic Tröger's base (TB)-derived bis-naphthalimides 1 and 2 and the TB derivative 6, characterized by X-ray crystallography, are presented. The enantiomers of 1 and 2 are separated by cation-exchange chromatography on Sephadex C25 using sodium (-)-dibenzoyl-l-tartarate as the chiral mobile phase. The binding of enantiomers with salmon testes (st)-DNA and synthetic polynucleotides are studied by a variety of spectroscopic methods including UV/vis absorbance, circular dichroism, linear dichroism, and ethidium bromide displacement assays, which demonstrated binding of these compounds to the DNA grooves with very high affinity (K ∼ 10(6) M(-1)) and preferential binding of (-)-enantiomer. In all cases, binding to DNA resulted in a significant stabilization of the double-helical structure of DNA against thermal denaturation. Compound (±)-2 and its enantiomers possessed significantly higher binding affinity for double-stranded DNA compared to 1, possibly due to the presence of the methyl group, which allows favorable hydrophobic and van der Waals interactions with DNA. The TB derivatives exhibited marked preference for AT rich sequences, where the binding affinities follow the order (-)-enantiomer > (±) > (+)-enantiomer. The compounds exhibited significant photocleavage of plasmid DNA upon visible light irradiation and are rapidly internalized into malignant cell lines.

Haptoglobin preferentially binds β but not α subunits cross-linked hemoglobin tetramers with minimal effects on ligand and redox reactions.

PubMed

Jia, Yiping; Wood, Francine; Buehler, Paul W; Alayash, Abdu I

2013-01-01

Human hemoglobin (Hb) and haptoglobin (Hp) exhibit an extremely high affinity for each other, and the dissociation of Hb tetramers into dimers is generally believed to be a prerequisite for complex formation. We have investigated Hp interactions with native Hb, αα, and ββ cross-linked Hb (ααXLHb and ββXLHb, respectively), and rapid kinetics of Hb ligand binding as well as the redox reactivity in the presence of and absence of Hp. The quaternary conformation of ββ subunit cross-linking results in a higher binding affinity than that of αα subunit cross-linked Hb. However, ββ cross-linked Hb exhibits a four fold slower association rate constant than the reaction rate of unmodified Hb with Hp. The Hp contact regions in the Hb dimer interfaces appear to be more readily exposed in ββXLHb than ααXLHb. In addition, apart from the functional changes caused by chemical modifications, Hp binding does not induce appreciable effects on the ligand binding and redox reactions of ββXLHb. Our findings may therefore be relevant to the design of safer Hb-based oxygen therapeutics by utilizing this preferential binding of ββXLHb to Hp. This may ultimately provide a safe oxidative inactivation and clearance pathway for chemically modified Hbs in circulation.

Intrinsic Thermodynamics and Structures of 2,4- and 3,4-Substituted Fluorinated Benzenesulfonamides Binding to Carbonic Anhydrases.

PubMed

Zubrienė, Asta; Smirnov, Alexey; Dudutienė, Virginija; Timm, David D; Matulienė, Jurgita; Michailovienė, Vilma; Zakšauskas, Audrius; Manakova, Elena; Gražulis, Saulius; Matulis, Daumantas

2017-01-20

The goal of rational drug design is to understand structure-thermodynamics correlations in order to predict the chemical structure of a drug that would exhibit excellent affinity and selectivity for a target protein. In this study we explored the contribution of added functionalities of benzenesulfonamide inhibitors to the intrinsic binding affinity, enthalpy, and entropy for recombinant human carbonic anhydrases (CA) CA I, CA II, CA VII, CA IX, CA XII, and CA XIII. The binding enthalpies of compounds possessing similar chemical structures and affinities were found to be very different, spanning a range from -90 to +10 kJ mol -1 , and are compensated by a similar opposing entropy contribution. The intrinsic parameters of binding were determined by subtracting the linked protonation reactions. The sulfonamide group pK a values of the compounds were measured spectrophotometrically, and the protonation enthalpies were measured by isothermal titration calorimetry (ITC). Herein we describe the development of meta- or ortho-substituted fluorinated benzenesulfonamides toward the highly potent compound 10 h, which exhibits an observed dissociation constant value of 43 pm and an intrinsic dissociation constant value of 1.1 pm toward CA IX, an anticancer target that is highly overexpressed in various tumors. Fluorescence thermal shift assays, ITC, and X-ray crystallography were all applied in this work. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Determination of malachite green in aquatic products based on magnetic molecularly imprinted polymers.

PubMed

Lin, Zheng-zhong; Zhang, Hong-yuan; Peng, Ai-hong; Lin, Yi-dong; Li, Lu; Huang, Zhi-yong

2016-06-01

Magnetic molecularly imprinted polymers (MMIPs) were synthesized through precipitation polymerization using malachite green (MG) as template, methacrylic acid as monomer, ethylene dimethacrylate as crosslinker, and Fe3O4 magnetite as magnetic component. MMIPs were characterized by scanning electron microscopy, Fourier transform infrared spectrometry, and vibrating sample magnetometry. Under the optimum condition, the MMIPs obtained exhibited quick binding kinetics and high affinity to MG in the solution. Scatchard plot analysis revealed that the MMIPs contained only one type of binding site with dissociation constant of 24.0 μg mL(-1). The selectivity experiment confirmed that the MMIPs exhibited higher selective binding capacity for MG than its structurally related compound (e.g., crystal violet). As a sorbent for the extraction of MG in sample preparation, MMIPs together with the absorbed analytes could easily be separated from the sample matrix with an external magnet. After elution with methanol/acetic acid (9:1, v/v), MG in the eluent was determined by high-performance liquid chromatography coupled with UV detector with recoveries of 94.0-115%. Results indicated that the as-prepared MMIPs are promising materials for MG analysis in aquatic products. Copyright © 2016 Elsevier Ltd. All rights reserved.

Challenging the Metallothionein (MT) Gene of Biomphalaria glabrata: Unexpected Response Patterns Due to Cadmium Exposure and Temperature Stress.

PubMed

Niederwanger, Michael; Dvorak, Martin; Schnegg, Raimund; Pedrini-Martha, Veronika; Bacher, Katharina; Bidoli, Massimo; Dallinger, Reinhard

2017-08-11

Metallothioneins (MTs) are low-molecular-mass, cysteine-rich, metal binding proteins. In most animal species, they are involved in metal homeostasis and detoxification, and provide protection from oxidative stress. Gastropod MTs are highly diversified, exhibiting unique features and adaptations like metal specificity and multiplications of their metal binding domains. Here, we show that the MT gene of Biomphalaria glabrata , one of the largest MT genes identified so far, is composed in a unique way. The encoding for an MT protein has a three-domain structure and a C-terminal, Cys-rich extension. Using a bioinformatic approach involving structural and in silico analysis of putative transcription factor binding sites (TFBs), we found that this MT gene consists of five exons and four introns. It exhibits a regulatory promoter region containing three metal-responsive elements (MREs) and several TFBs with putative involvement in environmental stress response, and regulation of gene expression. Quantitative real-time polymerase chain reaction (qRT-PCR) data indicate that the MT gene is not inducible by cadmium (Cd) nor by temperature challenges (heat and cold), despite significant Cd uptake within the midgut gland and the high Cd tolerance of metal-exposed snails.

Challenging the Metallothionein (MT) Gene of Biomphalaria glabrata: Unexpected Response Patterns Due to Cadmium Exposure and Temperature Stress

PubMed Central

Dvorak, Martin; Schnegg, Raimund; Pedrini-Martha, Veronika; Bacher, Katharina; Bidoli, Massimo; Dallinger, Reinhard

2017-01-01

Metallothioneins (MTs) are low-molecular-mass, cysteine-rich, metal binding proteins. In most animal species, they are involved in metal homeostasis and detoxification, and provide protection from oxidative stress. Gastropod MTs are highly diversified, exhibiting unique features and adaptations like metal specificity and multiplications of their metal binding domains. Here, we show that the MT gene of Biomphalaria glabrata, one of the largest MT genes identified so far, is composed in a unique way. The encoding for an MT protein has a three-domain structure and a C-terminal, Cys-rich extension. Using a bioinformatic approach involving structural and in silico analysis of putative transcription factor binding sites (TFBs), we found that this MT gene consists of five exons and four introns. It exhibits a regulatory promoter region containing three metal-responsive elements (MREs) and several TFBs with putative involvement in environmental stress response, and regulation of gene expression. Quantitative real-time polymerase chain reaction (qRT-PCR) data indicate that the MT gene is not inducible by cadmium (Cd) nor by temperature challenges (heat and cold), despite significant Cd uptake within the midgut gland and the high Cd tolerance of metal-exposed snails. PMID:28800079

Photophysical Characterization and BSA Interaction of Direct Ring Carboxy Functionalized Symmetrical squaraine Dyes

NASA Astrophysics Data System (ADS)

Saikiran, Maryala; Pandey, Shyam S.; Hayase, Shuzi; Kato, Tamaki

2017-11-01

A series of far-red sensitive symmetrical squaraine dyes bearing direct -COOH functionalized indole ring were synthesized, characterized and subjected to photophysical investigations. These symmetrical squaraine dyes were then subjected to investigate their interaction with bovine serum albumin (BSA) in Phosphate buffer solutions. All the squaraine dyes under investigation exhibit intense and sharp optical absorption mainly in the far-red wavelength region from 550 nm -700 nm having very high molar extinction coefficients from 1.3 × 105 dm3.mol-1.cm-1. A very small Stokes shift of 10-17 nm indicates the rigid conformational structure of squaraine chromophore. Interaction of these dyes with BSA leads to not only enhanced emission intensity but also bathochromically shifted absorption maximum due to formation of dye-BSA conjugate. These dyes bind strongly with BSA having about an order of magnitude higher binding constant as compared to the reported squaraine dyes. Amongst the symmetrical squaraine dyes investigated in this work one bearing substituents like trifluorobutyl as alkyl chain at N-position of indole ring and carboxylic acid on benzene ring at the terminal (SQ-26) exhibited highest association with the BSA having very high binding constant 8.01 × 106 M-1.

Nickel-quinolones interaction. Part 4. Structure and biological evaluation of nickel(II)-enrofloxacin complexes compared to zinc(II) analogues.

PubMed

Skyrianou, Kalliopi C; Psycharis, Vassilis; Raptopoulou, Catherine P; Kessissoglou, Dimitris P; Psomas, George

2011-01-01

The nickel(II) complexes with the second-generation quinolone antibacterial agent enrofloxacin in the presence or absence of the nitrogen-donor heterocyclic ligands 1,10-phenanthroline, 2,2'-bipyridine or pyridine have been synthesized and characterized. Enrofloxacin acts as bidentate ligand coordinated to Ni(II) ion through the ketone oxygen and a carboxylato oxygen. The crystal structure of (1,10-phenanthroline)bis(enrofloxacinato)nickel(II) has been determined by X-ray crystallography. UV study of the interaction of the complexes with calf-thymus DNA (CT DNA) has shown that they bind to CT DNA and bis(pyridine)bis(enrofloxacinato)nickel(II) exhibits the highest binding constant to CT DNA. The cyclic voltammograms of the complexes have shown that in the presence of CT DNA the complexes can bind to CT DNA by the intercalative binding mode which has also been verified by DNA solution viscosity measurements. Competitive study with ethidium bromide (EB) has shown that the complexes can displace the DNA-bound EB indicating that they bind to DNA in strong competition with EB. The complexes exhibit good binding propensity to human or bovine serum albumin protein having relatively high binding constant values. The biological properties of the complexes have been evaluated in comparison to the corresponding Zn(II) enrofloxacinato complexes as well as Ni(II) complexes with the first-generation quinolone oxolinic acid. Copyright © 2010 Elsevier Inc. All rights reserved.

Molecular characterization and functional analysis of pheromone binding protein 1 from Cydia pomonella (L.).

PubMed

Tian, Z; Zhang, Y

2016-12-01

A full-length cDNA encoding Cydia pomonella pheromone binding protein 1 (CpomPBP1) was cloned and characterized. CpomPBP1, possessing the typical characteristics of lepidopteran odorant binding proteins, was detected to be specifically expressed in the antennae of male and female moths at the mRNA and protein level. Soluble recombinant CpomPBP1 was subjected to in vitro binding to analyse its binding properties and to search for potentially active semiochemicals. A competitive binding assay showed that three 12-carbon ligands, codlemone, 1-dodecanol and E,E-2,4-dodecadienal, were able to bind to CpomPBP1 in decreasing order of affinity. Moreover, unlike the wild-type CpomPBP1, the C-terminus truncated CpomPBP1 exhibited high affinity to ligands even in an acidic environment, suggesting that the C-terminus plays a role in preventing ligands from binding to CpomPBP1 in a lower pH environment. © 2016 The Royal Entomological Society.

Functional somatostatin receptors on a rat pancreatic acinar cell line

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

Viguerie, N.; Tahiri-Jouti, N.; Esteve, J.P.

1988-07-01

Somatostatin receptors from a rat pancreatic acinar cell line, AR4-2J, were characterized biochemically, structurally, and functionally. Binding of {sup 125}I-(Tyr{sup 11})Somatostatin to AR4-2J cells was saturable, exhibiting a single class of high-affinity binding sites with a maximal binding capacity of 258 {plus minus} 20 fmol/10{sup 6} cells. Somatostatin receptor structure was analyzed by covalently cross-linking {sup 125}I-(Tyr{sup 11})somatostatin to its plasma membrane receptors. Gel electrophoresis and autoradiography of cross-linked proteins revealed a peptide containing the somatostatin receptor. Somatostatin inhibited vasoactive intestinal peptide (VIP)-stimulated adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP) formation in a dose-dependent manner. The concentration of somatostatin that caused half-maximal inhibitionmore » of cAMP formation was close to the receptor affinity for somatostatin. Pertussis toxin pretreatment of AR4-2J cells prevented somatostatin inhibition of VIP-stimulated cAMP formation as well as somatostatin binding. The authors conclude that AR4-2J cells exhibit functional somatostatin receptors that retain both specificity and affinity of the pancreatic acinar cell somatostatin receptors and act via the pertussis toxin-sensitive guanine nucleotide-binding protein N{sub i} to inhibit adenylate cyclase.« less

Identification of distant drug off-targets by direct superposition of binding pocket surfaces.

PubMed

Schumann, Marcel; Armen, Roger S

2013-01-01

Correctly predicting off-targets for a given molecular structure, which would have the ability to bind a large range of ligands, is both particularly difficult and important if they share no significant sequence or fold similarity with the respective molecular target ("distant off-targets"). A novel approach for identification of off-targets by direct superposition of protein binding pocket surfaces is presented and applied to a set of well-studied and highly relevant drug targets, including representative kinases and nuclear hormone receptors. The entire Protein Data Bank is searched for similar binding pockets and convincing distant off-target candidates were identified that share no significant sequence or fold similarity with the respective target structure. These putative target off-target pairs are further supported by the existence of compounds that bind strongly to both with high topological similarity, and in some cases, literature examples of individual compounds that bind to both. Also, our results clearly show that it is possible for binding pockets to exhibit a striking surface similarity, while the respective off-target shares neither significant sequence nor significant fold similarity with the respective molecular target ("distant off-target").

Identification of Distant Drug Off-Targets by Direct Superposition of Binding Pocket Surfaces

PubMed Central

Schumann, Marcel; Armen, Roger S.

2013-01-01

Correctly predicting off-targets for a given molecular structure, which would have the ability to bind a large range of ligands, is both particularly difficult and important if they share no significant sequence or fold similarity with the respective molecular target (“distant off-targets”). A novel approach for identification of off-targets by direct superposition of protein binding pocket surfaces is presented and applied to a set of well-studied and highly relevant drug targets, including representative kinases and nuclear hormone receptors. The entire Protein Data Bank is searched for similar binding pockets and convincing distant off-target candidates were identified that share no significant sequence or fold similarity with the respective target structure. These putative target off-target pairs are further supported by the existence of compounds that bind strongly to both with high topological similarity, and in some cases, literature examples of individual compounds that bind to both. Also, our results clearly show that it is possible for binding pockets to exhibit a striking surface similarity, while the respective off-target shares neither significant sequence nor significant fold similarity with the respective molecular target (“distant off-target”). PMID:24391782

Switch control pocket inhibitors of p38-MAP kinase. Durable type II inhibitors that do not require binding into the canonical ATP hinge region.

PubMed

Ahn, Yu Mi; Clare, Michael; Ensinger, Carol L; Hood, Molly M; Lord, John W; Lu, Wei-Ping; Miller, David F; Patt, William C; Smith, Bryan D; Vogeti, Lakshminarayana; Kaufman, Michael D; Petillo, Peter A; Wise, Scott C; Abendroth, Jan; Chun, Lawrence; Clark, Robin; Feese, Michael; Kim, Hidong; Stewart, Lance; Flynn, Daniel L

2010-10-01

Switch control pocket inhibitors of p38-alpha kinase are described. Durable type II inhibitors were designed which bind to arginines (Arg67 or Arg70) that function as key residues for mediating phospho-threonine 180 dependant conformational fluxing of p38-alpha from an inactive type II state to an active type I state. Binding to Arg70 in particular led to potent inhibitors, exemplified by DP-802, which also exhibited high kinase selectivity. Binding to Arg70 obviated the requirement for binding into the ATP Hinge region. X-ray crystallography revealed that DP-802 and analogs induce an enhanced type II conformation upon binding to either the unphosphorylated or the doubly phosphorylated form of p38-alpha kinase. Copyright © 2010 Elsevier Ltd. All rights reserved.

Binding-Induced Fluorescence of Serotonin Transporter Ligands: A Spectroscopic and Structural Study of 4-(4-(Dimethylamino)phenyl)-1-methylpyridinium (APP+) and APP+ Analogues

PubMed Central

2014-01-01

The binding-induced fluorescence of 4-(4-(dimethylamino)-phenyl)-1-methylpyridinium (APP+) and two new serotonin transporter (SERT)-binding fluorescent analogues, 1-butyl-4-[4-(1-dimethylamino)phenyl]-pyridinium bromide (BPP+) and 1-methyl-4-[4-(1-piperidinyl)phenyl]-pyridinium (PPP+), has been investigated. Optical spectroscopy reveals that these probes are highly sensitive to their chemical microenvironment, responding to variations in polarity with changes in transition energies and responding to changes in viscosity or rotational freedom with emission enhancements. Molecular docking calculations reveal that the probes are able to access the nonpolar and conformationally restrictive binding pocket of SERT. As a result, the probes exhibit previously not identified binding-induced turn-on emission that is spectroscopically distinct from dyes that have accumulated intracellularly. Thus, binding and transport dynamics of SERT ligands can be resolved both spatially and spectroscopically. PMID:24460204

Sugar-binding and crystallographic studies of an arabinose-binding protein mutant (Met108Leu) that exhibits enhanced affinity and altered specificity.

PubMed

Vermersch, P S; Lemon, D D; Tesmer, J J; Quiocho, F A

1991-07-16

In addition to hydrogen bonds, van der Waals forces contribute to the affinity of protein-carbohydrate interactions. Nonpolar van der Waals contacts in the complexes of the L-arabinose-binding protein (ABP) with monosaccharides have been studied by means of site-directed mutagenesis, equilibrium and rapid kinetic binding techniques, and X-ray crystallography. ABP, a periplasmic transport receptor of Escherichia coli, binds L-arabinose, D-galactose, and D-fucose with preferential affinity in the order of Ara greater than Gal much greater than Fuc. Well-refined, high-resolution structures of ABP complexed with the three sugars revealed that the structural differences in the ABP-sugar complexes are localized around C5 of the sugars, where the equatorial H of Ara has been substituted for CH3 (Fuc) or CH2OH (Gal). The side chain of Met108 undergoes a sterically dictated, ligand-specific, conformational change to optimize nonpolar interactions between its methyl group and the sugar. We found that the Met108Leu ABP binds Gal tighter than wild-type ABP binds Ara and exhibits a preference for ligand in the order of Gal much greater than Fuc greater than Ara. The differences in affinity can be attributed to differences in the dissociation rates of the ABP-sugar complexes. We have refined at better than 1.7-A resolution the crystal structures of the Met108Leu ABP complexed with each of the sugars and offer a molecular explanation for the altered binding properties.

Aza-Bambusurils En Route to Anion Transporters.

PubMed

Singh, Mandeep; Solel, Ephrath; Keinan, Ehud; Reany, Ofer

2016-06-20

Previous calculations of anion binding with various bambusuril analogs predicted that the replacement of oxygen by nitrogen atoms to produce semiaza-bambus[6]urils would award these new cavitands with multiple anion binding properties. This study validates the hypothesis by efficient synthesis, crystallography, thermogravimetric analysis and calorimetry. These unique host molecules are easily accessible from the corresponding semithio-bambusurils in a one-pot reaction, which converts a single anion receptor into a potential anion channel. Solid-state structures exhibit simultaneous accommodation of three anions, linearly positioned within the cavity along the main symmetry axis. The ability to hold anions at a short distance of about 4 Å is reminiscent of natural chloride channels in E. coli, which exhibit similar distances between their adjacent anion binding sites. The calculated transition-state energy for double-anion movement through the channel suggests that although these host-guest complexes are thermodynamically stable they enjoy high kinetic flexibility to render them efficient anion channels. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The mRNA-bound proteome of the early fly embryo

PubMed Central

Wessels, Hans-Hermann; Imami, Koshi; Baltz, Alexander G.; Kolinski, Marcin; Beldovskaya, Anastasia; Selbach, Matthias; Small, Stephen; Ohler, Uwe; Landthaler, Markus

2016-01-01

Early embryogenesis is characterized by the maternal to zygotic transition (MZT), in which maternally deposited messenger RNAs are degraded while zygotic transcription begins. Before the MZT, post-transcriptional gene regulation by RNA-binding proteins (RBPs) is the dominant force in embryo patterning. We used two mRNA interactome capture methods to identify RBPs bound to polyadenylated transcripts within the first 2 h of Drosophila melanogaster embryogenesis. We identified a high-confidence set of 476 putative RBPs and confirmed RNA-binding activities for most of 24 tested candidates. Most proteins in the interactome are known RBPs or harbor canonical RBP features, but 99 exhibited previously uncharacterized RNA-binding activity. mRNA-bound RBPs and TFs exhibit distinct expression dynamics, in which the newly identified RBPs dominate the first 2 h of embryonic development. Integrating our resource with in situ hybridization data from existing databases showed that mRNAs encoding RBPs are enriched in posterior regions of the early embryo, suggesting their general importance in posterior patterning and germ cell maturation. PMID:27197210

High-oleic canola oil consumption enriches LDL particle cholesteryl oleate content and reduces LDL proteoglycan binding in humans.

PubMed

Jones, Peter J H; MacKay, Dylan S; Senanayake, Vijitha K; Pu, Shuaihua; Jenkins, David J A; Connelly, Philip W; Lamarche, Benoît; Couture, Patrick; Kris-Etherton, Penny M; West, Sheila G; Liu, Xiaoran; Fleming, Jennifer A; Hantgan, Roy R; Rudel, Lawrence L

2015-02-01

Oleic acid consumption is considered cardio-protective according to studies conducted examining effects of the Mediterranean diet. However, animal models have shown that oleic acid consumption increases LDL particle cholesteryl oleate content which is associated with increased LDL-proteoglycan binding and atherosclerosis. The objective was to examine effects of varying oleic, linoleic and docosahexaenoic acid consumption on human LDL-proteoglycan binding in a non-random subset of the Canola Oil Multi-center Intervention Trial (COMIT) participants. COMIT employed a randomized, double-blind, five-period, cross-over trial design. Three of the treatment oil diets: 1) a blend of corn/safflower oil (25:75); 2) high oleic canola oil; and 3) DHA-enriched high oleic canola oil were selected for analysis of LDL-proteoglycan binding in 50 participants exhibiting good compliance. LDL particles were isolated from frozen plasma by gel filtration chromatography and LDL cholesteryl esters quantified by mass-spectrometry. LDL-proteoglycan binding was assessed using surface plasmon resonance. LDL particle cholesterol ester fatty acid composition was sensitive to the treatment fatty acid compositions, with the main fatty acids in the treatments increasing in the LDL cholesterol esters. The corn/safflower oil and high-oleic canola oil diets lowered LDL-proteoglycan binding relative to their baseline values (p = 0.0005 and p = 0.0012, respectively). At endpoint, high-oleic canola oil feeding resulted in lower LDL-proteoglycan binding than corn/safflower oil (p = 0.0243) and DHA-enriched high oleic canola oil (p = 0.0249), although high-oleic canola oil had the lowest binding at baseline (p = 0.0344). Our findings suggest that high-oleic canola oil consumption in humans increases cholesteryl oleate percentage in LDL, but in a manner not associated with a rise in LDL-proteoglycan binding. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Yeast mitochondrial HMG proteins: DNA-binding properties of the most evolutionarily divergent component of mitochondrial nucleoids.

PubMed

Bakkaiova, Jana; Marini, Victoria; Willcox, Smaranda; Nosek, Jozef; Griffith, Jack D; Krejci, Lumir; Tomaska, Lubomir

2015-12-08

Yeast mtDNA is compacted into nucleoprotein structures called mitochondrial nucleoids (mt-nucleoids). The principal mediators of nucleoid formation are mitochondrial high-mobility group (HMG)-box containing (mtHMG) proteins. Although these proteins are some of the fastest evolving components of mt-nucleoids, it is not known whether the divergence of mtHMG proteins on the level of their amino acid sequences is accompanied by diversification of their biochemical properties. In the present study we performed a comparative biochemical analysis of yeast mtHMG proteins from Saccharomyces cerevisiae (ScAbf2p), Yarrowia lipolytica (YlMhb1p) and Candida parapsilosis (CpGcf1p). We found that all three proteins exhibit relatively weak binding to intact dsDNA. In fact, ScAbf2p and YlMhb1p bind quantitatively to this substrate only at very high protein to DNA ratios and CpGcf1p shows only negligible binding to dsDNA. In contrast, the proteins exhibit much higher preference for recombination intermediates such as Holliday junctions (HJ) and replication forks (RF). Therefore, we hypothesize that the roles of the yeast mtHMG proteins in maintenance and compaction of mtDNA in vivo are in large part mediated by their binding to recombination/replication intermediates. We also speculate that the distinct biochemical properties of CpGcf1p may represent one of the prerequisites for frequent evolutionary tinkering with the form of the mitochondrial genome in the CTG-clade of hemiascomycetous yeast species. © 2016 Authors.

High resolution mapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgG1 variants with improved binding to the Fc gamma R.

PubMed

Shields, R L; Namenuk, A K; Hong, K; Meng, Y G; Rae, J; Briggs, J; Xie, D; Lai, J; Stadlen, A; Li, B; Fox, J A; Presta, L G

2001-03-02

Immunoglobulin G (IgG) Fc receptors play a critical role in linking IgG antibody-mediated immune responses with cellular effector functions. A high resolution map of the binding site on human IgG1 for human Fc gamma RI, Fc gamma RIIA, Fc gamma RIIB, Fc gamma RIIIA, and FcRn receptors has been determined. A common set of IgG1 residues is involved in binding to all Fc gamma R; Fc gamma RII and Fc gamma RIII also utilize residues outside this common set. In addition to residues which, when altered, abrogated binding to one or more of the receptors, several residues were found that improved binding only to specific receptors or simultaneously improved binding to one type of receptor and reduced binding to another type. Select IgG1 variants with improved binding to Fc gamma RIIIA exhibited up to 100% enhancement in antibody-dependent cell cytotoxicity using human effector cells; these variants included changes at residues not found at the binding interface in the IgG/Fc gamma RIIIA co-crystal structure (Sondermann, P., Huber, R., Oosthuizen, V., and Jacob, U. (2000) Nature 406, 267-273). These engineered antibodies may have important implications for improving antibody therapeutic efficacy.

The Src SH2 domain interacts dynamically with the focal adhesion kinase binding site as demonstrated by paramagnetic NMR spectroscopy.

PubMed

Lindfors, Hanna E; Drijfhout, Jan Wouter; Ubbink, Marcellus

2012-06-01

The interaction between the tyrosine kinases Src and focal adhesion kinase (FAK) is a key step in signaling processes from focal adhesions. The phosphorylated tyrosine residue 397 in FAK is able to bind the Src SH2 domain. To establish the extent of the FAK binding motif, the binding affinity of the SH2 domain for phosphorylated and unphosphorylated FAK-derived peptides of increasing length was determined and compared with that of the internal Src SH2 binding site. It is shown that the FAK peptides have higher affinity than the internal binding site and that seven negative residues adjacent to the core SH2 binding motif increase the binding constant 30-fold. A rigid spin-label incorporated in the FAK peptides was used to establish on the basis of paramagnetic relaxation enhancement whether the peptide-protein complex is well defined. A large spread of the paramagnetic effects on the surface of the SH2 domain suggests that the peptide-protein complex exhibits dynamics, despite the high affinity of the peptide. The strong electrostatic interaction between the positive side of the SH2 domain and the negative peptide results in a high affinity but may also favor a dynamic interaction. Copyright © 2012 Wiley Periodicals, Inc.

Compact Biocompatible Quantum Dots Functionalized for Cellular Imaging

PubMed Central

Liu, Wenhao; Howarth, Mark; Greytak, Andrew B.; Zheng, Yi; Nocera, Daniel G.; Ting, Alice Y.; Bawendi, Moungi G.

2009-01-01

We present a family of water-soluble quantum dots (QDs) that exhibit low nonspecific binding to cells, small hydrodynamic diameter, tunable surface charge, high quantum yield, and good solution stability across a wide pH range. These QDs are amenable to covalent modification via simple carbodiimide coupling chemistry, which is achieved by functionalizing the surface of QDs with a new class of heterobifunctional ligands incorporating dihydrolipoic acid, a short poly(ethylene glycol) (PEG) spacer, and an amine or carboxylate terminus. The covalent attachment of molecules is demonstrated by appending a rhodamine dye to form a QD-dye conjugate exhibiting fluorescence resonance energy transfer (FRET). High-affinity labeling is demonstrated by covalent attachment of streptavidin, thus enabling the tracking of biotinylated epidermal growth factor (EGF) bound to EGF receptor on live cells. In addition, QDs solubilized with the heterobifunctional ligands retain their metal-affinity driven conjugation chemistry with polyhistidine-tagged proteins. This dual functionality is demonstrated by simultaneous covalent attachment of a rhodamine FRET acceptor and binding of polyhistidine-tagged streptavidin on the same nanocrystal to create a targeted QD, which exhibits dual-wavelength emission. Such emission properties could serve as the basis for ratiometric sensing of the cellular receptor’s local chemical environment. PMID:18177042

Slow ligand-induced conformational switch increases the catalytic rate in Plasmodium falciparum hypoxanthine guanine xanthine phosphoribosyltransferase.

PubMed

Roy, Sourav; Karmakar, Tarak; Prahlada Rao, Vasudeva S; Nagappa, Lakshmeesha K; Balasubramanian, Sundaram; Balaram, Hemalatha

2015-05-01

P. falciparum (Pf) hypoxanthine guanine xanthine phosphoribosyltransferase (HGXPRT) exhibits a unique mechanism of activation where the enzyme switches from a low activity (unactivated) to a high activity (activated) state upon pre-incubation with substrate/products. Xanthine phosphoribosylation by unactivated PfHGXPRT exhibits a lag phase, the duration of which reduces with an increase in concentration of the enzyme or substrate, PRPP·Mg(2+). Activated PfHGXPRT does not display the lag phase and exhibits a ten-fold drop in the Km value for PRPP·Mg(2+). These observations suggest the involvement of ligand-mediated oligomerization and conformational changes in the process of activation. The dipeptide Leu-Lys in the PPi binding site of human and T. gondii HG(X)PRT that facilitates PRPP·Mg(2+) binding by isomerization from trans to cis conformation is conserved in PfHGXPRT. Free energy calculations using the well-tempered metadynamics technique show the ligand-free enzyme to be more stable when this dipeptide is in the trans conformation than in the cis conformation. The high rotational energy barrier observed for the conformational change from experimental and computational studies permits delineation of the activation mechanism.

Structural Basis for High Affinity Volatile Anesthetic Binding in a Natural 4-helix Bundle Protein

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

Liu,R.; Loll, P.; Eckenhoff, R.

2005-01-01

Physiologic sites for inhaled anesthetics are presumed to be cavities within transmembrane 4-{alpha}-helix bundles of neurotransmitter receptors, but confirmation of binding and structural detail of such sites remains elusive. To provide such detail, we screened soluble proteins containing this structural motif, and found only one that exhibited evidence of strong anesthetic binding. Ferritin is a 24-mer of 4-{alpha}-helix bundles; both halothane and isoflurane bind with K{sub A} values of {approx}10{sup 5} M{sup -1, } higher than any previously reported inhaled anesthetic-protein interaction. The crystal structures of the halothane/apoferritin and isoflurane/apoferritin complexes were determined at 1.75 Angstroms resolution, revealing a commonmore » anesthetic binding pocket within an interhelical dimerization interface. The high affinity is explained by several weak polar contacts and an optimal host/guest packing relationship. Neither the acidic protons nor ether oxygen of the anesthetics contribute to the binding interaction. Compared with unliganded apoferritin, the anesthetic produced no detectable alteration of structure or B factors. The remarkably high affinity of the anesthetic/apoferritin complex implies greater selectivity of protein sites than previously thought, and suggests that direct protein actions may underlie effects at lower than surgical levels of anesthetic, including loss of awareness.« less

High Structural Resolution Hydroxyl Radical Protein Footprinting Reveals an Extended Robo1-Heparin Binding Interface*

PubMed Central

Li, Zixuan; Moniz, Heather; Wang, Shuo; Ramiah, Annapoorani; Zhang, Fuming; Moremen, Kelley W.; Linhardt, Robert J.; Sharp, Joshua S.

2015-01-01

Interaction of transmembrane receptors of the Robo family and the secreted protein Slit provides important signals in the development of the central nervous system and regulation of axonal midline crossing. Heparan sulfate, a sulfated linear polysaccharide modified in a complex variety of ways, serves as an essential co-receptor in Slit-Robo signaling. Previous studies have shown that closely related heparin octasaccharides bind to Drosophila Robo directly, and surface plasmon resonance analysis revealed that Robo1 binds more tightly to full-length unfractionated heparin. For the first time, we utilized electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting to identify two separate binding sites for heparin interaction with Robo1: one binding site at the previously identified site for heparin dp8 and a second binding site at the N terminus of Robo1 that is disordered in the x-ray crystal structure. Mutagenesis of the identified N-terminal binding site exhibited a decrease in binding affinity as measured by surface plasmon resonance and heparin affinity chromatography. Footprinting also indicated that heparin binding induces a minor change in the conformation and/or dynamics of the Ig2 domain, but no major conformational changes were detected. These results indicate a second low affinity binding site in the Robo-Slit complex as well as suggesting the role of the Ig2 domain of Robo1 in heparin-mediated signal transduction. This study also marks the first use of electron transfer dissociation-based high spatial resolution hydroxyl radical protein footprinting, which shows great utility for the characterization of protein-carbohydrate complexes. PMID:25752613

Sequestration of U(VI) from Acidic, Alkaline, and High Ionic-Strength Aqueous Media by Functionalized Magnetic Mesoporous Silica Nanoparticles: Capacity and Binding Mechanisms

EPA Science Inventory

Uranium (VI) exhibits little adsorption onto sediment minerals in acidic, alkaline or high ionic-strength aqueous media that often occur in U mining or contaminated sites, which makes U(VI) very mobile and difficult to sequester. In this work, magnetic mesoporous silica nanoparti...

The Yersiniabactin-Associated ATP Binding Cassette Proteins YbtP and YbtQ Enhance Escherichia coli Fitness during High-Titer Cystitis

PubMed Central

Koh, Eun-Ik; Hung, Chia S.

2016-01-01

The Yersinia high-pathogenicity island (HPI) is common to multiple virulence strategies used by Escherichia coli strains associated with urinary tract infection (UTI). Among the genes in this island are ybtP and ybtQ, encoding distinctive ATP binding cassette (ABC) proteins associated with iron(III)-yersiniabactin import in Yersinia pestis. In this study, we compared the impact of ybtPQ on a model E. coli cystitis strain during in vitro culture and experimental murine infections. A ybtPQ-null mutant exhibited no growth defect under standard culture conditions, consistent with nonessentiality in this background. A growth defect phenotype was observed and genetically complemented in vitro during iron(III)-yersiniabactin-dependent growth. Following inoculation into the bladders of C3H/HEN and C3H/HeOuJ mice, this strain exhibited a profound, 106-fold competitive infection defect in the subgroup of mice that progressed to high-titer bladder infections. These results identify a virulence role for YbtPQ in the highly inflammatory microenvironment characteristic of high-titer cystitis. The profound competitive defect may relate to the apparent selection of Yersinia HPI-positive E. coli in uncomplicated clinical UTIs. PMID:26883590

Simple method provides resolution of albumin, lipoprotein, free fraction, and chylomicron to enhance the utility of protein binding assays.

PubMed

Brockman, Adam H; Oller, Haley R; Moreau, Benoît; Kriksciukaite, Kristina; Bilodeau, Mark T

2015-02-12

Medicinal chemists have been encouraged in recent years to embrace high speed protein binding assays. These methods employ dialysis membranes in 96-well format or spin filters. Membrane-based methods do not separate lipoprotein binding from albumin binding and introduce interference despite membrane binding controls. Ultracentrifugation methods, in contrast, do not introduce interference if density gradients can be avoided and they resolve lipoprotein from albumin. A new generation of compact, fast ultracentrifuges facilitates the rapid and fully informative separation of plasma into albumin, albumin/fatty acid complex, lipoprotein, protein-free, and chylomicron fractions with no need of salt or sugar density gradients. We present a simple and fast ultracentrifuge method here for two platinum compounds and a taxane that otherwise bound irreversibly to dialysis membranes and which exhibited distinctive lipoprotein binding behaviors. This new generation of ultracentrifugation methods underscores a need to further discuss protein binding assessments as they relate to medicinal chemistry efforts.

Lipid-binding proteins modulate ligand-dependent trans-activation by peroxisome proliferator-activated receptors and localize to the nucleus as well as the cytoplasm.

PubMed

Helledie, T; Antonius, M; Sorensen, R V; Hertzel, A V; Bernlohr, D A; Kølvraa, S; Kristiansen, K; Mandrup, S

2000-11-01

Peroxisome proliferator-activated receptors (PPARs) are activated by a variety of fatty acids, eicosanoids, and hypolipidemic and insulin-sensitizing drugs. Many of these compounds bind avidly to members of a family of small lipid-binding proteins, the fatty acid-binding proteins (FABPs). Fatty acids are activated to CoA esters, which bind with high affinity to the acyl-CoA-binding protein (ACBP). Thus, the availability of known and potential PPAR ligands may be regulated by lipid-binding proteins. In this report we show by transient transfection of CV-1 cells that coexpression of ACBP and adipocyte lipid-binding protein (ALBP) exerts a ligand- and PPAR subtype-specific attenuation of PPAR-mediated trans-activation, suggesting that lipid-binding proteins, when expressed at high levels, may function as negative regulators of PPAR activation by certain ligands. Expression of ACBP, ALBP, and keratinocyte lipid-binding protein (KLBP) is induced during adipocyte differentiation, a process during which PPARgamma plays a prominent role. We present evidence that endogenous ACBP, ALBP, and KLBP not only localize to the cytoplasm but also exhibit a prominent nuclear localization in 3T3-L1 adipocytes. In addition, forced expression of ACBP, ALBP, and KLBP in CV-1 cells resulted in a substantial accumulation of all three proteins in the nucleus. These results suggest that lipid-binding proteins, contrary to the general assumption, may exert their action in the nucleus as well as in the cytoplasm.

Synthesis and characterization of time-resolved fluorescence probes for evaluation of competitive binding to melanocortin receptors.

PubMed

Alleti, Ramesh; Vagner, Josef; Dehigaspitiya, Dilani Chathurika; Moberg, Valerie E; Elshan, N G R D; Tafreshi, Narges K; Brabez, Nabila; Weber, Craig S; Lynch, Ronald M; Hruby, Victor J; Gillies, Robert J; Morse, David L; Mash, Eugene A

2013-09-01

Probes for use in time-resolved fluorescence competitive binding assays at melanocortin receptors based on the parental ligands MSH(4), MSH(7), and NDP-α-MSH were prepared by solid phase synthesis methods, purified, and characterized. The saturation binding of these probes was studied using HEK-293 cells engineered to overexpress the human melanocortin 4 receptor (hMC4R) as well as the human cholecystokinin 2 receptor (hCCK2R). The ratios of non-specific binding to total binding approached unity at high concentrations for each probe. At low probe concentrations, receptor-mediated binding and uptake was discernable, and so probe concentrations were kept as low as possible in determining Kd values. The Eu-DTPA-PEGO-MSH(4) probe exhibited low specific binding relative to non-specific binding, even at low nanomolar concentrations, and was deemed unsuitable for use in competition binding assays. The Eu-DTPA-PEGO probes based on MSH(7) and NDP-α-MSH exhibited Kd values of 27±3.9nM and 4.2±0.48nM, respectively, for binding with hMC4R. These probes were employed in competitive binding assays to characterize the interactions of hMC4R with monovalent and divalent MSH(4), MSH(7), and NDP-α-MSH constructs derived from squalene. Results from assays with both probes reflected only statistical enhancements, suggesting improper ligand spacing on the squalene scaffold for the divalent constructs. The Ki values from competitive binding assays that employed the MSH(7)-based probe were generally lower than the Ki values obtained when the probe based on NDP-α-MSH was employed, which is consistent with the greater potency of the latter probe. The probe based on MSH(7) was also competed with monovalent, divalent, and trivalent MSH(4) constructs that previously demonstrated multivalent binding in competitive binding assays against a variant of the probe based on NDP-α-MSH. Results from these assays confirm multivalent binding, but suggest a more modest increase in avidity for these MSH(4) constructs than was previously reported. Copyright © 2013 Elsevier Ltd. All rights reserved.

Interaction of SR 33557 with skeletal muscle calcium channel blocker receptors in the baboon: characterization of its binding sites

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

Sol-Rolland, J.; Joseph, M.; Rinaldi-Carmona, M.

1991-05-01

A procedure for the isolation of primate skeletal microsomal membranes was initiated. Membranes exhibited specific enzymatic markers such as 5'-nucleotidase, Ca{sup 2}{sup +},Mg({sup 2}{sup +})-adenosine triphosphatase and an ATP-dependent calcium uptake. Baboon skeletal microsomes bound specifically with high-affinity potent Ca{sup 2}{sup +} channel blockers such as dihydropyridine, phenylalkylamine and benzothiazepine derivatives. Scatchard analysis of equilibrium binding assays with ({sup 3}H)(+)-PN 200-110, ({sup 3}H)(-)-desmethoxyverapamil (( {sup 3}H)(-)-D888) and ({sup 3}H)-d-cis-dilitiazem were consistent with a single class of binding sites for the three radioligands. The pharmacological profile of SR 33557, an original compound with calcium antagonist properties, was investigated using radioligand bindingmore » studies. SR 33557 totally inhibited the specific binding of the three main classes of Ca{sup 2}{sup +} channel effectors and interacted allosterically with them. In addition, SR 33557 bound with high affinity to a homogeneous population of binding sites in baboon skeletal muscle.« less

Why does high pressure destroy co-non-solvency of PNIPAm in aqueous methanol?

PubMed

de Oliveira, Tiago E; Netz, Paulo A; Mukherji, Debashish; Kremer, Kurt

2015-11-28

It is well known that poly(N-isopropylacrylamide) (PNIPAm) exhibits an interesting, yet puzzling, phenomenon of co-non-solvency. Co-non-solvency occurs when two competing good solvents for PNIPAm, such as water and alcohol, are mixed together. As a result, the same PNIPAm collapses within intermediate mixing ratios. This complex conformational transition is driven by preferential binding of methanol with PNIPAm. Interestingly, co-non-solvency can be destroyed when applying high hydrostatic pressures. In this work, using a large scale molecular dynamics simulation employing high pressures, we propose a microscopic picture behind the suppression of the co-non-solvency phenomenon. Based on thermodynamic and structural analysis, our results suggest that the preferential binding of methanol with PNIPAm gets partially lost at high pressures, making the background fluid reasonably homogeneous for the polymer. This is consistent with the hypothesis that the co-non-solvency phenomenon is driven by preferential binding and is not based on depletion effects.

High-affinity 3H-substance P binding to longitudinal muscle membranes of the guinea pig small intestine.

PubMed

Buck, S H; Maurin, Y; Burks, T F; Yamamura, H I

1984-01-30

The binding of 3H-substance P (3H-SP) to longitudinal muscle membranes of the guinea pig small intestine has been characterized. The binding of 3H-SP exhibited a high affinity (Kd = 0.5nM). It was saturable (Bmax = 2 fmoles/mg tissue), reversible, and temperature-dependent. Kinetic studies and competition of 3H-SP binding by unlabeled SP yielded Kd and Ki values, respectively, which were in good agreement with the Kd calculated from saturation studies. The binding of 3H-SP appeared to be dependent on the presence of divalent cations in the incubation buffer. It was displaced by SP and various analogs and fragments in the rank order of SP greater than SP-(2-11) = SP-(3-11) greater than Nle11- SP = physalaemin greater than SP-(4-11) greater than SP-(5-11) greater than eledoisin much greater than SP-(7-11). Our results indicate that 3H-SP binds in longitudinal muscle of the guinea pig small intestine to a biologically relevant receptor which in many respects resembles the SP receptor characterized in the brain and the salivary gland of the rat.

Alpha-amylase inhibitor, CS-1036 binds to serum amylase in a concentration-dependent and saturable manner.

PubMed

Honda, Tomohiro; Kaneno-Urasaki, Yoko; Ito, Takashi; Kimura, Takako; Matsushima, Nobuko; Okabe, Hiromi; Yamasaki, Atsushi; Izumi, Takashi

2014-03-01

(2R,3R,4R)-4-hydroxy-2-(hydroxymethyl)pyrrolidin-3-yl 4-O-(6-deoxy-β-D-glucopyranosyl)-α-D-glucopyranoside (CS-1036), which is an α-amylase inhibitor, exhibited biphasic and sustained elimination with a long t1/2 (18.4-30.0 hours) in rats and monkeys, but exhibited a short t1/2 (3.7-7.9 hours) in humans. To clarify the species differences in the t1/2, the plasma protein binding of CS-1036 was evaluated by ultrafiltration. A concentration-dependent and saturable plasma protein binding of CS-1036 was observed in rats and monkeys with the dissociation rate constant (KD) of 8.95 and 27.2 nM, and maximal binding capacity (Bmax) of 52.8 and 22.1 nM, respectively. By the assessments of the recombinant amylase and immunoprecipitation, the major binding protein of CS-1036 in rats was identified as salivary amylase (KD 5.64 nM). CS-1036 also showed concentration-dependent and saturable binding to human salivary and pancreatic amylase, with similar binding affinity in rats. However, the protein binding of CS-1036 was constant in human plasma (≤10.2%) due to the lower serum amylase level compared with rats and monkeys. From the calculation of the unbound fraction (fu) in plasma based on in vitro KD and Bmax, the dose-dependent increase in fu after oral administration is speculated to lead to a dose-dependent increase in total body clearance and a high area under the curve/dose at lower doses, such as 0.3 mg/kg in rats.

FK506-Binding Proteins and Their Diverse Functions.

PubMed

Tong, Mingming; Jiang, Yu

2015-01-01

FK506 binding proteins (FKBPs) are a family of highly conserved proteins in eukaryotes. The prototype of this protein family, FKBP12, is the binding partner for immunosuppressive drugs FK506 and rapamycin. FKBP12 functions as a cis/trans peptidyl prolyl isomerase (PPIase) that catalyzes interconversion between prolyl cis/trans conformations. Members of the FKBP family contain one or several PPIase domains, which do not always exhibit PPIase activity yet are all essential for their function. FKBPs are involved in diverse cellular functions including protein folding, cellular signaling, apoptosis and transcription. They elicit their function through direct binding and altering conformation of their target proteins, hence acting as molecular switches. In this review, we provide a general summary for the structures and diverse functions of FKBPs found in mammalian cells.

An antibody reactive to the Gly63–Lys68 epitope of NT-proBNP exhibits O-glycosylation-independent binding

PubMed Central

Lee, Yujean; Kim, Hyori; Chung, Junho

2014-01-01

The N-terminal fragment of prohormone brain natriuretic peptide (NT-proBNP) is a commonly used biomarker for the diagnosis of congestive heart failure, although its biological function is not well known. NT-proBNP exhibits heavy O-linked glycosylation, and it is quite difficult to develop an antibody that exhibits glycosylation-independent binding. We developed an antibody that binds to the recombinant NT-proBNP protein and its deglycosylated form with similar affinities in an enzyme immunoassay. The epitope was defined as Gly63–Lys68 based on mimetic peptide screening, site-directed mutagenesis and a competition assay with a peptide mimotope. The nearest O-glycosylation residues are Thr58 and Thr71; therefore, four amino acid residues intervene between the epitope and those residues in both directions. In conclusion, we report that an antibody reactive to Gly63–Lys68 of NT-proBNP exhibits O-glycosylation-independent binding. PMID:25236766

Investigating isoindoline, tetrahydroisoquinoline, and tetrahydrobenzazepine scaffolds for their sigma receptor binding properties.

PubMed

Linkens, Kathryn; Schmidt, Hayden R; Sahn, James J; Kruse, Andrew C; Martin, Stephen F

2018-05-10

Substituted norbenzomorphans are known to display high affinity and selectivity for the two sigma receptor (σR) subtypes. In order to study the effects of simplifying the structures of these compounds, a scaffold hopping strategy was used to design several novel sets of substituted isoindolines, tetrahydroisoquinolines and tetrahydro-2-benzazepines. The binding affinities of these new compounds for the sigma 1 (σ1R) and sigma 2 (σ2R) receptors were determined, and some analogs were identified that exhibit high affinity (K i  ≤ 25 nM) and significant selectivity (>10-fold) for σ1R or σ2R. The preferred binding modes of selected compounds for the σ1R are predicted by modeling studies, and the nature of substituents on the aromatic ring and the nitrogen atom of the bicyclic skeleton appears to affect the preferred binding orientation of σ1R-preferring ligands. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Localization in human interleukin 2 of the binding site to the alpha chain (p55) of the interleukin 2 receptor.

PubMed Central

Sauvé, K; Nachman, M; Spence, C; Bailon, P; Campbell, E; Tsien, W H; Kondas, J A; Hakimi, J; Ju, G

1991-01-01

Human interleukin 2 (IL-2) analogs with defined amino acid substitutions were used to identify specific residues that interact with the 55-kDa subunit (p55) or alpha chain of the human IL-2 receptor. Analog proteins containing specific substitutions for Lys-35, Arg-38, Phe-42, or Lys-43 were inactive in competitive binding assays for p55. All of these analogs retained substantial competitive binding to the intermediate-affinity p70 subunit (beta chain) of the receptor complex. The analogs varied in ability to interact with the high-affinity p55/p70 receptor. Despite the lack of binding to p55, all analogs exhibited significant biological activity, as assayed on the murine CTLL cell line. The dissociation constants of Arg-38 and Phe-42 analogs for p70 were consistent with intermediate-affinity binding; the Kd values were not significantly affected by the presence of p55 in binding to the high-affinity IL-2 receptor complex. These results confirm the importance of the B alpha-helix in IL-2 as the locus for p55-receptor binding and support a revised model of IL-2-IL-2 receptor interaction. PMID:2052547

HIP1 and HIP12 display differential binding to F-actin, AP2, and clathrin. Identification of a novel interaction with clathrin light chain.

PubMed

Legendre-Guillemin, Valerie; Metzler, Martina; Charbonneau, Martine; Gan, Lu; Chopra, Vikramjit; Philie, Jacynthe; Hayden, Michael R; McPherson, Peter S

2002-05-31

Huntingtin-interacting protein 1 (HIP1) and HIP12 are orthologues of Sla2p, a yeast protein with essential functions in endocytosis and regulation of the actin cytoskeleton. We now report that HIP1 and HIP12 are major components of the clathrin coat that interact but differ in their ability to bind clathrin and the clathrin adaptor AP2. HIP1 contains a clathrin-box and AP2 consensus-binding sites that display high affinity binding to the terminal domain of the clathrin heavy chain and the ear domain of the AP2 alpha subunit, respectively. These consensus sites are poorly conserved in HIP12 and correspondingly, HIP12 does not bind to AP2 nor does it demonstrate high affinity clathrin binding. Moreover, HIP12 co-sediments with F-actin in contrast to HIP1, which exhibits no interaction with actin in vitro. Despite these differences, both proteins efficiently stimulate clathrin assembly through their central helical domain. Interestingly, in both HIP1 and HIP12, this domain binds directly to the clathrin light chain. Our data suggest that HIP1 and HIP12 play related yet distinct functional roles in clathrin-mediated endocytosis.

Computational and experimental studies of the interaction between phospho-peptides and the C-terminal domain of BRCA1

NASA Astrophysics Data System (ADS)

Anisimov, Victor M.; Ziemys, Arturas; Kizhake, Smitha; Yuan, Ziyan; Natarajan, Amarnath; Cavasotto, Claudio N.

2011-11-01

The C-terminal domain of BRCA1(BRCT) is involved in the DNA repair pathway by recognizing the pSXXF motif in interacting proteins. It has been reported that short peptides containing this motif bind to BRCA1(BRCT) in the micromolar range with high specificity. In this work, the binding of pSXXF peptides has been studied computationally and experimentally in order to characterize their interaction with BRCA1(BRCT). Elucidation of the contacts that drive the protein-ligand interaction is critical for the development of high affinity small-molecule BRCA1 inhibitors. Molecular dynamics (MD) simulations revealed the key role of threonine at the peptide P+2 position in providing structural rigidity to the ligand in the bound state. The mutation at P+1 had minor effects. Peptide extension at the N-terminal position with the naphthyl amino acid exhibited a modest increase in binding affinity, what could be explained by the dispersion interaction of the naphthyl side-chain with a hydrophobic patch. Three in silico end-point methods were considered for the calculation of binding free energy. The Molecular Mechanics Poisson-Boltzmann Surface Area and the Solvated Interaction Energy methods gave reasonable agreement with experimental data, exhibiting a Pearlman predictive index of 0.71 and 0.78, respectively. The MM-quantum mechanics-surface area method yielded improved results, which was characterized by a Pearlman index of 0.78. The correlation coefficients were 0.59, 0.61 and 0.69, respectively. The ability to apply a QM level of theory within an end-point binding free energy protocol may provide a way for a consistent improvement of accuracy in computer-aided drug design.

Integrated In Silico-In Vitro Identification and Characterization of the SH3-Mediated Interaction between Human PTTG and its Cognate Partners in Medulloblastoma.

PubMed

Liu, Jiangang; Wang, Dapeng; Li, Yanyan; Yao, Hui; Zhang, Nan; Zhang, Xuewen; Zhong, Fangping; Huang, Yulun

2018-06-01

The human pituitary tumor-transforming gene is an oncogenic protein which serves as a central hub in the cellular signaling network of medulloblastoma. The protein contains two vicinal PxxP motifs at its C terminus that are potential binding sites of peptide-recognition SH3 domains. Here, a synthetic protocol that integrated in silico analysis and in vitro assay was described to identify the SH3-binding partners of pituitary tumor-transforming gene in the gene expression profile of medulloblastoma. In the procedure, a variety of structurally diverse, non-redundant SH3 domains with high gene expression in medulloblastoma were compiled, and their three-dimensional structures were either manually retrieved from the protein data bank database or computationally modeled through bioinformatics technique. The binding capability of these domains towards the two PxxP-containing peptides m1p: 161 LGPPSPVK 168 and m2p: 168 KMPSPPWE 175 of pituitary tumor-transforming gene were ranked by structure-based scoring and fluorescence-based assay. Consequently, a number of SH3 domains, including MAP3K and PI3K, were found to have moderate or high affinity for m1p and/or m2p. Interestingly, the two overlapping peptides exhibits a distinct binding profile to these identified domain partners, suggesting that the binding selectivity of m1p and m2p is optimized across the medulloblastoma expression spectrum by competing for domain candidates. In addition, two redesigned versions of m1p peptide ware obtained via a structure-based rational mutation approach, which exhibited an increased affinity for the domain as compared to native peptide.

γ-secretase binding sites in aged and Alzheimer's disease human cerebrum: the choroid plexus as a putative origin of CSF Aβ.

PubMed

Liu, Fei; Xue, Zhi-Qin; Deng, Si-Hao; Kun, Xiong; Luo, Xue-Gang; Patrylo, Peter R; Rose, Gregory M; Cai, Huaibin; Struble, Robert G; Cai, Yan; Yan, Xiao-Xin

2013-05-01

Deposition of β -amyloid (Aβ) peptides, cleavage products of β-amyloid precursor protein (APP) by β-secretase-1 (BACE1) and γ-secretase, is a neuropathological hallmark of Alzheimer's disease (AD). γ-Secretase inhibition is a therapeutical anti-Aβ approach, although changes in the enzyme's activity in AD brain are unclear. Cerebrospinal fluid (CSF) Aβ peptides are thought to derive from brain parenchyma and thus may serve as biomarkers for assessing cerebral amyloidosis and anti-Aβ efficacy. The present study compared active γ-secretase binding sites with Aβ deposition in aged and AD human cerebrum, and explored the possibility of Aβ production and secretion by the choroid plexus (CP). The specific binding density of [(3) H]-L-685,458, a radiolabeled high-affinity γ-secretase inhibitor, in the temporal neocortex and hippocampal formation was similar for AD and control cases with similar ages and post-mortem delays. The CP in post-mortem samples exhibited exceptionally high [(3) H]-L-685,458 binding density, with the estimated maximal binding sites (Bmax) reduced in the AD relative to control groups. Surgically resected human CP exhibited APP, BACE1 and presenilin-1 immunoreactivity, and β-site APP cleavage enzymatic activity. In primary culture, human CP cells also expressed these amyloidogenic proteins and released Aβ40 and Aβ42 into the medium. Overall, our results suggest that γ-secretase activity appears unaltered in the cerebrum in AD and is not correlated with regional amyloid plaque pathology. The CP appears to be a previously unrecognised non-neuronal contributor to CSF Aβ, probably at reduced levels in AD. © 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

γ-Secretase binding sites in aged and Alzheimer’s disease human cerebrum: The choroid plexus as a putative origin of CSF Aβ

PubMed Central

Liu, Fei; Xue, Zhi-Qin; Deng, Si-Hao; Kun, Xiong; Luo, Xue-Gang; Patrylo, Peter R.; Rose, Gregory M.; Cai, Huaibin; Struble, Robert G.; Cai, Yan; Yan, Xiao-Xin

2013-01-01

Deposition of β-amyloid (Aβ) peptides, cleavage products of β-amyloid precursor protein (APP) by β-secretase-1 (BACE1) and γ-secretase, is a neuropathological hallmark of Alzheimer’s disease (AD). γ-Secretase inhibition is a therapeutical anti-Aβ approach, although less is clear about the change of the enzyme’s activity in AD brain. Cerebrospinal fluid (CSF) Aβ peptides are considered to derive from brain parenchyma, thus may serve as biomarkers for assessing cerebral amyloidosis and anti-Aβ efficacy. The present study compared active γ-secretase binding sites with Aβ deposition in aged and AD human cerebrum, and explored a possibility of Aβ production and secretion by the choroid plexus (CP). Specific binding density of [3H]-L-685,458, a radiolabeled high affinity γ-secretase inhibitor, in the temporal neocortex and hippocampal formation was similar for AD and control cases with comparable ages and postmortem delays. The CP in postmortem samples exhibited exceptionally high [3H]-L-685,458 binding density, with the estimated maximal binding sites (Bmax) reduced in the AD relative to control groups. Surgically resected human CP exhibited APP, BACE1 and presenilin-1 immunoreactivity, and β-site APP cleavage enzymatic activity. In primary culture, human CP cells also expressed these amyloidogenic proteins but released Aβ40 and Aβ42 into the medium. These results suggest that γ-secretase activity appears not altered in the cerebrum in AD related to aged control, nor correlated with regional amyloid plaque pathology. The choroid plexus appears to represent a novel non-neuronal source in the brain that may contribute Aβ into cerebrospinal fluid, probably at reduced levels in AD. PMID:23432732

De novo design and engineering of functional metal and porphyrin-binding protein domains

NASA Astrophysics Data System (ADS)

Everson, Bernard H.

In this work, I describe an approach to the rational, iterative design and characterization of two functional cofactor-binding protein domains. First, a hybrid computational/experimental method was developed with the aim of algorithmically generating a suite of porphyrin-binding protein sequences with minimal mutual sequence information. This method was explored by generating libraries of sequences, which were then expressed and evaluated for function. One successful sequence is shown to bind a variety of porphyrin-like cofactors, and exhibits light- activated electron transfer in mixed hemin:chlorin e6 and hemin:Zn(II)-protoporphyrin IX complexes. These results imply that many sophisticated functions such as cofactor binding and electron transfer require only a very small number of residue positions in a protein sequence to be fixed. Net charge and hydrophobic content are important in determining protein solubility and stability. Accordingly, rational modifications were made to the aforementioned design procedure in order to improve its overall success rate. The effects of these modifications are explored using two `next-generation' sequence libraries, which were separately expressed and evaluated. Particular modifications to these design parameters are demonstrated to effectively double the purification success rate of the procedure. Finally, I describe the redesign of the artificial di-iron protein DF2 into CDM13, a single chain di-Manganese four-helix bundle. CDM13 acts as a functional model of natural manganese catalase, exhibiting a kcat of 0.08s-1 under steady-state conditions. The bound manganese cofactors have a reduction potential of +805 mV vs NHE, which is too high for efficient dismutation of hydrogen peroxide. These results indicate that as a high-potential manganese complex, CDM13 may represent a promising first step toward a polypeptide model of the Oxygen Evolving Complex of the photosynthetic enzyme Photosystem II.

Effect of B-ring substitution pattern on binding mode of propionamide selective androgen receptor modulators.

PubMed

Bohl, Casey E; Wu, Zengru; Chen, Jiyun; Mohler, Michael L; Yang, Jun; Hwang, Dong Jin; Mustafa, Suni; Miller, Duane D; Bell, Charles E; Dalton, James T

2008-10-15

Selective androgen receptor modulators (SARMs) are essentially prostate sparing androgens, which provide therapeutic potential in osteoporosis, male hormone replacement, and muscle wasting. Herein we report crystal structures of the androgen receptor (AR) ligand-binding domain (LBD) complexed to a series of potent synthetic nonsteroidal SARMs with a substituted pendant arene referred to as the B-ring. We found that hydrophilic B-ring para-substituted analogs exhibit an additional region of hydrogen bonding not seen with steroidal compounds and that multiple halogen substitutions affect the B-ring conformation and aromatic interactions with Trp741. This information elucidates interactions important for high AR binding affinity and provides new insight for structure-based drug design.

Thermochemistry of the specific binding of C12 surfactants to bovine serum albumin.

PubMed

Nielsen, A D; Borch, K; Westh, P

2000-06-15

The specific binding to bovine serum albumin (BSA) of anionic and non-ionic surfactants with C12 acyl chains has been studied by high sensitivity isothermal titration calorimetry. This method proved particularly effective in resolving the binding of anionic surfactants into separate classes of sites with different affinity. For sodium dodecylsulfate (SDS) the measured binding curves could be rationalized as association to two classes (high affinity/low affinity) of sites comprising, respectively, three and six similar (i.e. thermodynamically equivalent), independent sites. Changes in the thermodynamic functions enthalpy, standard free energy, standard entropy and heat capacity could be discerned for each class of binding site, as well as for micelle formation. These data suggest that binding to low affinity sites (in analogy with micelle formation) exhibits energetic parameters; in particular, a large negative change in heat capacity, which is characteristic of hydrophobic interactions. The thermodynamics of high affinity binding, on the other hand, is indicative of other dominant forces; most likely electrostatic interactions. Other anionic ligands investigated (laurate and dodecyl benzylsulfonate) showed a behavior similar to SDS, the most significant difference being the high affinity binding of the alkylbenzyl sulfonate. For this ligand, the thermodynamic data is indicative of a more loosely associated complex than for SDS and laurate. BSA was found to bind one or two of the non-ionic surfactants (NIS) hepta- or penta(ethylene glycol) monododecyl ether (C12EO7 and C12EO5) with binding constants about three orders of magnitude lower than for SDS. Hence, the free energy of the surfactant in the weakly bound BSA-NIS complex is only slightly favored over the micellar state. The binding process is characterized by very large exothermic enthalpy changes (larger than for the charged surfactants) and a large, positive increment in heat capacity. These observations cannot be reconciled with a molecular picture based on simple hydrophobic condensation onto non-polar patches on the protein surface.

Helicobacter pylori Strains from Duodenal Ulcer Patients Exhibit Mixed babA/B Genotypes with Low Levels of BabA Adhesin and Lewis b Binding.

PubMed

Saberi, Samaneh; Schmidt, Alexej; Eybpoosh, Sana; Esmaili, Maryam; Talebkhan, Yeganeh; Mohajerani, Nazanin; Oghalaie, Akbar; Eshagh Hosseini, Mahmoud; Mohagheghi, Mohammad Ali; Bugaytova, Jeanna; Borén, Thomas; Mohammadi, Marjan

2016-10-01

BabA is a Helicobacter pylori cell surface adhesin, which binds to the ABO/Le(b) histo-blood group antigens (Le(b)) and serves as a virulence factor. H. pylori single colonies were isolated from 156 [non-ulcer dyspepsia (NUD) = 97, duodenal ulcer (DU) = 34, gastric cancer (GC) = 25)] patients. babA and babB genes were evaluated by gene/locus-specific PCR. BabA protein expression and Le(b) binding activity were determined by immunoblotting and ELISA, respectively. The combined categorization of H. pylori strains based on high, low or no levels of BabA expression and Le(b) binding, produced 4 groups: (I) BabA-high/Le(b)-high (36 %), (II) BabA-low/Le(b)-low (26 %), (III) BabA-neg/Le(b)-low (30 %) and (IV) BabA-neg/Le(b)-neg (8 %) strains. The majority (63 %) of the BabA-low/Le(b)-low strains exhibited mixed babA/B genotypes as compared to merely 18 % of the BabA-high/Le(b)-high, 15 % of the BabA-neg/Le(b)-neg and 11 % of the BabA-neg/Le(b)-low (P = 0.0001) strains. In contrast to NUD strains, the great majority (70 %) of DU strains were BabA-low/Le(b)-low (11 %, P = 0.0001), which compared to NUD strains, enhanced the risk of DU by 18.8-fold. In parallel, infection with babA/B mixed genotype strains amplified the risk of DU by 3.6-fold (vs. babA-positive: P = 0.01) to 6.9-fold (vs. babA-negative: P = 0.007). Here, we show higher prevalence of mixed babA/B genotypes among BabA-low/Le(b)-low clinical strains. Recombination of babA and babB genes across their loci may yield lower BabA expression and lower Le(b) binding activity. We conclude that H. pylori strains with lower Le(b) binding activity are better adapted for colonization of the gastric metaplastic patches in the duodenum and enhance the risk of duodenal ulcers.

Cryptic glucocorticoid receptor-binding sites pervade genomic NF-κB response elements.

PubMed

Hudson, William H; Vera, Ian Mitchelle S de; Nwachukwu, Jerome C; Weikum, Emily R; Herbst, Austin G; Yang, Qin; Bain, David L; Nettles, Kendall W; Kojetin, Douglas J; Ortlund, Eric A

2018-04-06

Glucocorticoids (GCs) are potent repressors of NF-κB activity, making them a preferred choice for treatment of inflammation-driven conditions. Despite the widespread use of GCs in the clinic, current models are inadequate to explain the role of the glucocorticoid receptor (GR) within this critical signaling pathway. GR binding directly to NF-κB itself-tethering in a DNA binding-independent manner-represents the standing model of how GCs inhibit NF-κB-driven transcription. We demonstrate that direct binding of GR to genomic NF-κB response elements (κBREs) mediates GR-driven repression of inflammatory gene expression. We report five crystal structures and solution NMR data of GR DBD-κBRE complexes, which reveal that GR recognizes a cryptic response element between the binding footprints of NF-κB subunits within κBREs. These cryptic sequences exhibit high sequence and functional conservation, suggesting that GR binding to κBREs is an evolutionarily conserved mechanism of controlling the inflammatory response.

In Silico Prediction and In Vitro Characterization of Multifunctional Human RNase3

PubMed Central

Kuo, Ping-Hsueh; Chen, Chien-Jung; Chang, Hsiu-Hui; Fang, Shun-lung; Wu, Wei-Shuo; Lai, Yiu-Kay; Pai, Tun-Wen; Chang, Margaret Dah-Tsyr

2013-01-01

Human ribonucleases A (hRNaseA) superfamily consists of thirteen members with high-structure similarities but exhibits divergent physiological functions other than RNase activity. Evolution of hRNaseA superfamily has gained novel functions which may be preserved in a unique region or domain to account for additional molecular interactions. hRNase3 has multiple functions including ribonucleolytic, heparan sulfate (HS) binding, cellular binding, endocytic, lipid destabilization, cytotoxic, and antimicrobial activities. In this study, three putative multifunctional regions, 34RWRCK38 (HBR1), 75RSRFR79 (HBR2), and 101RPGRR105 (HBR3), of hRNase3 have been identified employing in silico sequence analysis and validated employing in vitro activity assays. A heparin binding peptide containing HBR1 is characterized to act as a key element associated with HS binding, cellular binding, and lipid binding activities. In this study, we provide novel insights to identify functional regions of hRNase3 that may have implications for all hRNaseA superfamily members. PMID:23484086

Intrinsic disorder mediates the diverse regulatory functions of the Cdk inhibitor p21

PubMed Central

Wang, Yuefeng; Fisher, John C.; Mathew, Rose; Ou, Li; Otieno, Steve; Sublett, Jack; Xiao, Limin; Chen, Jianhan; Roussel, Martine F.; Kriwacki, Richard W.

2011-01-01

Traditionally, well-defined three-dimensional structure was thought to be essential for protein function. However, myriad biological functions are performed by highly dynamic, intrinsically disordered proteins (IDPs). IDPs often fold upon binding their biological targets and frequently exhibit “binding diversity” by targeting multiple ligands. We sought to understand the physical basis of IDP binding diversity and herein report that the cyclin-dependent kinase (Cdk) inhibitor, p21Cip1, adaptively binds to and inhibits the various Cdk/cyclin complexes that regulate eukaryotic cell division. Based on results from NMR spectroscopy, and biochemical and cellular assays, we show that structural adaptability of a helical sub-domain within p21 termed LH enables two other sub-domains termed D1 and D2 to specifically bind conserved surface features of the cyclin and Cdk subunits, respectively, within otherwise structurally distinct Cdk/cyclin complexes. Adaptive folding upon binding is likely to mediate the diverse biological functions of the thousands of IDPs present in eukaryotes. PMID:21358637

In silico evolution of the Drosophila gap gene regulatory sequence under elevated mutational pressure.

PubMed

Chertkova, Aleksandra A; Schiffman, Joshua S; Nuzhdin, Sergey V; Kozlov, Konstantin N; Samsonova, Maria G; Gursky, Vitaly V

2017-02-07

Cis-regulatory sequences are often composed of many low-affinity transcription factor binding sites (TFBSs). Determining the evolutionary and functional importance of regulatory sequence composition is impeded without a detailed knowledge of the genotype-phenotype map. We simulate the evolution of regulatory sequences involved in Drosophila melanogaster embryo segmentation during early development. Natural selection evaluates gene expression dynamics produced by a computational model of the developmental network. We observe a dramatic decrease in the total number of transcription factor binding sites through the course of evolution. Despite a decrease in average sequence binding energies through time, the regulatory sequences tend towards organisations containing increased high affinity transcription factor binding sites. Additionally, the binding energies of separate sequence segments demonstrate ubiquitous mutual correlations through time. Fewer than 10% of initial TFBSs are maintained throughout the entire simulation, deemed 'core' sites. These sites have increased functional importance as assessed under wild-type conditions and their binding energy distributions are highly conserved. Furthermore, TFBSs within close proximity of core sites exhibit increased longevity, reflecting functional regulatory interactions with core sites. In response to elevated mutational pressure, evolution tends to sample regulatory sequence organisations with fewer, albeit on average, stronger functional transcription factor binding sites. These organisations are also shaped by the regulatory interactions among core binding sites with sites in their local vicinity.

Structural Insights into the Affinity of Cel7A Carbohydrate-binding Module for Lignin*

PubMed Central

Strobel, Kathryn L.; Pfeiffer, Katherine A.; Blanch, Harvey W.; Clark, Douglas S.

2015-01-01

The high cost of hydrolytic enzymes impedes the commercial production of lignocellulosic biofuels. High enzyme loadings are required in part due to their non-productive adsorption to lignin, a major component of biomass. Despite numerous studies documenting cellulase adsorption to lignin, few attempts have been made to engineer enzymes to reduce lignin binding. In this work, we used alanine-scanning mutagenesis to elucidate the structural basis for the lignin affinity of Trichoderma reesei Cel7A carbohydrate binding module (CBM). T. reesei Cel7A CBM mutants were produced with a Talaromyces emersonii Cel7A catalytic domain and screened for their binding to cellulose and lignin. Mutation of aromatic and polar residues on the planar face of the CBM greatly decreased binding to both cellulose and lignin, supporting the hypothesis that the cellulose-binding face is also responsible for lignin affinity. Cellulose and lignin affinity of the 31 mutants were highly correlated, although several mutants displayed selective reductions in lignin or cellulose affinity. Four mutants with increased cellulose selectivity (Q2A, H4A, V18A, and P30A) did not exhibit improved hydrolysis of cellulose in the presence of lignin. Further reduction in lignin affinity while maintaining a high level of cellulose affinity is thus necessary to generate an enzyme with improved hydrolysis capability. This work provides insights into the structural underpinnings of lignin affinity, identifies residues amenable to mutation without compromising cellulose affinity, and informs engineering strategies for family one CBMs. PMID:26209638

Novel water soluble NIR dyes: does charge matter?

NASA Astrophysics Data System (ADS)

Patonay, Gabor; Henary, Maged; Beckford, Garfield; Daube, Alison

2012-03-01

Near-Infrared (NIR) dyes are used as reporters, probes or markers in the biological and medical field. NIR dyes can be useful for investigating and characterizing biomolecular interactions or imaging which is possible because biological mammalian tissue has a low absorption window in the NIR region. Biomolecules such as proteins are known to bind to NIR dyes. Upon binding NIR dyes often exhibit spectral changes that can be used for characterizing the binding event. Serum albumins may be responsible for in vivo transport of NIR dyes. Studying this binding event can be useful when correlated to in vivo behavior of the NIR dye. The studies presented here use spectroscopic methods to investigate how NIR dyes that may be used in imaging, biological or bioanalytical applications bind to proteins, such as serum albumins. Our research group systematically synthesized several NIR dyes that have varying hydrophobicity, chromophore size and charge. During these investigations we developed novel NIR cyanine fluorophores having varying aqueous solubility and a variety of net charges. The binding properties of the carbocyanines change when charged or hydrophobic moieties are systematically varied. One of the properties we put a special emphasis on is what we call residual hydrophobicity of the NIR dye molecule which is defined as the unmasked (by the charged moieties) hydrophobicity of the molecule. Residual hydrophobicity may be responsible for binding the otherwise highly water soluble NIR dye to hydrophobic pockets of biomolecules. High residual hydrophobicity of a highly water soluble dye can be disadvantageous during biological, medical or similar applications.

The evolution of filamin – A protein domain repeat perspective

PubMed Central

Light, Sara; Sagit, Rauan; Ithychanda, Sujay S.; Qin, Jun; Elofsson, Arne

2013-01-01

Particularly in higher eukaryotes, some protein domains are found in tandem repeats, performing broad functions often related to cellular organization. For instance, the eukaryotic protein filamin interacts with many proteins and is crucial for the cytoskeleton. The functional properties of long repeat domains are governed by the specific properties of each individual domain as well as by the repeat copy number. To provide better understanding of the evolutionary and functional history of repeating domains, we investigated the mode of evolution of the filamin domain in some detail. Among the domains that are common in long repeat proteins, sushi and spectrin domains evolve primarily through cassette tandem duplications while scavenger and immunoglobulin repeats appear to evolve through clustered tandem duplications. Additionally, immunoglobulin and filamin repeats exhibit a unique pattern where every other domain shows high sequence similarity. This pattern may be the result of tandem duplications, serve to avert aggregation between adjacent domains or it is the result of functional constraints. In filamin, our studies confirm the presence of interspersed integrin binding domains in vertebrates, while invertebrates exhibit more varied patterns, including more clustered integrin binding domains. The most notable case is leech filamin, which contains a 20 repeat expansion and exhibits unique dimerization topology. Clearly, invertebrate filamins are varied and contain examples of similar adjacent integrin-binding domains. Given that invertebrate integrin shows more similarity to the weaker filamin binder, integrin β3, it is possible that the distance between integrin-binding domains is not as crucial for invertebrate filamins as for vertebrates. PMID:22414427

The evolution of filamin-a protein domain repeat perspective.

PubMed

Light, Sara; Sagit, Rauan; Ithychanda, Sujay S; Qin, Jun; Elofsson, Arne

2012-09-01

Particularly in higher eukaryotes, some protein domains are found in tandem repeats, performing broad functions often related to cellular organization. For instance, the eukaryotic protein filamin interacts with many proteins and is crucial for the cytoskeleton. The functional properties of long repeat domains are governed by the specific properties of each individual domain as well as by the repeat copy number. To provide better understanding of the evolutionary and functional history of repeating domains, we investigated the mode of evolution of the filamin domain in some detail. Among the domains that are common in long repeat proteins, sushi and spectrin domains evolve primarily through cassette tandem duplications while scavenger and immunoglobulin repeats appear to evolve through clustered tandem duplications. Additionally, immunoglobulin and filamin repeats exhibit a unique pattern where every other domain shows high sequence similarity. This pattern may be the result of tandem duplications, serve to avert aggregation between adjacent domains or it is the result of functional constraints. In filamin, our studies confirm the presence of interspersed integrin binding domains in vertebrates, while invertebrates exhibit more varied patterns, including more clustered integrin binding domains. The most notable case is leech filamin, which contains a 20 repeat expansion and exhibits unique dimerization topology. Clearly, invertebrate filamins are varied and contain examples of similar adjacent integrin-binding domains. Given that invertebrate integrin shows more similarity to the weaker filamin binder, integrin β3, it is possible that the distance between integrin-binding domains is not as crucial for invertebrate filamins as for vertebrates. Copyright © 2012 Elsevier Inc. All rights reserved.

[Changes in the secondary and tertiary structure of serum albumin in interactions with ligands of various structures].

PubMed

Trinus, F P; Braver-Chernobul'skaia, B S; Luĭk, A I; Boldeskul, A E; Velichko, A N

1984-01-01

High affinity interactions between blood serum albumin and five substances of various chemical structure, exhibiting distinct physiological activity, were accompanied by alterations in the protein tertiary structure, while the albumin secondary structure was involved in conformational transformation after less effective affinity binding.

Glycosylphosphatidylinositol-anchored high density lipoprotein–binding protein 1 plays a critical role in the lipolytic processing of chylomicrons

PubMed Central

Beigneux, Anne P.; Davies, Brandon S. J.; Gin, Peter; Weinstein, Michael M.; Farber, Emily; Qiao, Xin; Peale, Franklin; Bunting, Stuart; Walzem, Rosemary L.; Wong, Jinny S.; Blaner, William S.; Ding, Zhi-Ming; Melford, Kristan; Wongsiriroj, Nuttaporn; Shu, Xiao; de Sauvage, Fred; Ryan, Robert O.; Fong, Loren G.; Bensadoun, André; Young, Stephen G.

2007-01-01

Summary The triglycerides in chylomicrons are hydrolyzed by lipoprotein lipase (LpL) along the luminal surface of the capillaries. However, the endothelial cell molecule that facilitates chylomicron processing by LpL has not yet been defined. Here, we show that glycosylphosphatidylinositol-anchored high density lipoprotein–binding protein 1 (GPIHBP1) plays a critical role in the lipolytic processing of chylomicrons. Gpihbp1-deficient mice exhibit a striking accumulation of chylomicrons in the plasma, even on a low-fat diet, resulting in milky plasma and plasma triglyceride levels as high as 5,000 mg/dl. Normally, Gpihbp1 is expressed highly in heart and adipose tissue, the same tissues that express high levels of LpL. In these tissues, GPIHBP1 is located on the luminal face of the capillary endothelium. Expression of GPIHBP1 in cultured cells confers the ability to bind both LpL and chylomicrons. These studies strongly suggest that GPIHBP1 is an important platform for the LpL-mediated processing of chylomicrons in capillaries. PMID:17403372

Cell-Selective Biological Activity of Rhodium Metalloinsertors Correlates with Subcellular Localization

PubMed Central

Komor, Alexis C.; Schneider, Curtis J.; Weidmann, Alyson G.; Barton, Jacqueline K.

2013-01-01

Deficiencies in the mismatch repair (MMR) pathway are associated with several types of cancers, as well as resistance to commonly used chemotherapeutics. Rhodium metalloinsertors have been found to bind DNA mismatches with high affinity and specificity in vitro, and also exhibit cell-selective cytotoxicity, targeting MMR-deficient cells over MMR-proficient cells. Ten distinct metalloinsertors with varying lipophilicities have been synthesized and their mismatch binding affinities and biological activities determined. Although DNA photocleavage experiments demonstrate that their binding affinities are quite similar, their cell-selective antiproliferative and cytotoxic activities vary significantly. Inductively coupled plasma mass spectrometry (ICP-MS) experiments have uncovered a relationship between the subcellular distribution of these metalloinsertors and their biological activities. Specifically, we find that all of our metalloinsertors localize in the nucleus at sufficient concentrations for binding to DNA mismatches. However, the metalloinsertors with high rhodium localization in the mitochondria show toxicity that is not selective for MMR-deficient cells, whereas metalloinsertors with less mitochondrial rhodium show activity that is highly selective for MMR-deficient versus proficient cells. This work supports the notion that specific targeting of the metalloinsertors to nuclear DNA gives rise to their cell-selective cytotoxic and antiproliferative activities. The selectivity in cellular targeting depends upon binding to mismatches in genomic DNA. PMID:23137296

Strong Ligand-Protein Interactions Derived from Diffuse Ligand Interactions with Loose Binding Sites.

PubMed

Marsh, Lorraine

2015-01-01

Many systems in biology rely on binding of ligands to target proteins in a single high-affinity conformation with a favorable ΔG. Alternatively, interactions of ligands with protein regions that allow diffuse binding, distributed over multiple sites and conformations, can exhibit favorable ΔG because of their higher entropy. Diffuse binding may be biologically important for multidrug transporters and carrier proteins. A fine-grained computational method for numerical integration of total binding ΔG arising from diffuse regional interaction of a ligand in multiple conformations using a Markov Chain Monte Carlo (MCMC) approach is presented. This method yields a metric that quantifies the influence on overall ligand affinity of ligand binding to multiple, distinct sites within a protein binding region. This metric is essentially a measure of dispersion in equilibrium ligand binding and depends on both the number of potential sites of interaction and the distribution of their individual predicted affinities. Analysis of test cases indicates that, for some ligand/protein pairs involving transporters and carrier proteins, diffuse binding contributes greatly to total affinity, whereas in other cases the influence is modest. This approach may be useful for studying situations where "nonspecific" interactions contribute to biological function.

Evaluation of [11C]TAZA for amyloid β plaque imaging in postmortem human Alzheimer's disease brain region and whole body distribution in rodent PET/CT.

PubMed

Pan, Min-Liang; Mukherjee, Meenakshi T; Patel, Himika H; Patel, Bhavin; Constantinescu, Cristian C; Mirbolooki, M Reza; Liang, Christopher; Mukherjee, Jogeshwar

2016-04-01

Alzheimer's disease (AD) is a neurodegenerative disease characterized by Aβ plaques in the brain. The aim of this study was to evaluate the effectiveness of a novel radiotracer, 4-[(11) C]methylamino-4'-N,N-dimethylaminoazobenzene ([(11)C]TAZA), for binding to Aβ plaques in postmortem human brain (AD and normal control (NC)). Radiosyntheses of [(11)C]TAZA, related [(11)C]Dalene ((11)C-methylamino-4'-dimethylaminostyrylbenzene), and reference [(11)C]PIB were carried out using [(11)C]methyltriflate prepared from [(11) C]CO(2) and purified using HPLC. In vitro binding affinities were carried out in human AD brain homogenate with Aβ plaques labeled with [(3) H]PIB. In vitro autoradiography studies with the three radiotracers were performed on hippocampus of AD and NC brains. PET/CT studies were carried out in normal rats to study brain and whole body distribution. The three radiotracers were produced in high radiochemical yields (>40%) and had specific activities >37 GBq/μmol. TAZA had an affinity, K(i) = 0.84 nM and was five times more potent than PIB. [(11)C]TAZA bound specifically to Aβ plaques present in AD brains with gray matter to white matter ratios >20. [(11)C]TAZA was displaced by PIB (>90%), suggesting similar binding site for [(11)C]TAZA and [(11)C]PIB. [(11)C]TAZA exhibited slow kinetics of uptake in the rat brain and whole body images showed uptake in interscapular brown adipose tissue (IBAT). Binding in brain and IBAT were affected by preinjection of atomoxetine, a norepinephrine transporter blocker. [(11)C]TAZA exhibited high binding to Aβ plaques in human AD hippocampus. Rat brain kinetics was slow and peripheral binding to IBAT needs to be further evaluated. © 2016 Wiley Periodicals, Inc.

High-Mobility Group Chromatin Proteins 1 and 2 Functionally Interact with Steroid Hormone Receptors To Enhance Their DNA Binding In Vitro and Transcriptional Activity in Mammalian Cells

PubMed Central

Boonyaratanakornkit, Viroj; Melvin, Vida; Prendergast, Paul; Altmann, Magda; Ronfani, Lorenza; Bianchi, Marco E.; Taraseviciene, Laima; Nordeen, Steven K.; Allegretto, Elizabeth A.; Edwards, Dean P.

1998-01-01

We previously reported that the chromatin high-mobility group protein 1 (HMG-1) enhances the sequence-specific DNA binding activity of progesterone receptor (PR) in vitro, thus providing the first evidence that HMG-1 may have a coregulatory role in steroid receptor-mediated gene transcription. Here we show that HMG-1 and the highly related HMG-2 stimulate DNA binding by other steroid receptors, including estrogen, androgen, and glucocorticoid receptors, but have no effect on DNA binding by several nonsteroid nuclear receptors, including retinoid acid receptor (RAR), retinoic X receptor (RXR), and vitamin D receptor (VDR). As highly purified recombinant full-length proteins, all steroid receptors tested exhibited weak binding affinity for their optimal palindromic hormone response elements (HREs), and the addition of purified HMG-1 or -2 substantially increased their affinity for HREs. Purified RAR, RXR, and VDR also exhibited little to no detectable binding to their cognate direct repeat HREs but, in contrast to results with steroid receptors, the addition of HMG-1 or HMG-2 had no stimulatory effect. Instead, the addition of purified RXR enhanced RAR and VDR DNA binding through a heterodimerization mechanism and HMG-1 or HMG-2 had no further effect on DNA binding by RXR-RAR or RXR-VDR heterodimers. HMG-1 and HMG-2 (HMG-1/-2) themselves do not bind to progesterone response elements, but in the presence of PR they were detected as part of an HMG-PR-DNA ternary complex. HMG-1/-2 can also interact transiently in vitro with PR in the absence of DNA; however, no direct protein interaction was detected with VDR. These results, taken together with the fact that PR can bend its target DNA and that HMG-1/-2 are non-sequence-specific DNA binding proteins that recognize DNA structure, suggest that HMG-1/-2 are recruited to the PR-DNA complex by the combined effect of transient protein interaction and DNA bending. In transient-transfection assays, coexpression of HMG-1 or HMG-2 increased PR-mediated transcription in mammalian cells by as much as 7- to 10-fold without altering the basal promoter activity of target reporter genes. This increase in PR-mediated gene activation by coexpression of HMG-1/-2 was observed in different cell types and with different target promoters, suggesting a generality to the functional interaction between HMG-1/-2 and PR in vivo. Cotransfection of HMG-1 also increased reporter gene activation mediated by other steroid receptors, including glucocorticoid and androgen receptors, but it had a minimal influence on VDR-dependent transcription in vivo. These results support the conclusion that HMG-1/-2 are coregulatory proteins that increase the DNA binding and transcriptional activity of the steroid hormone class of receptors but that do not functionally interact with certain nonsteroid classes of nuclear receptors. PMID:9671457

Tetrazepam: a benzodiazepine which dissociates sedation from other benzodiazepine activities. II. In vitro and in vivo interactions with benzodiazepine binding sites

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

Keane, P.E.; Bachy, A.; Morre, M.

1988-05-01

Tetrazepam is a 1,4-benzodiazepine (BZD) derivative which, in rodents, appears to have very little sedative and ataxic effects. In an attempt to identify the molecular mechanisms underlying this particular pharmacological profile we examined the interaction of tetrazepam with BZD binding sites. Tetrazepam interacted competitively with central and peripheral BZD binding sites and exhibited comparable affinities for both sites. Tetrazepam was approximately one-seventh as potent as diazepam at the central receptor and as potent as diazepam at the peripheral binding site. Tetrazepam did not distinguish type I from type II central BZD receptors, as evidenced by comparable affinities for the cerebellarmore » and hippocampal receptors. In vitro autoradiographic studies showed that tetrazepam displaced (3H)flunitrazepam from rat brain membranes without any clear regional specificity. Like all BZD receptor agonists, tetrazepam exhibited a gamma-aminobutyric acid shift, a photoaffinity shift and potentiated the binding of 35S-t-butyl-bicyclophosphorothionate to rat brain membranes. However, the latter effect was observed at relatively high concentrations of tetrazepam. In vivo, tetrazepam displaced specifically bound (3H)flunitrazepam from mouse brain (ID50, 37 mg/kg p.o. vs 3.5 mg/kg p.o. for diazepam) and from mouse kidney (ID50, 38 mg/kg p.o. vs. 21 mg/kg p.o. for diazepam). It is concluded that tetrazepam is a BZD receptor agonist; the molecular mechanisms which underly the low sedative potential of the drug cannot at present be explained by a particular interaction with either central or peripheral BZD binding sites, but may be related to the drug's relatively weak effect on 35S-t-butyl-bicyclophosphorothionate binding.« less

Development of melanoma-targeted polymer micelles by conjugation of a Melanocortin 1 Receptor (MC1R) specific ligand

PubMed Central

Barkey, Natalie M.; Tafreshi, Narges K.; Josan, Jatinder S.; De Silva, Channa R.; Sill, Kevin N.; Hruby, Victor J.; Gillies, Robert J.; Morse, David L.; Vagner, Josef

2012-01-01

The incidence of malignant melanoma is rising faster than that of any other cancer in the United States. Due to its high expression on the surface of melanomas, MC1R has been investigated as a target for selective imaging and therapeutic agents against melanoma. Eight ligands were screened against cell lines engineered to over-express MC1R, MC4R or MC5R. Of these, compound 1 (4-phenylbutyryl-His-Dphe-Arg-Trp-NH2) exhibited high (0.2 nM) binding affinity for MC1R, and low (high nM) affinities for MC4R and MC5R. Subsequently functionalization of the ligand at the C-terminus with an alkyne for use in Cu-catalyzed click chemistry was shown not to affect the binding affinity. Finally, formation of the targeted-polymer, as well as the targeted micelle formulation, also resulted in constructs with low nM binding affinity. PMID:22011200

Development of melanoma-targeted polymer micelles by conjugation of a melanocortin 1 receptor (MC1R) specific ligand.

PubMed

Barkey, Natalie M; Tafreshi, Narges K; Josan, Jatinder S; De Silva, Channa R; Sill, Kevin N; Hruby, Victor J; Gillies, Robert J; Morse, David L; Vagner, Josef

2011-12-08

The incidence of malignant melanoma is rising faster than that of any other cancer in the United States. Because of its high expression on the surface of melanomas, MC1R has been investigated as a target for selective imaging and therapeutic agents against melanoma. Eight ligands were screened against cell lines engineered to overexpress MC1R, MC4R, or MC5R. Of these, compound 1 (4-phenylbutyryl-His-dPhe-Arg-Trp-NH(2)) exhibited high (0.2 nM) binding affinity for MC1R and low (high nanomolar) affinities for MC4R and MC5R. Functionalization of the ligand at the C-terminus with an alkyne for use in Cu-catalyzed click chemistry was shown not to affect the binding affinity. Finally, formation of the targeted polymer, as well as the targeted micelle formulation, also resulted in constructs with low nanomolar binding affinity.

Selective binding of choline by a phosphate-coordination-based triple helicate featuring an aromatic box.

PubMed

Jia, Chuandong; Zuo, Wei; Yang, Dong; Chen, Yanming; Cao, Liping; Custelcean, Radu; Hostaš, Jiří; Hobza, Pavel; Glaser, Robert; Wang, Yao-Yu; Yang, Xiao-Juan; Wu, Biao

2017-10-16

In nature, proteins have evolved sophisticated cavities tailored for capturing target guests selectively among competitors of similar size, shape, and charge. The fundamental principles guiding the molecular recognition, such as self-assembly and complementarity, have inspired the development of biomimetic receptors. In the current work, we report a self-assembled triple anion helicate (host 2) featuring a cavity resembling that of the choline-binding protein ChoX, as revealed by crystal and density functional theory (DFT)-optimized structures, which binds choline in a unique dual-site-binding mode. This similarity in structure leads to a similarly high selectivity of host 2 for choline over its derivatives, as demonstrated by the NMR and fluorescence competition experiments. Furthermore, host 2 is able to act as a fluorescence displacement sensor for discriminating choline, acetylcholine, L-carnitine, and glycine betaine effectively.The choline-binding protein ChoX exhibits a synergistic dual-site binding mode that allows it to discriminate choline over structural analogues. Here, the authors design a biomimetic triple anion helicate receptor whose selectivity for choline arises from a similar binding mechanism.

Food proteins and maturation of small intestinal microvillus membranes (MVM). III. Food protein binding and MVM proteins in rats from newborn to young adult age.

PubMed

Stern, M; Gellermann, B; Wieser, H

1990-10-01

To investigate postnatal maturational profiles of functional and biochemical properties of rat small intestinal microvillus membranes (MVM), we did a longitudinal study in rats from birth to the age of 12 weeks. In parallel, we studied binding of cow's milk proteins and of the wheat gliadin peptide B 3142, as well as MVM proteins (SDS-PAGE). Changes in MVM fluidity and lipid composition exhibited early (0-4 weeks) and intermediate and late (6-12 weeks) patterns, as has been published earlier. Postnatal changes of food protein and peptide binding occurred early during the observation period, not related to weaning. There was not much further change in binding after 6-8 weeks. Developmental profiles of MVM protein and some lipid changes resembled, but did not equal, changes in food protein binding. We conclude that changes in MVM biochemical composition affect MVM binding characteristics. In particular, high molecular weight MVM proteins (susceptible to trypsin treatment) appear to play a role in postnatal maturational differences in MVM food protein binding.

The role of attention in binding visual features in working memory: evidence from cognitive ageing.

PubMed

Brown, Louise A; Brockmole, James R

2010-10-01

Two experiments were conducted to assess the costs of attentional load during a feature (colour-shape) binding task in younger and older adults. Experiment 1 showed that a demanding backwards counting task, which draws upon central executive/general attentional resources, reduced binding to a greater extent than individual feature memory, but the effect was no greater in older than in younger adults. Experiment 2 showed that presenting memory items sequentially rather than simultaneously, such that items are required to be maintained while new representations are created, selectively affects binding performance in both age groups. Although this experiment exhibited an age-related binding deficit overall, both age groups were affected by the attention manipulation to an equal extent. While a role for attentional processes in colour-shape binding was apparent across both experiments, manipulations of attention exerted equal effects in both age groups. We therefore conclude that age-related binding deficits neither emerge nor are exacerbated under conditions of high attentional load. Implications for theories of visual working memory and cognitive ageing are discussed.

Synthesis of photothermal nanocomposites and their application to antibacterial assays

NASA Astrophysics Data System (ADS)

Yang, Ning; Wang, Chun; Wang, Xiaoyu; Li, Lidong

2018-04-01

In this work, we report a novel gold nanorod (AuNR)-based nanocomposite that shows strong binding to bacterium and high antibacterial efficiency. The AuNRs were used as a photothermal material to transform near-infrared radiation (NIR) into heat. We selected poly (acrylic acid) to modify the surface of the AuNRs based on a simple self-assembly method. After conjugation of the bacterium-binding molecule vancomycin, the nanocomposites were capable of efficiently gathering on the cell walls of bacteria. The nanocomposites exhibited a high bacterial inhibition capability owing to NIR-induced heat generation in situ. Therefore, the prepared photothermal nanocomposites show great potential for use in antibacterial assays.

A novel anti-GPC3 monoclonal antibody (YP7) | Center for Cancer Research

Cancer.gov

Glypican-3 (GPC3) is an emerging therapeutic target in hepatoma. A novel anti-GPC3 monoclonal antibody (YP7) has been generated through a combination of peptide immunization and high-throughput flow cytometry screening. YP7 binds cell-surface-associated GPC3 with high affinity and exhibits significant hepatoma xenograft growth inhibition in nude mice. The new antibody may have

Direct binding to antigen-coated beads refines the specificity and cross-reactivity of four monoclonal antibodies that recognize polymorphic epitopes of HLA class I molecules

PubMed Central

Hilton, Hugo G; Parham, Peter

2013-01-01

Monoclonal antibodies with specificity for HLA class I determinants of HLA were originally characterized using serological assays in which the targets were cells expressing 3-6 HLA class I variants. Because of this complexity, the specificities of the antibodies were defined indirectly by correlation. Here we use a direct binding assay, in which the targets are synthetic beads coated with one of 111 HLA class I variants, representing the full range of HLA-A, -B and -C variation. We studied one monoclonal antibody with monomorphic specificity (W6/32) and four with polymorphic specificity (MA2.1, PA2.1, BB7.2 and BB7.1) and compared the results with those obtained previously. W6/32 reacted with all HLA class I variants. MA2.1 exhibits high specificity for HLA-A*02, -B*57 and -B*58, but also exhibited cross-reactivity with HLA-A*11 and -B*15:16. At low concentration (1μg/ml) PA2.1 and BB7.2 were both specific for HLA-A*02 and -A*69, and at high concentration (50μg/ml) exhibited significant cross-reactions with HLA-A*68, -A*23, and -A*24. BB7.1 exhibits specificity for HLA-B*07 and -B*42, as previously described, but reacts equally well with HLA-B*81, a rare allotype defined some 16 years after the description of BB7.1. The results obtained with cell-based and bead-based assays are consistent and, in combination with amino acid sequence comparison, increase understanding of the polymorphic epitopes recognized by the MA2.1, PA2.1, BB7.2 and BB7.1 antibodies. Comparison of two overlapping but distinctive bead sets from two sources gave similar results, but the overall levels of binding were significantly different. Several weaker reactions were observed with only one of the bead sets. PMID:23510417

Residues in the H+ Translocation Site Define the pKa for Sugar Binding to LacY†

PubMed Central

Smirnova, Irina; Kasho, Vladimir; Sugihara, Junichi; Choe, Jun-Yong; Kaback, H. Ronald

2009-01-01

A remarkably high pKa of approximately 10.5 has been determined for sugar-binding affinity to the lactose permease of Escherichia coli (LacY), indicating that, under physiological conditions, substrate binds to fully protonated LacY. We have now systematically tested site-directed replacements for the residues involved in sugar binding, as well as H+ translocation and coupling, in order to determine which residues may be responsible for this alkaline pKa. Mutations in the sugar-binding site (Glu126, Trp151, Glu269) markedly decrease affinity for sugar but do not alter the pKa for binding. In contrast, replacements for residues involved in H+ translocation (Arg302, Tyr236, His322, Asp240, Glu325, Lys319) exhibit pKa values for sugar binding that are either shifted toward neutral pH or independent of pH. Values for the apparent dissociation constant for sugar binding (Kdapp) increase greatly for all mutants except neutral replacements for Glu325 or Lys319, which are characterized by remarkably high affinity sugar binding (i.e., low Kdapp) from pH 5.5 to pH 11. The pH dependence of the on- and off-rate constants for sugar binding measured directly by stopped-flow fluorometry implicates koff as a major factor for the affinity change at alkaline pH and confirms the effects of pH on Kdapp inferred from steady-state fluorometry. These results indicate that the high pKa for sugar binding by wild-type LacY cannot be ascribed to any single amino acid residue but appears to reside within a complex of residues involved in H+ translocation. There is structural evidence for water bound in this complex, and the water could be the site of protonation responsible for the pH dependence of sugar binding. PMID:19689129

Transcriptome Sequencing Identified Genes and Gene Ontologies Associated with Early Freezing Tolerance in Maize

PubMed Central

Li, Zhao; Hu, Guanghui; Liu, Xiangfeng; Zhou, Yao; Li, Yu; Zhang, Xu; Yuan, Xiaohui; Zhang, Qian; Yang, Deguang; Wang, Tianyu; Zhang, Zhiwu

2016-01-01

Originating in a tropical climate, maize has faced great challenges as cultivation has expanded to the majority of the world's temperate zones. In these zones, frost and cold temperatures are major factors that prevent maize from reaching its full yield potential. Among 30 elite maize inbred lines adapted to northern China, we identified two lines of extreme, but opposite, freezing tolerance levels—highly tolerant and highly sensitive. During the seedling stage of these two lines, we used RNA-seq to measure changes in maize whole genome transcriptome before and after freezing treatment. In total, 19,794 genes were expressed, of which 4550 exhibited differential expression due to either treatment (before or after freezing) or line type (tolerant or sensitive). Of the 4550 differently expressed genes, 948 exhibited differential expression due to treatment within line or lines under freezing condition. Analysis of gene ontology found that these 948 genes were significantly enriched for binding functions (DNA binding, ATP binding, and metal ion binding), protein kinase activity, and peptidase activity. Based on their enrichment, literature support, and significant levels of differential expression, 30 of these 948 genes were selected for quantitative real-time PCR (qRT-PCR) validation. The validation confirmed our RNA-Seq-based findings, with squared correlation coefficients of 80% and 50% in the tolerance and sensitive lines, respectively. This study provided valuable resources for further studies to enhance understanding of the molecular mechanisms underlying maize early freezing response and enable targeted breeding strategies for developing varieties with superior frost resistance to achieve yield potential. PMID:27774095

The Structural Basis for Recognition of the PreQ0 Metabolite by an Unusually Small Riboswitch Aptamer Domain*S⃞♦

PubMed Central

Spitale, Robert C.; Torelli, Andrew T.; Krucinska, Jolanta; Bandarian, Vahe; Wedekind, Joseph E.

2009-01-01

Riboswitches are RNA elements that control gene expression through metabolite binding. The preQ1 riboswitch exhibits the smallest known ligand-binding domain and is of interest for its economical organization and high affinity interactions with guanine-derived metabolites required to confer tRNA wobbling. Here we present the crystal structure of a preQ1 aptamer domain in complex with its precursor metabolite preQ0. The structure is highly compact with a core that features a stem capped by a well organized decaloop. The metabolite is recognized within a deep pocket via Watson-Crick pairing with C15. Additional hydrogen bonds are made to invariant bases U6 and A29. The ligand-bound state confers continuous helical stacking throughout the core fold, thus providing a platform to promote Watson-Crick base pairing between C9 of the decaloop and the first base of the ribosome-binding site, G33. The structure offers insight into the mode of ribosome-binding site sequestration by a minimal RNA fold stabilized by metabolite binding and has implications for understanding the molecular basis by which bacterial genes are regulated. PMID:19261617

A Single Amino Acid Substitution in the Active Site of Escherichia coli Aspartate Transcarbamoylase Prevents the Allosteric Transition

PubMed Central

Stieglitz, Kimberly A.; Pastra-Landis, Styliani C.; Xia, Jiarong; Tsuruta, Hiro; Kantrowitz, Evan R.

2005-01-01

Modeling of the tetrahedral intermediate within the active site of Escherichia coli aspartate transcarbamoylase revealed a specific interaction with the side chain of Gln137, an interaction not previously observed in the structure of the X-ray enzyme in the presence of N-phosphonacetyl-L-aspartate (PALA). Previous site-specific mutagenesis experiments showed that when Gln137 was replaced by alanine, the resulting mutant enzyme (Q137A) exhibited approximately 50-fold less activity than the wild-type enzyme, exhibited no homotropic cooperativity, and the binding of both carbamoyl phosphate and aspartate were extremely compromised. To elucidate the structural alterations in the mutant enzyme that might lead to such pronounced changes in kinetic and binding properties, the Q137A enzyme was studied by time-resolved small-angle X-ray scattering and its structure was determined in the presence of PALA to 2.7Å resolution. Time-resolved small-angle X-ray scattering established that the natural substrates, carbamoyl phosphate and L-aspartate, do not induce in the Q137A enzyme the same conformational changes as observed for the wild-type enzyme, although the scattering pattern of the Q137A and wild-type enzymes in the presence of PALA were identical. The overall structure of the Q137A enzyme is similar to that of the R-state structure of wild-type enzyme with PALA bound. However, there are differences in the manner by which the Q137A enzyme coordinates PALA, especially in the side chain positions of Arg105 and His134. The replacement of Gln137 by Ala also has a dramatic effect on the electrostatics of the active site. These data taken together suggest that the side chain of Gln137 in the wild-type enzyme is required for the binding of carbamoyl phosphate in the proper orientation so as to induce conformational changes required for the creation of the high-affinity aspartate binding site. The inability of carbamoyl phosphate to create the high-affinity binding site in the Q137A enzyme results in an enzyme locked in the low activity low affinity T state. These results emphasize the absolute requirement of the binding of carbamoyl phosphate for the creation of the high-affinity aspartate binding site and for inducing the homotropic cooperativity in aspartate transcarbamoylase. PMID:15890205

Binding Properties of General Odorant Binding Proteins from the Oriental Fruit Moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae)

PubMed Central

Li, Guangwei; Chen, Xiulin; Li, Boliao; Zhang, Guohui; Li, Yiping; Wu, Junxiang

2016-01-01

Background The oriental fruit moth Grapholita molesta is a host-switching pest species. The adults highly depend on olfactory cues in locating optimal host plants and oviposition sites. Odorant binding proteins (OBPs) are thought to be responsible for recognizing and transporting hydrophobic odorants across the aqueous sensillum lymph to stimulate the odorant receptors (ORs) within the antennal sensilla and activate the olfactory signal transduction pathway. Exploring the physiological function of these OBPs could facilitate understanding insect chemical communications. Methodology/Principal Finding Two antennae-specific general OBPs (GOBPs) of G. molesta were expressed and purified in vitro. The binding affinities of G. molesta GOBP1 and 2 (GmolGOBP1 and 2) for sex pheromone components and host plant volatiles were measured by fluorescence ligand-binding assays. The distribution of GmolGOBP1 and 2 in the antennal sensillum were defined by whole mount fluorescence immunohistochemistry (WM-FIHC) experiments. The binding sites of GmolGOBP2 were predicted using homology modeling, molecular docking and site-directed mutagenesis. Both GmolGOBP1 and 2 are housing in sensilla basiconica and with no differences in male and female antennae. Recombinant GmolGOBP1 (rGmolGOBP1) exhibited broad binding properties towards host plant volatiles and sex pheromone components; rGmolGOBP2 could not effectively bind host plant volatiles but showed specific binding affinity with a minor sex pheromone component dodecanol. We chose GmolGOBP2 and dodecanol for further homology modeling, molecular docking, and site-directed mutagenesis. Binding affinities of mutants demonstrated that Thr9 was the key binding site and confirmed dodecanol bonding to protein involves a hydrogen bond. Combined with the pH effect on binding affinities of rGmolGOBP2, ligand binding and release of GmolGOBP2 were related to a pH-dependent conformational transition. Conclusion Two rGmolGOBPs exhibit different binding characteristics for tested ligands. rGmolGOBP1 has dual functions in recognition of host plant volatiles and sex pheromone components, while rGmolGOBP2 is mainly involved in minor sex pheromone component dodecanol perception. This study also provides empirical evidence for the predicted functions of key amino acids in recombinant protein ligand-binding characteristics. PMID:27152703

A High Affinity Red Fluorescence and Colorimetric Probe for Amyloid β Aggregates

NASA Astrophysics Data System (ADS)

Rajasekhar, K.; Narayanaswamy, Nagarjun; Murugan, N. Arul; Kuang, Guanglin; Ågren, Hans; Govindaraju, T.

2016-04-01

A major challenge in the Alzheimer’s disease (AD) is its timely diagnosis. Amyloid β (Aβ) aggregates have been proposed as the most viable biomarker for the diagnosis of AD. Here, we demonstrate hemicyanine-based benzothiazole-coumarin (TC) as a potential probe for the detection of highly toxic Aβ42 aggregates through switch-on, enhanced (~30 fold) red fluorescence (Emax = 654 nm) and characteristic colorimetric (light red to purple) optical outputs. Interestingly, TC exhibits selectivity towards Aβ42 fibrils compared to other abnormal protein aggregates. TC probe show nanomolar binding affinity (Ka = 1.72 × 107 M-1) towards Aβ42 aggregates and also displace ThT bound to Aβ42 fibrils due to its high binding affinity. The Aβ42 fibril-specific red-shift in the absorption spectra of TC responsible for the observed colorimetric optical output has been attributed to micro-environment change around the probe from hydrophilic-like to hydrophobic-like nature. The binding site, binding energy and changes in optical properties observed for TC upon interaction with Aβ42 fibrils have been further validated by molecular docking and time dependent density functional theory studies.

The structure of cell adhesion molecule uvomorulin. Insights into the molecular mechanism of Ca2+-dependent cell adhesion.

PubMed Central

Ringwald, M; Schuh, R; Vestweber, D; Eistetter, H; Lottspeich, F; Engel, J; Dölz, R; Jähnig, F; Epplen, J; Mayer, S

1987-01-01

We have determined the amino acid sequence of the Ca2+-dependent cell adhesion molecule uvomorulin as it appears on the cell surface. The extracellular part of the molecule exhibits three internally repeated domains of 112 residues which are most likely generated by gene duplication. Each of the repeated domains contains two highly conserved units which could represent putative Ca2+-binding sites. Secondary structure predictions suggest that the putative Ca2+-binding units are located in external loops at the surface of the protein. The protein sequence exhibits a single membrane-spanning region and a cytoplasmic domain. Sequence comparison reveals extensive homology to the chicken L-CAM. Both uvomorulin and L-CAM are identical in 65% of their entire amino acid sequence suggesting a common origin for both CAMs. Images Fig. 1. Fig. 4. Fig. 7. PMID:3501370

Part I: an x-ray scattering study of cholera toxin penetration and induced phase transformations in lipid membranes.

PubMed

Miller, C E; Majewski, J; Watkins, E B; Kuhl, T L

2008-07-01

Cholera toxin is a highly efficient biotoxin, which is frequently used as a tool to investigate protein-membrane interactions and as a reporter for membrane rafts. Cholera toxin binds selectively to gangliosides with highest affinity to GM(1). However, the mechanism by which cholera toxin crosses the membrane remains unresolved. Using x-ray reflectivity and grazing incidence diffraction, we have been able to monitor the binding and penetration of cholera toxin into a model lipid monolayer containing the receptor GM(1) at the air-water interface. Very high toxin coverage was obtained allowing precise measurements of how toxin binding alters lipid packing. Grazing incidence x-ray diffraction revealed the coexistence of two monolayer phases after toxin binding. The first was identical to the monolayer before toxin binding. In regions where toxin was bound, a second membrane phase exhibited a decrease in order as evidenced by a larger area per molecule and tilt angle with concomitant thinning of the monolayer. These results demonstrate that cholera toxin binding induces the formation of structurally distinct, less ordered domains in gel phases. Furthermore, the largest decrease in lateral order to the monolayer occurred at low pH, supporting a low endosomal pH in the infection pathway. Surprisingly, at pH = 8 toxin penetration by the binding portion of the toxin, the B(5) pentamer, was also observed.

Multichannel imaging to quantify four classes of pharmacokinetic distribution in tumors

PubMed Central

Bhatnagar, Sumit; Deschenes, Emily; Liao, Jianshan; Cilliers, Cornelius; Thurber, Greg M.

2014-01-01

Low and heterogeneous delivery of drugs and imaging agents to tumors results in decreased efficacy and poor imaging results. Systemic delivery involves a complex interplay of drug properties and physiological factors, and heterogeneity in the tumor microenvironment makes predicting and overcoming these limitations exceptionally difficult. Theoretical models have indicated that there are four different classes of pharmacokinetic behavior in tissue, depending on the fundamental steps in distribution. In order to study these limiting behaviors, we used multichannel fluorescence microscopy and stitching of high-resolution images to examine the distribution of four agents in the same tumor microenvironment. A validated generic partial differential equation model with a graphical user interface was used to select fluorescent agents exhibiting these four classes of behavior, and the imaging results agreed with predictions. BODIPY-FL exhibited higher concentrations in tissue with high blood flow, cetuximab gave perivascular distribution limited by permeability, high plasma protein and target binding resulted in diffusion-limited distribution for Hoechst 33342, and Integrisense 680 was limited by the number of binding sites in the tissue. Together, the probes and simulations can be used to investigate distribution in other tumor models, predict tumor drug distribution profiles, and design and interpret in vivo experiments. PMID:25048378

A single-ligand ultra-microporous MOF for precombustion CO2 capture and hydrogen purification.

PubMed

Nandi, Shyamapada; De Luna, Phil; Daff, Thomas D; Rother, Jens; Liu, Ming; Buchanan, William; Hawari, Ayman I; Woo, Tom K; Vaidhyanathan, Ramanathan

2015-12-01

Metal organic frameworks (MOFs) built from a single small ligand typically have high stability, are rigid, and have syntheses that are often simple and easily scalable. However, they are normally ultra-microporous and do not have large surface areas amenable to gas separation applications. We report an ultra-microporous (3.5 and 4.8 Å pores) Ni-(4-pyridylcarboxylate)2 with a cubic framework that exhibits exceptionally high CO2/H2 selectivities (285 for 20:80 and 230 for 40:60 mixtures at 10 bar, 40°C) and working capacities (3.95 mmol/g), making it suitable for hydrogen purification under typical precombustion CO2 capture conditions (1- to 10-bar pressure swing). It exhibits facile CO2 adsorption-desorption cycling and has CO2 self-diffusivities of ~3 × 10(-9) m(2)/s, which is two orders higher than that of zeolite 13X and comparable to other top-performing MOFs for this application. Simulations reveal a high density of binding sites that allow for favorable CO2-CO2 interactions and large cooperative binding energies. Ultra-micropores generated by a small ligand ensures hydrolytic, hydrostatic stabilities, shelf life, and stability toward humid gas streams.

Novel crown-ether-methylenediphosphonotetrathioate hybrids as Zn(II) chelators.

PubMed

Meltzer, Diana; Gottlieb, Hugo E; Amir, Aviran; Shimon, Linda J W; Fischer, Bilha

2015-12-28

Hybrids of methylenediphosphonotetrathioate and crown-ether (MDPT-CE) were synthesized forming 7-,8-,9-,10- and 13-membered rings. Both 7- and 13-membered ring-containing compounds were found to be highly stable to air-oxidation for at least four weeks. These hybrids bind Zn(II) by both MDPT and CE moieties, forming a 2 : 1 L : Zn(II) complex. Interestingly, the 13-membered ring MDPT-CE showing a high affinity to Zn(II) (Ka 3 ± 0.5 × 10(6) mol(-2) L(2)) does not bind Li(I) or Na(I). The 13-Membered MDPT-CE hybrid is a promising water-soluble, air-stable, high-affinity Zn(II)-chelator, exhibiting selectivity to Zn(II) vs. Mg(II), Na(I), and Li(I).

Lack of conventional oxygen-linked proton and anion binding sites does not impair allosteric regulation of oxygen binding in dwarf caiman hemoglobin

PubMed Central

Fago, Angela; Malte, Hans; Storz, Jay F.; Gorr, Thomas A.

2013-01-01

In contrast to other vertebrate hemoglobins (Hbs) whose high intrinsic O2 affinities are reduced by red cell allosteric effectors (mainly protons, CO2, organic phosphates, and chloride ions), crocodilian Hbs exhibit low sensitivity to organic phosphates and high sensitivity to bicarbonate (HCO3−), which is believed to augment Hb-O2 unloading during diving and postprandial alkaline tides when blood HCO3− levels and metabolic rates increase. Examination of α- and β-globin amino acid sequences of dwarf caiman (Paleosuchus palpebrosus) revealed a unique combination of substitutions at key effector binding sites compared with other vertebrate and crocodilian Hbs: β82Lys→Gln, β143His→Val, and β146His→Tyr. These substitutions delete positive charges and, along with other distinctive changes in residue charge and polarity, may be expected to disrupt allosteric regulation of Hb-O2 affinity. Strikingly, however, P. palpebrosus Hb shows a strong Bohr effect, and marked deoxygenation-linked binding of organic phosphates (ATP and DPG) and CO2 as carbamate (contrasting with HCO3− binding in other crocodilians). Unlike other Hbs, it polymerizes to large complexes in the oxygenated state. The highly unusual properties of P. palpebrosus Hb align with a high content of His residues (potential sites for oxygenation-linked proton binding) and distinctive surface Cys residues that may form intermolecular disulfide bridges upon polymerization. On the basis of its singular properties, P. palpebrosus Hb provides a unique opportunity for studies on structure-function coupling and the evolution of compensatory mechanisms for maintaining tissue O2 delivery in Hbs that lack conventional effector-binding residues. PMID:23720132

Laminar and regional distribution of galanin binding sites in cat and monkey visual cortex determined by in vitro receptor autoradiography

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

Rosier, A.M.; Vandesande, F.; Orban, G.A.

1991-03-08

The distribution of galanin (GAL) binding sites in the visual cortex of cat and monkey was determined by autoradiographic visualization of ({sup 125}I)-GAL binding to tissue sections. Binding conditions were optimized and, as a result, the binding was saturable and specific. In cat visual cortex, GAL binding sites were concentrated in layers I, IVc, V, and VI. Areas 17, 18, and 19 exhibited a similar distribution pattern. In monkey primary visual cortex, the highest density of GAL binding sites was observed in layers II/III, lower IVc, and upper V. Layers IVA and VI contained moderate numbers of GAL binding sites,more » while layer I and the remaining parts of layer IV displayed the lowest density. In monkey secondary visual cortex, GAL binding sites were mainly concentrated in layers V-VI. Layer IV exhibited a moderate density, while the supragranular layers contained the lowest proportion of GAL binding sites. In both cat and monkey, we found little difference between regions subserving central and those subserving peripheral vision. Similarities in the distribution of GAL and acetylcholine binding sites are discussed.« less

Lysine Decarboxylase with an Enhanced Affinity for Pyridoxal 5-Phosphate by Disulfide Bond-Mediated Spatial Reconstitution.

PubMed

Sagong, Hye-Young; Kim, Kyung-Jin

2017-01-01

Lysine decarboxylase (LDC) catalyzes the decarboxylation of l-lysine to produce cadaverine, an important industrial platform chemical for bio-based polyamides. However, due to high flexibility at the pyridoxal 5-phosphate (PLP) binding site, use of the enzyme for cadaverine production requires continuous supplement of large amounts of PLP. In order to develop an LDC enzyme from Selenomonas ruminantium (SrLDC) with an enhanced affinity for PLP, we introduced an internal disulfide bond between Ala225 and Thr302 residues with a desire to retain the PLP binding site in a closed conformation. The SrLDCA225C/T302C mutant showed a yellow color and the characteristic UV/Vis absorption peaks for enzymes with bound PLP, and exhibited three-fold enhanced PLP affinity compared with the wild-type SrLDC. The mutant also exhibited a dramatically enhanced LDC activity and cadaverine conversion particularly under no or low PLP concentrations. Moreover, introduction of the disulfide bond rendered SrLDC more resistant to high pH and temperature. The formation of the introduced disulfide bond and the maintenance of the PLP binding site in the closed conformation were confirmed by determination of the crystal structure of the mutant. This study shows that disulfide bond-mediated spatial reconstitution can be a platform technology for development of enzymes with enhanced PLP affinity.

Highly selective on-off fluorescence recognition of Fe3+ based on a coumarin derivative and its application in live-cell imaging

NASA Astrophysics Data System (ADS)

Warrier, Sona; Kharkar, Prashant S.

2018-01-01

A novel coumarin chemosensor, 7-hydroxy-2-oxo-N-(pyridin-2-ylmethyl)chromene-3-carboxamide (Probe 1), demonstrated significant selectivity towards Fe3+ ions. Probe 1 exhibited high fluorescence emission profile at 447 nm, excellent selectivity towards Fe3+ over other biologically important metal ions (Al3+, Ba2+, Co2+, Cu2+, Zn2+, Cd2+, Hg2+, Pb2+ and Sn2+). Interestingly, there was 30-fold decrease in fluorescence intensity upon Fe3+ binding. The limit of detection of Fe3+ was found to be 0.76 μM ( 40 ppb). Probe 1 also exhibited high potential as an intracellular chemosensor for Fe3+.

A Dual-Specific Targeting Approach Based on the Simultaneous Recognition of Duplex and Quadruplex Motifs.

PubMed

Nguyen, Thi Quynh Ngoc; Lim, Kah Wai; Phan, Anh Tuân

2017-09-20

Small-molecule ligands targeting nucleic acids have been explored as potential therapeutic agents. Duplex groove-binding ligands have been shown to recognize DNA in a sequence-specific manner. On the other hand, quadruplex-binding ligands exhibit high selectivity between quadruplex and duplex, but show limited discrimination between different quadruplex structures. Here we propose a dual-specific approach through the simultaneous application of duplex- and quadruplex-binders. We demonstrated that a quadruplex-specific ligand and a duplex-specific ligand can simultaneously interact at two separate binding sites of a quadruplex-duplex hybrid harbouring both quadruplex and duplex structural elements. Such a dual-specific targeting strategy would combine the sequence specificity of duplex-binders and the strong binding affinity of quadruplex-binders, potentially allowing the specific targeting of unique quadruplex structures. Future research can be directed towards the development of conjugated compounds targeting specific genomic quadruplex-duplex sites, for which the linker would be highly context-dependent in terms of length and flexibility, as well as the attachment points onto both ligands.

Harmane and harmalan are bioactive components of classical clonidine-displacing substance.

PubMed

Parker, Christine A; Anderson, Neil J; Robinson, Emma S J; Price, Rhiannon; Tyacke, Robin J; Husbands, Stephen M; Dillon, Michael P; Eglen, Richard M; Hudson, Alan L; Nutt, David J; Crump, Matthew P; Crosby, John

2004-12-28

Elucidation of the structure of the endogenous ligand(s) for imidazoline binding sites, clonidine-displacing substance (CDS), has been a major goal for many years. Crude CDS from bovine lung was purified by reverse-phase high-pressure liquid chromatography. Electrospray mass spectrometry (ESMS) and nuclear magnetic resonance ((1)H NMR) analysis revealed the presence of L-tryptophan and 1-carboxy-1-methyltetrahydro-beta-carboline in the active CDS extract. Competition radioligand binding studies, however, failed to show displacement of specific [(3)H]clonidine binding to rat brain membranes for either compound. Further purification of the bovine lung extract allowed the isolation of the beta-carbolines harmane and harmalan as confirmed by ESMS, (1)H NMR, and comparison with synthetic standards. Both compounds exhibited a high (nanomolar) affinity for both type 1 and type 2 imidazoline binding sites, and the synthetic standards were shown to coelute with the active classical CDS extracts. We therefore propose that the beta-carbolines harmane and harmalan represent active components of classical CDS. The identification of these compounds will allow us to establish clear physiological roles for CDS.

Characterization of rodent liver and kidney AVP receptors: pharmacologic evidence for species differences.

PubMed

Tahara, A; Tsukada, J; Ishii, N; Tomura, Y; Wada, K; Kusayama, T; Yatsu, T; Uchida, W; Tanaka, A

1999-10-22

Radioligand binding studies with [3H]vasopressin (AVP) were used to determine the affinities of AVP receptor agonists and antagonists for mouse liver and kidney plasma membrane preparations. Both membrane preparations exhibited one class of high-affinity binding site. AVP ligand binding inhibition studies confirmed that mouse liver binding sites belong to the V1A subtype while kidney binding sites belong to the V2 receptor subtype. The affinity of each ligand for mouse V1A receptors was very similar to that for rat V1A receptors, showing differences in Ki values of less than 3-fold. In contrast, several peptide (d(CH2)5Tyr(Me)AVP) and nonpeptide (OPC-21268 and SR 49059) ligands had different affinities for mouse and rat kidney V2 receptors, with differences in Ki values ranging from 14- to 17-fold. These results indicate that mouse and rat kidney V2 receptors show significant pharmacologic differences.

Notch-Jagged complex structure implicates a catch bond in tuning ligand sensitivity

DOE PAGES

Luca, Vincent C.; Kim, Byoung Choul; Ge, Chenghao; ...

2017-03-02

Notch receptor activation initiates cell fate decisions and is distinctive in its reliance on mechanical force and protein glycosylation. The 2.5-angstrom-resolution crystal structure of the extracellular interacting region of Notch1 complexed with an engineered, high-affinity variant of Jagged1 (Jag1) reveals a binding interface that extends ~120 angstroms along five consecutive domains of each protein. O-Linked fucose modifications on Notch1 epidermal growth factor–like (EGF) domains 8 and 12 engage the EGF3 and C2 domains of Jag1, respectively, and different Notch1 domains are favored in binding to Jag1 than those that bind to the Delta-like 4 ligand. Jag1 undergoes conformational changes uponmore » Notch binding, exhibiting catch bond behavior that prolongs interactions in the range of forces required for Notch activation. In conclusion, this mechanism enables cellular forces to regulate binding, discriminate among Notch ligands, and potentiate Notch signaling.« less

Notch-Jagged complex structure implicates a catch bond in tuning ligand sensitivity

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

Luca, Vincent C.; Kim, Byoung Choul; Ge, Chenghao

Notch receptor activation initiates cell fate decisions and is distinctive in its reliance on mechanical force and protein glycosylation. The 2.5-angstrom-resolution crystal structure of the extracellular interacting region of Notch1 complexed with an engineered, high-affinity variant of Jagged1 (Jag1) reveals a binding interface that extends ~120 angstroms along five consecutive domains of each protein. O-Linked fucose modifications on Notch1 epidermal growth factor–like (EGF) domains 8 and 12 engage the EGF3 and C2 domains of Jag1, respectively, and different Notch1 domains are favored in binding to Jag1 than those that bind to the Delta-like 4 ligand. Jag1 undergoes conformational changes uponmore » Notch binding, exhibiting catch bond behavior that prolongs interactions in the range of forces required for Notch activation. In conclusion, this mechanism enables cellular forces to regulate binding, discriminate among Notch ligands, and potentiate Notch signaling.« less

Isolation of copper-binding proteins from activated sludge culture.

PubMed

Fukushi, K; Kato, S; Antsuki, T; Omura, T

2001-01-01

Six copper-binding microbial proteins were isolated from activated sludge cultures grown on media containing copper at various concentrations. Molecular weights among isolated proteins were ranged from 1.3k to 1 74k dalton. Isolated proteins were compared for their copper binding capabilities. Proteins isolated from cultures grown in the presence of copper in the growth media exhibited higher copper binding capabilities than those isolated from the culture grown in the absence of copper. The highest metal uptake of 61.23 (mol copper/mol protein) was observed by a protein isolated from a culture grown with copper at a concentration of 0.25 mM. This isolated protein (CBP2) had a molecular weight of 24k dalton. Other protein exhibited copper binding capability of 4.8-32.5 (mol copper/mol protein).

Differential affinities of molindone, metoclopramide and domperidone for classes of [3H]spiroperidol binding sites in rat striatum: evidence for pharmacologically distinct classes of receptors.

PubMed

Rosenfeld, M R; Dvorkin, B; Klein, P N; Makman, M H

1982-03-04

Rat striatum contains two populations of dopaminergic [3H]spiroperidol binding sites. The two populations are similar in their affinities for chlorpromazine and dopamine. Only one population, that with a somewhat higher affinity for spiroperidol itself, exhibits high affinity for the selective D2 antagonists molindone, metoclopramide and domperidone. Hence, this population may represent D2 receptor sites. The other larger population may represent either a separate class of receptor sites or a different form of D2 receptor sites.

A Colorimetric and Fluorescent Chemosensor of Fe3+ Based on an Asymmetrical Squarylium Dye

NASA Astrophysics Data System (ADS)

Lu, Min; Wang, Yue; Li, Ying; Li, Zhongyu; Xu, Song; Yao, Chao

2018-05-01

A novel asymmetrical squarylium-dye-based dual signaling probe was synthesized and found to exhibit colorimetric and fluorescent properties on selective binding towards Fe3+ in ethanol/water (4:1, v/v) solution. The binding constant was determined to be 7.69 × 102 M-1, and the detection limit of SQ was 9.158 × 10-6 M. Most importantly, the 1:2 stoichiometry of the host-guest complexation was confirmed by Job's method. Moreover, the high sensing ability of the receptor towards Fe3+ was also investigated by the electrochemical technique.

Discrimination against RNA Backbones by a ssDNA Binding Protein.

PubMed

Lloyd, Neil R; Wuttke, Deborah S

2018-05-01

Pot1 is the shelterin component responsible for the protection of the single-stranded DNA (ssDNA) overhang at telomeres in nearly all eukaryotic organisms. The C-terminal domain of the DNA-binding domain, Pot1pC, exhibits non-specific ssDNA recognition, achieved through thermodynamically equivalent alternative binding conformations. Given this flexibility, it is unclear how specificity for ssDNA over RNA, an activity required for biological function, is achieved. Examination of the ribose-position specificity of Pot1pC shows that ssDNA specificity is additive but not uniformly distributed across the ligand. High-resolution structures of several Pot1pC complexes with RNA-DNA chimeric ligands reveal Pot1pC discriminates against RNA by utilizing non-compensatory binding modes that feature significant rearrangement of the binding interface. These alternative conformations, accessed through both ligand and protein flexibility, recover much, but not all, of the binding energy, leading to the observed reduction in affinities. These findings suggest that intermolecular interfaces are remarkably sophisticated in their tuning of specificity toward flexible ligands. Copyright © 2018 Elsevier Ltd. All rights reserved.

Line Narrowing of Excited-State Transitions in Nonlinear Polarization Spectroscopy: Application to Water-Soluble Chlorophyll-Binding Protein

NASA Astrophysics Data System (ADS)

Schoth, Mario; Richter, Marten; Knorr, Andreas; Renger, Thomas

2012-04-01

The homogeneous linewidth of dye aggregates like photosynthetic light-harvesting complexes contains important information about energy transfer and relaxation times that is, however, masked by inhomogeneous broadening caused by static disorder. Whereas there exist line narrowing techniques for the study of low-energy exciton states, the homogeneous linewidth of the high-energy states is not so easy to decipher. Here we present a microscopic theory for nonlinear polarization spectroscopy in the frequency domain that contains a dynamic aggregate selection revealing the homogeneous linewidth of these states. The theory is applied to the water-soluble chlorophyll-binding protein for which the high-energy exciton state was predicted to exhibit a sub-100-fs lifetime.

Synthesis of a ratiometric fluorescent peptide sensor for the highly selective detection of Cd2+.

PubMed

Li, Yan; Li, Lianzhi; Pu, Xuewei; Ma, Guolin; Wang, Erqiong; Kong, Jinming; Liu, Zhipeng; Liu, Yangzhong

2012-06-15

A novel ratiometric fluorescent peptidyl chemosensor (Dansyl-Cys-Pro-Gly-Cys-Trp-NH(2), D-P5) for metal ions detection has been synthesized via Fmoc solid-phase peptide synthesis. The chemosensor exhibited a high selectivity for Cd(2+) over other metal ions including competitive transition and Group I and II metal ions in neutral pH. The fluorescence emission intensity of D-P5 was significantly enhanced in the presence of Cd(2+) by fluorescent resonance energy transfer (FRET) and chelation enhanced fluorescence (CHEF) effects. The binding stoichiometry, detection limit, binding affinity, reversibility and pH sensitivity of the sensor for Cd(2+) were investigated. Copyright © 2012 Elsevier Ltd. All rights reserved.

Binding of [3H] SR 49059, a potent nonpeptide vasopressin V1a antagonist, to rat and human liver membranes.

PubMed

Serradeil-Le Gal, C; Raufaste, D; Marty, E; Garcia, C; Maffrand, J P; Le Fur, G

1994-02-28

The new potent and selective nonpeptide vasopressin V1a antagonist, SR 49059, was tritiated and used for the characterization of rat and human liver AVP V1a receptors. Binding of [3H] SR 49059 was time-dependent, reversible and saturable. A single class of high affinity binding sites was identified with Kd values of 0.63 +/- 0.13 and 2.95 +/- 0.64 nM, in rat and human liver membranes, respectively. The maximal binding capacity (Bmax) was about 7 times higher in rat than in human liver preparations. The relative potencies of several AVP/oxytocin agonists or antagonists to inhibit [3H] SR 49059 binding confirmed that this ligand labeled a homogeneous population of sites with the expected AVP V1a profile. Furthermore, [3H] SR 49059 or unlabeled SR 49059 displayed only slight species differences between rat and human V1a receptors, whereas OPC-21268, another nonpeptide V1a antagonist, exhibited a high species-related potency with more than 500 fold higher affinity for rat than for human liver V1a receptors. Thus, [3H] SR 49059 is the first nonpeptide AVP V1a ligand reported having highly specific activity, stability, specificity and affinity. This makes it a suitable probe for labeling AVP V1a receptors in rat and also in human tissues.

Structural and Histone Binding Ability Characterizations of Human PWWP Domains

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

Wu, Hong; Zeng, Hong; Lam, Robert

2013-09-25

The PWWP domain was first identified as a structural motif of 100-130 amino acids in the WHSC1 protein and predicted to be a protein-protein interaction domain. It belongs to the Tudor domain 'Royal Family', which consists of Tudor, chromodomain, MBT and PWWP domains. While Tudor, chromodomain and MBT domains have long been known to bind methylated histones, PWWP was shown to exhibit histone binding ability only until recently. The PWWP domain has been shown to be a DNA binding domain, but sequence analysis and previous structural studies show that the PWWP domain exhibits significant similarity to other 'Royal Family' members,more » implying that the PWWP domain has the potential to bind histones. In order to further explore the function of the PWWP domain, we used the protein family approach to determine the crystal structures of the PWWP domains from seven different human proteins. Our fluorescence polarization binding studies show that PWWP domains have weak histone binding ability, which is also confirmed by our NMR titration experiments. Furthermore, we determined the crystal structures of the BRPF1 PWWP domain in complex with H3K36me3, and HDGF2 PWWP domain in complex with H3K79me3 and H4K20me3. PWWP proteins constitute a new family of methyl lysine histone binders. The PWWP domain consists of three motifs: a canonical {beta}-barrel core, an insertion motif between the second and third {beta}-strands and a C-terminal {alpha}-helix bundle. Both the canonical {beta}-barrel core and the insertion motif are directly involved in histone binding. The PWWP domain has been previously shown to be a DNA binding domain. Therefore, the PWWP domain exhibits dual functions: binding both DNA and methyllysine histones.« less

Fragment-Linking Approach Using (19)F NMR Spectroscopy To Obtain Highly Potent and Selective Inhibitors of β-Secretase.

PubMed

Jordan, John B; Whittington, Douglas A; Bartberger, Michael D; Sickmier, E Allen; Chen, Kui; Cheng, Yuan; Judd, Ted

2016-04-28

Fragment-based drug discovery (FBDD) has become a widely used tool in small-molecule drug discovery efforts. One of the most commonly used biophysical methods in detecting weak binding of fragments is nuclear magnetic resonance (NMR) spectroscopy. In particular, FBDD performed with (19)F NMR-based methods has been shown to provide several advantages over (1)H NMR using traditional magnetization-transfer and/or two-dimensional methods. Here, we demonstrate the utility and power of (19)F-based fragment screening by detailing the identification of a second-site fragment through (19)F NMR screening that binds to a specific pocket of the aspartic acid protease, β-secretase (BACE-1). The identification of this second-site fragment allowed the undertaking of a fragment-linking approach, which ultimately yielded a molecule exhibiting a more than 360-fold increase in potency while maintaining reasonable ligand efficiency and gaining much improved selectivity over cathepsin-D (CatD). X-ray crystallographic studies of the molecules demonstrated that the linked fragments exhibited binding modes consistent with those predicted from the targeted screening approach, through-space NMR data, and molecular modeling.

Local anesthetic and antiepileptic drug access and binding to a bacterial voltage-gated sodium channel

PubMed Central

Boiteux, Céline; Vorobyov, Igor; French, Robert J.; French, Christopher; Yarov-Yarovoy, Vladimir; Allen, Toby W.

2014-01-01

Voltage-gated sodium (Nav) channels are important targets in the treatment of a range of pathologies. Bacterial channels, for which crystal structures have been solved, exhibit modulation by local anesthetic and anti-epileptic agents, allowing molecular-level investigations into sodium channel-drug interactions. These structures reveal no basis for the “hinged lid”-based fast inactivation, seen in eukaryotic Nav channels. Thus, they enable examination of potential mechanisms of use- or state-dependent drug action based on activation gating, or slower pore-based inactivation processes. Multimicrosecond simulations of NavAb reveal high-affinity binding of benzocaine to F203 that is a surrogate for FS6, conserved in helix S6 of Domain IV of mammalian sodium channels, as well as low-affinity sites suggested to stabilize different states of the channel. Phenytoin exhibits a different binding distribution owing to preferential interactions at the membrane and water–protein interfaces. Two drug-access pathways into the pore are observed: via lateral fenestrations connecting to the membrane lipid phase, as well as via an aqueous pathway through the intracellular activation gate, despite being closed. These observations provide insight into drug modulation that will guide further developments of Nav inhibitors. PMID:25136136

Evaluation of Bacterial Expansin EXLX1 as a Cellulase Synergist for the Saccharification of Lignocellulosic Agro-Industrial Wastes

PubMed Central

Lin, Hui; Shen, Qi; Zhan, Ju-Mei; Wang, Qun; Zhao, Yu-Hua

2013-01-01

Various types of lignocellulosic wastes extensively used in biofuel production were provided to assess the potential of EXLX1 as a cellulase synergist. Enzymatic hydrolysis of natural wheat straw showed that all the treatments using mixtures of cellulase and an optimized amount of EXLX1, released greater quantities of sugars than those using cellulase alone, regardless of cellulase dosage and incubation time. EXLX1 exhibited different synergism and binding characteristics for different wastes, but this can be related to their lignocellulosic components. The cellulose proportion could be one of the important factors. However, when the cellulose proportion of different biomass samples exhibited no remarkable differences, a higher synergism of EXLX1 is prone to occur on these materials, with a high proportion of hemicellulose and a low proportion of lignin. The information could be favorable to assess whether EXLX1 is effective as a cellulase synergist for the hydrolysis of the used materials. Binding assay experiments further suggested that EXLX1 bound preferentially to alkali pretreated materials, as opposed to acid pretreated materials under the assay condition and the binding preference would be affected by incubation temperature. PMID:24086425

Synthesis and Structure–Activity Relationships of N-Benzyl Phenethylamines as 5-HT2A/2C Agonists

PubMed Central

2014-01-01

N-Benzyl substitution of 5-HT2A receptor agonists of the phenethylamine structural class of psychedelics (such as 4-bromo-2,5-dimethoxyphenethylamine, often referred to as 2C-B) confer a significant increase in binding affinity as well as functional activity of the receptor. We have prepared a series of 48 compounds with structural variations in both the phenethylamine and N-benzyl part of the molecule to determine the effects on receptor binding affinity and functional activity at 5-HT2A and 5-HT2C receptors. The compounds generally had high affinity for the 5-HT2A receptor with 8b having the highest affinity at 0.29 nM but with several other compounds also exhibiting subnanomolar binding affinities. The functional activity of the compounds was distributed over a wider range with 1b being the most potent at 0.074 nM. Most of the compounds exhibited low to moderate selectivity (1- to 40-fold) for the 5-HT2A receptor in the binding assays, although one compound 6b showed an impressive 100-fold selectivity for the 5-HT2A receptor. In the functional assay, selectivity was generally higher with 1b being more than 400-fold selective for the 5-HT2A receptor. PMID:24397362

Synthesis and structure-activity relationships of N-benzyl phenethylamines as 5-HT2A/2C agonists.

PubMed

Hansen, Martin; Phonekeo, Karina; Paine, James S; Leth-Petersen, Sebastian; Begtrup, Mikael; Bräuner-Osborne, Hans; Kristensen, Jesper L

2014-03-19

N-Benzyl substitution of 5-HT2A receptor agonists of the phenethylamine structural class of psychedelics (such as 4-bromo-2,5-dimethoxyphenethylamine, often referred to as 2C-B) confer a significant increase in binding affinity as well as functional activity of the receptor. We have prepared a series of 48 compounds with structural variations in both the phenethylamine and N-benzyl part of the molecule to determine the effects on receptor binding affinity and functional activity at 5-HT2A and 5-HT2C receptors. The compounds generally had high affinity for the 5-HT2A receptor with 8b having the highest affinity at 0.29 nM but with several other compounds also exhibiting subnanomolar binding affinities. The functional activity of the compounds was distributed over a wider range with 1b being the most potent at 0.074 nM. Most of the compounds exhibited low to moderate selectivity (1- to 40-fold) for the 5-HT2A receptor in the binding assays, although one compound 6b showed an impressive 100-fold selectivity for the 5-HT2A receptor. In the functional assay, selectivity was generally higher with 1b being more than 400-fold selective for the 5-HT2A receptor.

Gene polymorphism linked to increased asthma and IBD risk alters gasdermin-B structure, a sulfatide and phosphoinositide binding protein.

PubMed

Chao, Kinlin L; Kulakova, Liudmila; Herzberg, Osnat

2017-02-14

The exact function of human gasdermin-B (GSDMB), which regulates differentiation and growth of epithelial cells, is yet to be elucidated. In human epidermal growth factor receptor 2 (HER2)-positive breast cancer, GSDMB gene amplification and protein overexpression indicate a poor response to HER2-targeted therapy. Genome-wide association studies revealed a correlation between GSDMB SNPs and an increased susceptibility to Crohn's disease, ulcerative colitis, and asthma. The N- and C-terminal domains of all gasdermins possess lipid-binding and regulatory activities, respectively. Inflammatory caspases cleave gasdermin-D in the interdomain linker but not GSDMB. The cleaved N-terminal domain binds phosphoinositides and cardiolipin, forms membrane-disrupting pores, and executes pyroptosis. We show that both full-length GSDMB and the N-terminal domain bind to nitrocellulose membranes immobilized with phosphoinositides or sulfatide, but not with cardiolipin. In addition, the GSDMB N-terminal domain binds liposomes containing sulfatide. The crystal structure of the GSDMB C-terminal domain reveals the structural impact of the amino acids encoded by SNPs that are linked to asthma and inflammatory bowel disease (IBD). A loop that carries the polymorphism amino acids corresponding to healthy individuals (Gly299:Pro306) exhibits high conformational flexibility, whereas the loop carrying amino acids found in individuals with increased disease risk (Arg299:Ser306) exhibits a well-defined conformation and higher positive surface charge. Apoptotic executioner caspase-3, -6, and -7, but not the inflammatory caspases, cleave GSDMB at 88 DNVD 91 within the N-terminal domain. Selective sulfatide binding may indicate possible function for GSDMB in the cellular sulfatide transport.

Gene polymorphism linked to increased asthma and IBD risk alters gasdermin-B structure, a sulfatide and phosphoinositide binding protein

PubMed Central

Chao, Kinlin L.; Kulakova, Liudmila; Herzberg, Osnat

2017-01-01

The exact function of human gasdermin-B (GSDMB), which regulates differentiation and growth of epithelial cells, is yet to be elucidated. In human epidermal growth factor receptor 2 (HER2)-positive breast cancer, GSDMB gene amplification and protein overexpression indicate a poor response to HER2-targeted therapy. Genome-wide association studies revealed a correlation between GSDMB SNPs and an increased susceptibility to Crohn’s disease, ulcerative colitis, and asthma. The N- and C-terminal domains of all gasdermins possess lipid-binding and regulatory activities, respectively. Inflammatory caspases cleave gasdermin-D in the interdomain linker but not GSDMB. The cleaved N-terminal domain binds phosphoinositides and cardiolipin, forms membrane-disrupting pores, and executes pyroptosis. We show that both full-length GSDMB and the N-terminal domain bind to nitrocellulose membranes immobilized with phosphoinositides or sulfatide, but not with cardiolipin. In addition, the GSDMB N-terminal domain binds liposomes containing sulfatide. The crystal structure of the GSDMB C-terminal domain reveals the structural impact of the amino acids encoded by SNPs that are linked to asthma and inflammatory bowel disease (IBD). A loop that carries the polymorphism amino acids corresponding to healthy individuals (Gly299:Pro306) exhibits high conformational flexibility, whereas the loop carrying amino acids found in individuals with increased disease risk (Arg299:Ser306) exhibits a well-defined conformation and higher positive surface charge. Apoptotic executioner caspase-3, -6, and -7, but not the inflammatory caspases, cleave GSDMB at 88DNVD91 within the N-terminal domain. Selective sulfatide binding may indicate possible function for GSDMB in the cellular sulfatide transport. PMID:28154144

Autoradiographic localization of sigma receptor binding sites in guinea pig and rat central nervous system with (+)3H-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine

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

Gundlach, A.L.; Largent, B.L.; Snyder, S.H.

1986-06-01

(+)3H-3-PPP ((+)3H-3-(3-Hydroxyphenyl)-N-(1-propyl)-piperidine) binds with high affinity to brain membranes with a pharmacological profile consistent with that of sigma receptors. The distribution of (+)3H-3-PPP binding sites in brain and spinal cord of both guinea pig and rat has been determined by in vitro autoradiography with binding densities quantitated by computer-assisted densitometry. (+)3H-3-PPP binding to slide-mounted brain sections is saturable and displays high affinity and a pharmacological specificity very similar to sites labeled in homogenates. (+)3H-3-PPP binding sites are heterogeneously distributed. Highest concentrations of binding sites occur in spinal cord, particularly the ventral horn and dorsal root ganglia; the pons-medulla, associated withmore » the cranial nerve and pontine nuclei and throughout the brain stem reticular formation; the cerebellum, over the Purkinje cell layer; the midbrain, particularly the central gray and red nucleus; and hippocampus, over the pyramidal cell layer. Lowest levels are seen in the basal ganglia and parts of the thalamus, while all other areas, including hypothalamus and cerebral cortex, exhibit moderate grain densities. Quinolinic acid-induced lesions of the hippocampus indicate that (+)3H-3-PPP labels hippocampal pyramidal cells and granule cells in the dentate gyrus. Intrastriatal injection of ibotenic acid dramatically reduces (+)3H-3-PPP binding in this area, while injection of 6-hydroxydopamine produces a relatively slight decrease. The distribution of (+)3H-3-PPP binding sites does not correlate with the receptor distribution of any recognized neurotransmitter or neuropeptide, including dopamine. However, there is a notable similarity between the distribution of (+)3H-3-PPP sites and high-affinity binding sites for psychotomimetic opioids, such as the benzomorphan (+)SKF 10,047.« less

Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding.

PubMed

Munck Petersen, C; Nielsen, M S; Jacobsen, C; Tauris, J; Jacobsen, L; Gliemann, J; Moestrup, S K; Madsen, P

1999-02-01

We recently reported the isolation and sequencing of sortilin, a new putative sorting receptor that binds receptor-associated protein (RAP). The luminal N-terminus of sortilin comprises a consensus sequence for cleavage by furin, R41WRR44, which precedes a truncation originally found in sortilin isolated from human brain. We now show that the truncation results from cellular processing. Sortilin is synthesized as a proform which, in late Golgi compartments, is converted to the mature receptor by furin-mediated cleavage of a 44 residue N-terminal propeptide. We further demonstrate that the propeptide exhibits pH-dependent high affinity binding to fully processed sortilin, that the binding is competed for by RAP and the newly discovered sortilin ligand neurotensin, and that prevention of propeptide cleavage essentially prevents binding of RAP and neurotensin. The findings evidence that the propeptide sterically hinders ligands from gaining access to overlapping binding sites in prosortilin, and that cleavage and release of the propeptide preconditions sortilin for full functional activity. Although proteolytic processing is involved in the maturation of several receptors, the described exposure of previously concealed ligand-binding sites after furin-mediated cleavage of propeptide represents a novel mechanism in receptor activation.

Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding.

PubMed Central

Munck Petersen, C; Nielsen, M S; Jacobsen, C; Tauris, J; Jacobsen, L; Gliemann, J; Moestrup, S K; Madsen, P

1999-01-01

We recently reported the isolation and sequencing of sortilin, a new putative sorting receptor that binds receptor-associated protein (RAP). The luminal N-terminus of sortilin comprises a consensus sequence for cleavage by furin, R41WRR44, which precedes a truncation originally found in sortilin isolated from human brain. We now show that the truncation results from cellular processing. Sortilin is synthesized as a proform which, in late Golgi compartments, is converted to the mature receptor by furin-mediated cleavage of a 44 residue N-terminal propeptide. We further demonstrate that the propeptide exhibits pH-dependent high affinity binding to fully processed sortilin, that the binding is competed for by RAP and the newly discovered sortilin ligand neurotensin, and that prevention of propeptide cleavage essentially prevents binding of RAP and neurotensin. The findings evidence that the propeptide sterically hinders ligands from gaining access to overlapping binding sites in prosortilin, and that cleavage and release of the propeptide preconditions sortilin for full functional activity. Although proteolytic processing is involved in the maturation of several receptors, the described exposure of previously concealed ligand-binding sites after furin-mediated cleavage of propeptide represents a novel mechanism in receptor activation. PMID:9927419

Spleen size and plasma levels of mannose binding lectin in channel catfish Ictalurus punctatus families exhibiting different susceptibilities to Flavobacterium columnare and Edwardsiella ictaluri

USDA-ARS?s Scientific Manuscript database

Two major problems in the channel catfish (Ictalurus punctatus) aquaculture industry have been high disease losses to enteric septicemia of catfish (ESC), caused by the bacterium Edwardsiella ictaluri and columnaris disease, caused by the bacterium Flavobacterium columnare. Methods to control these...

Discovery of a novel general anesthetic chemotype using high-throughput screening.

PubMed

McKinstry-Wu, Andrew R; Bu, Weiming; Rai, Ganesha; Lea, Wendy A; Weiser, Brian P; Liang, David F; Simeonov, Anton; Jadhav, Ajit; Maloney, David J; Eckenhoff, Roderic G

2015-02-01

The development of novel anesthetics has historically been a process of combined serendipity and empiricism, with most recent new anesthetics developed via modification of existing anesthetic structures. Using a novel high-throughput screen employing the fluorescent anesthetic 1-aminoanthracene and apoferritin as a surrogate for on-pathway anesthetic protein target(s), we screened a 350,000 compound library for competition with 1-aminoanthracene-apoferritin binding. Hit compounds meeting structural criteria had their binding affinities for apoferritin quantified with isothermal titration calorimetry and were tested for γ-aminobutyric acid type A receptor binding using a flunitrazepam binding assay. Chemotypes with a strong presence in the top 700 and exhibiting activity via isothermal titration calorimetry were selected for medicinal chemistry optimization including testing for anesthetic potency and toxicity in an in vivo Xenopus laevis tadpole assay. Compounds with low toxicity and high potency were tested for anesthetic potency in mice. From an initial chemical library of more than 350,000 compounds, we identified 2,600 compounds that potently inhibited 1-aminoanthracene binding to apoferritin. A subset of compounds chosen by structural criteria (700) was successfully reconfirmed using the initial assay. Based on a strong presence in both the initial and secondary screens the 6-phenylpyridazin-3(2H)-one chemotype was assessed for anesthetic activity in tadpoles. Medicinal chemistry efforts identified four compounds with high potency and low toxicity in tadpoles, two were found to be effective novel anesthetics in mice. The authors demonstrate the first use of a high-throughput screen to successfully identify a novel anesthetic chemotype and show mammalian anesthetic activity for members of that chemotype.

Synthesis, iron binding and antimicrobial properties of hexadentate 3-hydroxypyridinones-terminated dendrimers.

PubMed

Zhou, Tao; Chen, Kai; Kong, Li-Min; Liu, Mu-Song; Ma, Yong-Min; Xie, Yuan-Yuan; Hider, Robert C

2018-05-30

Macromolecular chelators have potential applications in the medical area, for instance, in treatment of iron overload-related disorders and in the treatment of external infections. In this investigation, several novel iron(III)-selective hydroxypyridinone hexadentate-terminated first and second generation dendrimeric chelators were synthesized using a convergent strategy. Their iron chelating ability was demonstrated by UV/Visible spectrometry and high resolution mass spectrometry (HRMS). The iron binding affinities were also investigated by the competition with a fluorescent iron chelator CP691. The result indicated that these dendrimers possesses a high affinity for iron with a very high pFe 3+ value, which is close to that of an isolated hexadentate unit. These dendrimeric chelators were found to exhibit inhibitory effect on the growth of both Gram-positive and Gram-negative bacteria. Copyright © 2018 Elsevier Ltd. All rights reserved.

Visual feature binding in younger and older adults: encoding and suffix interference effects.

PubMed

Brown, Louise A; Niven, Elaine H; Logie, Robert H; Rhodes, Stephen; Allen, Richard J

2017-02-01

Three experiments investigated younger (18-25 yrs) and older (70-88 yrs) adults' temporary memory for colour-shape combinations (binding). We focused upon estimating the magnitude of the binding cost for each age group across encoding time (Experiment 1; 900/1500 ms), presentation format (Experiment 2; simultaneous/sequential), and interference (Experiment 3; control/suffix) conditions. In Experiment 1, encoding time did not differentially influence binding in the two age groups. In Experiment 2, younger adults exhibited poorer binding performance with sequential relative to simultaneous presentation, and serial position analyses highlighted a particular age-related difficulty remembering the middle item of a series (for all memory conditions). Experiments 1-3 demonstrated small to medium binding effect sizes in older adults across all encoding conditions, with binding less accurate than shape memory. However, younger adults also displayed negative effects of binding (small to large) in two of the experiments. Even when older adults exhibited a greater suffix interference effect in Experiment 3, this was for all memory types, not just binding. We therefore conclude that there is no consistent evidence for a visual binding deficit in healthy older adults. This relative preservation contrasts with the specific and substantial deficits in visual feature binding found in several recent studies of Alzheimer's disease.

Two distinct modes of metal ion binding in the nuclease active site of a viral DNA-packaging terminase: insight into the two-metal-ion catalytic mechanism

PubMed Central

Zhao, Haiyan; Lin, Zihan; Lynn, Anna Y.; Varnado, Brittany; Beutler, John A.; Murelli, Ryan P.; Le Grice, Stuart F. J.; Tang, Liang

2015-01-01

Many dsDNA viruses encode DNA-packaging terminases, each containing a nuclease domain that resolves concatemeric DNA into genome-length units. Terminase nucleases resemble the RNase H-superfamily nucleotidyltransferases in folds, and share a two-metal-ion catalytic mechanism. Here we show that residue K428 of a bacteriophage terminase gp2 nuclease domain mediates binding of the metal cofactor Mg2+. A K428A mutation allows visualization, at high resolution, of a metal ion binding mode with a coupled-octahedral configuration at the active site, exhibiting an unusually short metal-metal distance of 2.42 Å. Such proximity of the two metal ions may play an essential role in catalysis by generating a highly positive electrostatic niche to enable formation of the negatively charged pentacovalent phosphate transition state, and provides the structural basis for distinguishing Mg2+ from Ca2+. Using a metal ion chelator β-thujaplicinol as a molecular probe, we observed a second mode of metal ion binding at the active site, mimicking the DNA binding state. Arrangement of the active site residues differs drastically from those in RNase H-like nucleases, suggesting a drifting of the active site configuration during evolution. The two distinct metal ion binding modes unveiled mechanistic details of the two-metal-ion catalysis at atomic resolution. PMID:26450964

Characterization of Selective Exosite-Binding Inhibitors of Matrix Metalloproteinase 13 That Prevent Articular Cartilage Degradation in Vitro

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

Spicer, Timothy P.; Jiang, Jianwen; Taylor, Alexander B.

Matrix metalloproteinase 13 (MMP-13) has been shown to be the main collagenase responsible for degradation of articular cartilage during osteoarthritis and therefore represents a target for drug development. Here, as a result of high-throughput screening and structure$-$activity relationship studies, we identified a novel, highly selective class of MMP-13 inhibitors (compounds 1 (Q), 2 (Q1), and 3 (Q2)). Mechanistic characterization revealed a noncompetitive nature of these inhibitors with binding constants in the low micromolar range. Crystallographic analyses revealed two binding modes for compound 2 in the MMP-13 S 1' subsite and in an S 1/S 2* subsite. Type II collagen- andmore » cartilage-protective effects exhibited by compounds 1, 2, and 3 suggested that these compounds might be efficacious in future in vivo studies. Lastly, these compounds were also highly selective when tested against a panel of 30 proteases, which, in combination with a good CYP inhibition profile, suggested low off-target toxicity and drug$-$drug interactions in humans.« less

Characterization of Selective Exosite-Binding Inhibitors of Matrix Metalloproteinase 13 That Prevent Articular Cartilage Degradation in Vitro

DOE PAGES

Spicer, Timothy P.; Jiang, Jianwen; Taylor, Alexander B.; ...

2014-10-20

Matrix metalloproteinase 13 (MMP-13) has been shown to be the main collagenase responsible for degradation of articular cartilage during osteoarthritis and therefore represents a target for drug development. Here, as a result of high-throughput screening and structure$-$activity relationship studies, we identified a novel, highly selective class of MMP-13 inhibitors (compounds 1 (Q), 2 (Q1), and 3 (Q2)). Mechanistic characterization revealed a noncompetitive nature of these inhibitors with binding constants in the low micromolar range. Crystallographic analyses revealed two binding modes for compound 2 in the MMP-13 S 1' subsite and in an S 1/S 2* subsite. Type II collagen- andmore » cartilage-protective effects exhibited by compounds 1, 2, and 3 suggested that these compounds might be efficacious in future in vivo studies. Lastly, these compounds were also highly selective when tested against a panel of 30 proteases, which, in combination with a good CYP inhibition profile, suggested low off-target toxicity and drug$-$drug interactions in humans.« less

Hydroxylated polybrominated diphenyl ethers exhibit different activities on thyroid hormone receptors depending on their degree of bromination

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

Ren, Xiao-Min, E-mail: rxm200318@gmail.com; Guo, Liang-Hong, E-mail: LHGuo@rcees.ac.cn; Gao, Yu, E-mail: francesscototti@gmail.com

2013-05-01

Polybrominated diphenyl ethers (PBDEs) have been shown to disrupt thyroid hormone (TH) functions in experimental animals, and one of the proposed disruption mechanisms is direct binding of hydroxylated PBDE (OH-PBDE) to TH receptors (TRs). However, previous data on TH receptor binding and TH activity of OH-PBDEs were very limited and sometimes inconsistent. In the present paper, we examined the binding potency of ten OH-PBDEs with different degrees of bromination to TR using a fluorescence competitive binding assay. The results showed that the ten OH-PBDEs bound to TR with potency that correlated to their bromination level. We further examined their effectmore » on TR using a coactivator binding assay and GH3 cell proliferation assay. Different TR activities of OH-PBDEs were observed depending on their degree of bromination. Four low-brominated OH-PBDEs (2′-OH-BDE-28, 3′-OH-BDE-28, 5-OH-BDE-47, 6-OH-BDE-47) were found to be TR agonists, which recruited the coactivator peptide and enhanced GH3 cell proliferation. However, three high-brominated OH-PBDEs (3-OH-BDE-100, 3′-OH-BDE-154, 4-OH-BDE-188) were tested to be antagonists. Molecular docking was employed to simulate the interactions of OH-PBDEs with TR and identify the structural determinants for TR binding and activity. According to the docking results, low-brominated OH-PBDEs, which are weak binders but TR agonists, bind with TR at the inner side of its binding pocket, whereas high-brominated compounds, which are potent binders but TR antagonists, reside at the outer region. These results indicate that OH-PBDEs have different activities on TR (agonistic or antagonistic), possibly due to their different binding geometries with the receptor. - Highlights: ► Thyroid hormone (TH) activity of OH-PBDEs with different Br number was evaluated. ► Four different experimental approaches were employed to investigate the mechanism. ► Low-brominated OH-PBDEs were agonists, but high-brominated ones were antagonists. ► Low-brominated OH-PBDEs bind to TH receptor differently than high-brominated ones.« less

Highly selective on-off fluorescence recognition of Fe3+ based on a coumarin derivative and its application in live-cell imaging.

PubMed

Warrier, Sona; Kharkar, Prashant S

2018-01-05

A novel coumarin chemosensor, 7-hydroxy-2-oxo-N-(pyridin-2-ylmethyl)chromene-3-carboxamide (Probe 1), demonstrated significant selectivity towards Fe 3+ ions. Probe 1 exhibited high fluorescence emission profile at 447nm, excellent selectivity towards Fe 3+ over other biologically important metal ions (Al 3+ , Ba 2+ , Co 2+ , Cu 2+ , Zn 2+ , Cd 2+ , Hg 2+ , Pb 2+ and Sn 2+ ). Interestingly, there was ~30-fold decrease in fluorescence intensity upon Fe 3+ binding. The limit of detection of Fe 3+ was found to be 0.76μM (~40ppb). Probe 1 also exhibited high potential as an intracellular chemosensor for Fe 3+ . Copyright © 2017 Elsevier B.V. All rights reserved.

A highly selective and sensitive fluorescent chemosensor and its application for rapid on-site detection of Al3 +

NASA Astrophysics Data System (ADS)

Yue, Xiao-li; Wang, Zhao-qing; Li, Chao-rui; Yang, Zheng-yin

2018-03-01

In this paper, a simple naphthalene-based derivative (HL) has been designed and synthesized as a Al3 +-selective fluorescent chemosensor based on the PET mechanism. HL exhibited high selectivity and sensitivity towards Al3 + over other commonly coexisting metal ions in ethanol with a detection limit of 2.72 nM. The 1:1 binding stoichiometry of the complex (HL-Al3 +) was determined from the Job's plot based on fluorescence titrations and the ESI-MS spectrum data. Moreover, the binding site of HL with Al3 + was assured by the 1H NMR titration experiment. The binding constant (Ka) of the complex (HL-Al3 +) was calculated to be 5.06 × 104 M- 1 according to the Benesi-Hildebrand equation. In addition, the recognizing process of HL towards Al3 + was chemically reversible by adding Na2EDTA. Importantly, HL could directly and rapidly detect aluminum ion through the filter paper without resorting to additional instrumental analysis.

Application of Circular Dichroism Spectroscopy to the Analysis of the Interaction Between the Estrogen Receptor Alpha and Coactivators: The Case of Calmodulin.

PubMed

Miclet, Emeric; Bourgoin-Voillard, Sandrine; Byrne, Cillian; Jacquot, Yves

2016-01-01

The estrogen receptor α ligand-binding domain (ERα-LBD) binds the natural hormone 17β-estradiol (E2) to induce transcription and cell proliferation. This process occurs with the contribution of protein and peptide partners (also called coactivators) that can modulate the structure of ERα, and therefore its specificity of action. As with most transcription factors, ERα exhibits a high content of α helix, making it difficult to routinely run spectroscopic studies capable of deciphering the secondary structure of the different partners under binding conditions. Ca(2+)-calmodulin, a protein also highly structured in α-helix, is a key coactivator for ERα activity. Here, we show how circular dichroism can be used to study the interaction of ERα with Ca(2+)-calmodulin. Our approach allows the determination not only of the conformational changes induced upon complex formation but also the dissociation constant (K d) of this interaction.

Tracer diffusion in a sea of polymers with binding zones: mobile vs. frozen traps.

PubMed

Samanta, Nairhita; Chakrabarti, Rajarshi

2016-10-19

We use molecular dynamics simulations to investigate the tracer diffusion in a sea of polymers with specific binding zones for the tracer. These binding zones act as traps. Our simulations show that the tracer can undergo normal yet non-Gaussian diffusion under certain circumstances, e.g., when the polymers with traps are frozen in space and the volume fraction and the binding strength of the traps are moderate. In this case, as the tracer moves, it experiences a heterogeneous environment and exhibits confined continuous time random walk (CTRW) like motion resulting in a non-Gaussian behavior. Also the long time dynamics becomes subdiffusive as the number or the binding strength of the traps increases. However, if the polymers are mobile then the tracer dynamics is Gaussian but could be normal or subdiffusive depending on the number and the binding strength of the traps. In addition, with increasing binding strength and number of polymer traps, the probability of the tracer being trapped increases. On the other hand, removing the binding zones does not result in trapping, even at comparatively high crowding. Our simulations also show that the trapping probability increases with the increasing size of the tracer and for a bigger tracer with the frozen polymer background the dynamics is only weakly non-Gaussian but highly subdiffusive. Our observations are in the same spirit as found in many recent experiments on tracer diffusion in polymeric materials and question the validity of using Gaussian theory to describe diffusion in a crowded environment in general.

Estrogen receptor-independent catechol estrogen binding activity: protein binding studies in wild-type, Estrogen receptor-alpha KO, and aromatase KO mice tissues.

PubMed

Philips, Brian J; Ansell, Pete J; Newton, Leslie G; Harada, Nobuhiro; Honda, Shin-Ichiro; Ganjam, Venkataseshu K; Rottinghaus, George E; Welshons, Wade V; Lubahn, Dennis B

2004-06-01

Primary evidence for novel estrogen signaling pathways is based upon well-documented estrogenic responses not inhibited by estrogen receptor antagonists. In addition to 17beta-E2, the catechol estrogen 4-hydroxyestradiol (4OHE2) has been shown to elicit biological responses independent of classical estrogen receptors in estrogen receptor-alpha knockout (ERalphaKO) mice. Consequently, our research was designed to biochemically characterize the protein(s) that could be mediating the biological effects of catechol estrogens using enzymatically synthesized, radiolabeled 4-hydroxyestrone (4OHE1) and 4OHE2. Scatchard analyses identified a single class of high-affinity (K(d) approximately 1.6 nM), saturable cytosolic binding sites in several ERalphaKO estrogen-responsive tissues. Specific catechol estrogen binding was competitively inhibited by unlabeled catechol estrogens, but not by 17beta-E2 or the estrogen receptor antagonist ICI 182,780. Tissue distribution studies indicated significant binding differences both within and among various tissues in wild-type, ERalphaKO, and aromatase knockout female mice. Ligand metabolism experiments revealed extensive metabolism of labeled catechol estrogen, suggesting that catechol estrogen metabolites were responsible for the specific binding. Collectively, our data provide compelling evidence for the interaction of catechol estrogen metabolites with a novel binding protein that exhibits high affinity, specificity, and selective tissue distribution. The extensive biochemical characterization of this binding protein indicates that this protein may be a receptor, and thus may mediate ERalpha/beta-independent effects of catechol estrogens and their metabolites.

Characterization of [3H]LS-3-134, a Novel Arylamide Phenylpiperazine D3 Dopamine Receptor Selective Radioligand

PubMed Central

Rangel-Barajas, Claudia; Malik, Maninder; Taylor, Michelle; Neve, Kim A.; Mach, Robert H.; Luedtke, Robert R.

2014-01-01

LS-3-134 is a substituted N-phenylpiperazine derivative that has been reported to exhibit a) high-affinity binding (Ki value 0.2 nM) at human D3 dopamine receptors, b) >100-fold D3 vs. D2 dopamine receptor subtype binding selectivity and c) low-affinity binding (Ki values >5,000 nM) at sigma 1 and sigma 2 receptors. Based upon a forskolin-dependent activation of the adenylyl cyclase inhibition assay, LS-3-134 is a weak partial agonist at both D2 and D3 dopamine receptor subtypes (29% and 35% of full agonist activity, respectively). In this study, [3H]-labeled LS-3-134 was prepared and evaluated to further characterize its use as a D3 dopamine receptor selective radioligand. Kinetic and equilibrium radioligand binding studies were performed. This radioligand rapidly reaches equilibrium (10-15 min at 37°C) and binds with high affinity to both human (Kd = 0.06 ± 0.01 nM) and rat (Kd = 0.2 ± 0.02 nM) D3 receptors expressed in HEK-293 cells. Direct and competitive radioligand binding studies using rat caudate and nucleus accumbens tissue indicate that [3H]LS-3-134 selectively binds a homogeneous population of binding sites with a dopamine D3 receptor pharmacological profile. Based upon these studies we propose that [3H]LS-3-134 represents a novel D3 dopamine receptor selective radioligand that can be used for studying the expression and regulation of the D3 dopamine receptor subtype. PMID:25041389

Analysis and prediction of calcium-binding pockets from apo-protein structures exhibiting calcium-induced localized conformational changes

PubMed Central

Wang, Xue; Zhao, Kun; Kirberger, Michael; Wong, Hing; Chen, Guantao; Yang, Jenny J

2010-01-01

Calcium binding in proteins exhibits a wide range of polygonal geometries that relate directly to an equally diverse set of biological functions. The binding process stabilizes protein structures and typically results in local conformational change and/or global restructuring of the backbone. Previously, we established the MUG program, which utilized multiple geometries in the Ca2+-binding pockets of holoproteins to identify such pockets, ignoring possible Ca2+-induced conformational change. In this article, we first report our progress in the analysis of Ca2+-induced conformational changes followed by improved prediction of Ca2+-binding sites in the large group of Ca2+-binding proteins that exhibit only localized conformational changes. The MUGSR algorithm was devised to incorporate side chain torsional rotation as a predictor. The output from MUGSR presents groups of residues where each group, typically containing two to five residues, is a potential binding pocket. MUGSR was applied to both X-ray apo structures and NMR holo structures, which did not use calcium distance constraints in structure calculations. Predicted pockets were validated by comparison with homologous holo structures. Defining a “correct hit” as a group of residues containing at least two true ligand residues, the sensitivity was at least 90%; whereas for a “correct hit” defined as a group of residues containing at least three true ligand residues, the sensitivity was at least 78%. These data suggest that Ca2+-binding pockets are at least partially prepositioned to chelate the ion in the apo form of the protein. PMID:20512971

Physicochemical characteristics of structurally determined metabolite-protein and drug-protein binding events with respect to binding specificity.

PubMed

Korkuć, Paula; Walther, Dirk

2015-01-01

To better understand and ultimately predict both the metabolic activities as well as the signaling functions of metabolites, a detailed understanding of the physical interactions of metabolites with proteins is highly desirable. Focusing in particular on protein binding specificity vs. promiscuity, we performed a comprehensive analysis of the physicochemical properties of compound-protein binding events as reported in the Protein Data Bank (PDB). We compared the molecular and structural characteristics obtained for metabolites to those of the well-studied interactions of drug compounds with proteins. Promiscuously binding metabolites and drugs are characterized by low molecular weight and high structural flexibility. Unlike reported for drug compounds, low rather than high hydrophobicity appears associated, albeit weakly, with promiscuous binding for the metabolite set investigated in this study. Across several physicochemical properties, drug compounds exhibit characteristic binding propensities that are distinguishable from those associated with metabolites. Prediction of target diversity and compound promiscuity using physicochemical properties was possible at modest accuracy levels only, but was consistently better for drugs than for metabolites. Compound properties capturing structural flexibility and hydrogen-bond formation descriptors proved most informative in PLS-based prediction models. With regard to diversity of enzymatic activities of the respective metabolite target enzymes, the metabolites benzylsuccinate, hypoxanthine, trimethylamine N-oxide, oleoylglycerol, and resorcinol showed very narrow process involvement, while glycine, imidazole, tryptophan, succinate, and glutathione were identified to possess broad enzymatic reaction scopes. Promiscuous metabolites were found to mainly serve as general energy currency compounds, but were identified to also be involved in signaling processes and to appear in diverse organismal systems (digestive and nervous system) suggesting specific molecular and physiological roles of promiscuous metabolites.

Physicochemical characteristics of structurally determined metabolite-protein and drug-protein binding events with respect to binding specificity

PubMed Central

Korkuć, Paula; Walther, Dirk

2015-01-01

To better understand and ultimately predict both the metabolic activities as well as the signaling functions of metabolites, a detailed understanding of the physical interactions of metabolites with proteins is highly desirable. Focusing in particular on protein binding specificity vs. promiscuity, we performed a comprehensive analysis of the physicochemical properties of compound-protein binding events as reported in the Protein Data Bank (PDB). We compared the molecular and structural characteristics obtained for metabolites to those of the well-studied interactions of drug compounds with proteins. Promiscuously binding metabolites and drugs are characterized by low molecular weight and high structural flexibility. Unlike reported for drug compounds, low rather than high hydrophobicity appears associated, albeit weakly, with promiscuous binding for the metabolite set investigated in this study. Across several physicochemical properties, drug compounds exhibit characteristic binding propensities that are distinguishable from those associated with metabolites. Prediction of target diversity and compound promiscuity using physicochemical properties was possible at modest accuracy levels only, but was consistently better for drugs than for metabolites. Compound properties capturing structural flexibility and hydrogen-bond formation descriptors proved most informative in PLS-based prediction models. With regard to diversity of enzymatic activities of the respective metabolite target enzymes, the metabolites benzylsuccinate, hypoxanthine, trimethylamine N-oxide, oleoylglycerol, and resorcinol showed very narrow process involvement, while glycine, imidazole, tryptophan, succinate, and glutathione were identified to possess broad enzymatic reaction scopes. Promiscuous metabolites were found to mainly serve as general energy currency compounds, but were identified to also be involved in signaling processes and to appear in diverse organismal systems (digestive and nervous system) suggesting specific molecular and physiological roles of promiscuous metabolites. PMID:26442281

Robust, sensitive and facile method for detection of F-, CN- and Ac- anions

NASA Astrophysics Data System (ADS)

Madhusudhana Reddy, P.; Hsieh, Shih-Rong; Chen, Jem-Kun; Chang, Chi-Jung; Kang, Jing-Yuan; Chen, Chih-Hsien

2017-11-01

Sensing of F-, CN- and Ac- is important from the viewpoint of both medically and environmentally. Particularly, sensing of the anions in 100% water by a colorimetric chemical sensor is a highly difficult task as water molecules interfere the sensing mechanism. In this regard, sensor R1, having azo and nitrophenyl groups as signaling units and thiourea as a binding site was prepared. This sensor exclusively detected CN- ion over other testing anions in 30% aq. DMSO solution by exhibiting distinct spectral and visual color changes. However, in 15% aq. DMSO solution, R1 exhibited obvious spectral and color changes in response to F-, CN- and Ac-. On the other hand, we have also designed sensor, R2, having same signaling units of R1, but a different binding site of urea group. Surprisingly, in contrast to R1, R2 exhibited obvious spectral and color changes in 5% aq. DMSO solution only. Further, economically viable ;test stripes; were prepared in a facile mode to detect the CN- in 100% aqueous solution. Such stripes can serve as a practical colorimetric probe for ;in the field; detection of the ions and thus avoid additional expensive equipment.

Characterization of the DNA binding properties of polyomavirus capsid protein

NASA Technical Reports Server (NTRS)

Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

1993-01-01

The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.

Synergy of two low-affinity NLSs determines the high avidity of influenza A virus nucleoprotein NP for human importin α isoforms.

PubMed

Wu, Wei; Sankhala, Rajeshwer S; Florio, Tyler J; Zhou, Lixin; Nguyen, Nhan L T; Lokareddy, Ravi K; Cingolani, Gino; Panté, Nelly

2017-09-12

The influenza A virus nucleoprotein (NP) is an essential multifunctional protein that encapsidates the viral genome and functions as an adapter between the virus and the host cell machinery. NPs from all strains of influenza A viruses contain two nuclear localization signals (NLSs): a well-studied monopartite NLS1 and a less-characterized NLS2, thought to be bipartite. Through site-directed mutagenesis and functional analysis, we found that NLS2 is also monopartite and is indispensable for viral infection. Atomic structures of importin α bound to two variants of NLS2 revealed NLS2 primarily binds the major-NLS binding site of importin α, unlike NLS1 that associates with the minor NLS-pocket. Though peptides corresponding to NLS1 and NLS2 bind weakly to importin α, the two NLSs synergize in the context of the full length NP to confer high avidity for importin α7, explaining why the virus efficiently replicates in the respiratory tract that exhibits high levels of this isoform. This study, the first to functionally characterize NLS2, demonstrates NLS2 plays an important and unexpected role in influenza A virus infection. We propose NLS1 and NLS2 form a bipartite NLS in trans, which ensures high avidity for importin α7 while preventing non-specific binding to viral RNA.

Different roles suggested by sex-biased expression and pheromone binding affinity among three pheromone binding proteins in the pink rice borer, Sesamia inferens (Walker) (Lepidoptera: Noctuidae).

PubMed

Jin, Jun-Yan; Li, Zhao-Qun; Zhang, Ya-Nan; Liu, Nai-Yong; Dong, Shuang-Lin

2014-07-01

Pheromone binding proteins (PBPs) are thought to bind and transport hydrophobic sex pheromone molecules across the aqueous sensillar lymph to specific pheromone receptors on the dendritic membrane of olfactory neurons. A maximum of 3 PBP genes have been consistently identified in noctuid species, and each of them shares high identity with its counterparts in other species within the family. The functionality differences of the 3 proteins are poorly understood. In the present study, 3 PBP cDNAs (SinfPBP1, 2, 3) were identified from the pink rice borer, Sesamia inferens, for the first time. The quantitative real-time PCR indicated that the 3 PBPs displayed similar temporal but very different sex related expression profiles. Expression of SinfPBP1 and SinfPBP2 were highly and moderately male biased, respectively, while SinfPBP3 was slightly female biased, as SinfPBPs were expressed at very different levels (PBP1>PBP2≫PBP3) in male antennae, but at similar levels in female antennae. Furthermore, the 3 SinfPBPs displayed different ligand binding profiles in fluorescence competitive binding assays. SinfPBP1 exhibited high and similar binding affinities to all 3 sex pheromone components (Ki=0.72-1.60 μM), while SinfPBP2 showed selective binding to the alcohol and aldehyde components (Ki=0.78-1.71 μM), and SinfPBP3 showed no obvious binding to the 3 sex pheromone components. The results suggest that SinfPBP1 plays a major role in the reception of female sex pheromones in S. inferens, while SinfPBP3 plays a least role (if any) and SinfPBP2 functions as a recognizer of alcohol and aldehyde components. Copyright © 2014 Elsevier Ltd. All rights reserved.

A single-ligand ultra-microporous MOF for precombustion CO2 capture and hydrogen purification

PubMed Central

Nandi, Shyamapada; De Luna, Phil; Daff, Thomas D.; Rother, Jens; Liu, Ming; Buchanan, William; Hawari, Ayman I.; Woo, Tom K.; Vaidhyanathan, Ramanathan

2015-01-01

Metal organic frameworks (MOFs) built from a single small ligand typically have high stability, are rigid, and have syntheses that are often simple and easily scalable. However, they are normally ultra-microporous and do not have large surface areas amenable to gas separation applications. We report an ultra-microporous (3.5 and 4.8 Å pores) Ni-(4-pyridylcarboxylate)2 with a cubic framework that exhibits exceptionally high CO2/H2 selectivities (285 for 20:80 and 230 for 40:60 mixtures at 10 bar, 40°C) and working capacities (3.95 mmol/g), making it suitable for hydrogen purification under typical precombustion CO2 capture conditions (1- to 10-bar pressure swing). It exhibits facile CO2 adsorption-desorption cycling and has CO2 self-diffusivities of ~3 × 10−9 m2/s, which is two orders higher than that of zeolite 13X and comparable to other top-performing MOFs for this application. Simulations reveal a high density of binding sites that allow for favorable CO2-CO2 interactions and large cooperative binding energies. Ultra-micropores generated by a small ligand ensures hydrolytic, hydrostatic stabilities, shelf life, and stability toward humid gas streams. PMID:26824055

Multichannel imaging to quantify four classes of pharmacokinetic distribution in tumors.

PubMed

Bhatnagar, Sumit; Deschenes, Emily; Liao, Jianshan; Cilliers, Cornelius; Thurber, Greg M

2014-10-01

Low and heterogeneous delivery of drugs and imaging agents to tumors results in decreased efficacy and poor imaging results. Systemic delivery involves a complex interplay of drug properties and physiological factors, and heterogeneity in the tumor microenvironment makes predicting and overcoming these limitations exceptionally difficult. Theoretical models have indicated that there are four different classes of pharmacokinetic behavior in tissue, depending on the fundamental steps in distribution. In order to study these limiting behaviors, we used multichannel fluorescence microscopy and stitching of high-resolution images to examine the distribution of four agents in the same tumor microenvironment. A validated generic partial differential equation model with a graphical user interface was used to select fluorescent agents exhibiting these four classes of behavior, and the imaging results agreed with predictions. BODIPY-FL exhibited higher concentrations in tissue with high blood flow, cetuximab gave perivascular distribution limited by permeability, high plasma protein and target binding resulted in diffusion-limited distribution for Hoechst 33342, and Integrisense 680 was limited by the number of binding sites in the tissue. Together, the probes and simulations can be used to investigate distribution in other tumor models, predict tumor drug distribution profiles, and design and interpret in vivo experiments. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Plasma proteins of rainbow trout (Oncorhynchus mykiss) isolated by binding to lipopolysaccharide from Aeromonas salmonicida.

PubMed

Hoover, G J; el-Mowafi, A; Simko, E; Kocal, T E; Ferguson, H W; Hayes, M A

1998-07-01

In an attempt to find plasma proteins that might be involved in the constitutive resistance of rainbow trout to furunculosis, a disease caused by Aeromonas salmonicida (AS), we purified serum and plasma proteins based on their calcium- and carbohydrate-dependent affinity for A. salmonicida lipopolysaccharide (LPS) coupled to an epoxy-activated synthetic matrix (Toyopearl AF Epoxy 650M). A multimeric family of high molecular weight (96 to 200-kDa) LPS-binding proteins exhibiting both calcium and mannose dependent binding was isolated. Upon reduction the multimers collapsed to subunits of approximately 16-kDa as estimated by 1D-PAGE and exhibited pI values of 5.30 and 5.75 as estimated from 2D-PAGE. Their N-terminal sequences were related to rainbow trout ladderlectin (RT-LL), a Sepharose-binding protein. Polyclonal antibodies to the LPS-purified 16-kDa subunits recognized both the reduced 16-kDa subunits and the non-reduced multimeric forms. A calcium- and N-acetylglucosamine (GlcNAc)-dependent LPS-binding multimeric protein (approximately 207-kDa) composed of 34.5-kDa subunits was purified and found to be identical to trout serum amyloid P (SAP) by N-terminal sequence (DLQDLSGKVFV). A protein of 24-kDa, in reduced and non-reduced conditions, was isolated and had N-terminal sequence identity with a known C-reactive protein (CRP) homologue, C-polysaccharide-binding protein 2 (TCBP2) of rainbow trout. A novel calcium-dependent LPS-binding protein was purified and termed rainbow trout lectin 37 (RT-L37). This protein, composed of dimers, tetramers and pentamers of 37 kDa subunits (pI 5.50-6.10) with N-terminal sequence (IQE(D/N)GHAEAPGATTVLNEILR) showed no close homology to proteins known or predicted from cDNA sequences. These findings demonstrate that rainbow trout have several blood proteins with lectin properties for the LPS of A. salmonicida; the biological functions of these proteins in resistance to furunculosis are still unknown.

Hydroxylated polybrominated diphenyl ethers exhibit different activities on thyroid hormone receptors depending on their degree of bromination.

PubMed

Ren, Xiao-Min; Guo, Liang-Hong; Gao, Yu; Zhang, Bin-Tian; Wan, Bin

2013-05-01

Polybrominated diphenyl ethers (PBDEs) have been shown to disrupt thyroid hormone (TH) functions in experimental animals, and one of the proposed disruption mechanisms is direct binding of hydroxylated PBDE (OH-PBDE) to TH receptors (TRs). However, previous data on TH receptor binding and TH activity of OH-PBDEs were very limited and sometimes inconsistent. In the present paper, we examined the binding potency of ten OH-PBDEs with different degrees of bromination to TR using a fluorescence competitive binding assay. The results showed that the ten OH-PBDEs bound to TR with potency that correlated to their bromination level. We further examined their effect on TR using a coactivator binding assay and GH3 cell proliferation assay. Different TR activities of OH-PBDEs were observed depending on their degree of bromination. Four low-brominated OH-PBDEs (2'-OH-BDE-28, 3'-OH-BDE-28, 5-OH-BDE-47, 6-OH-BDE-47) were found to be TR agonists, which recruited the coactivator peptide and enhanced GH3 cell proliferation. However, three high-brominated OH-PBDEs (3-OH-BDE-100, 3'-OH-BDE-154, 4-OH-BDE-188) were tested to be antagonists. Molecular docking was employed to simulate the interactions of OH-PBDEs with TR and identify the structural determinants for TR binding and activity. According to the docking results, low-brominated OH-PBDEs, which are weak binders but TR agonists, bind with TR at the inner side of its binding pocket, whereas high-brominated compounds, which are potent binders but TR antagonists, reside at the outer region. These results indicate that OH-PBDEs have different activities on TR (agonistic or antagonistic), possibly due to their different binding geometries with the receptor. Copyright © 2013 Elsevier Inc. All rights reserved.

Design of a Highly Specific And Noninvasive Biosensor Suitable for Real-Time in Vivo Imaging of Mercury (II) Uptake

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

Chapleau, R.R.; Blomberg, R.; Ford, P.C.

2009-05-12

Mercury is a ubiquitous pollutant that when absorbed is extremely toxic to a wide variety of biochemical processes. Mercury (II) is a strong, invisible poison that is rapidly absorbed by tissues of the intestinal tract, kidneys, and liver upon ingestion. In this study, a novel fluorescence-based biosensor is presented that allows for the direct monitoring of the uptake and distribution of the metal under noninvasive in vivo conditions. With the introduction of a cysteine residue at position 205, located in close proximity to the chromophore, the green fluorescent protein (GFP) from Aequorea victoria was converted into a highly specific biosensormore » for this metal ion. The mutant protein exhibits a dramatic absorbance and fluorescence change upon mercuration at neutral pH. Absorbance and fluorescence properties with respect to the metal concentration exhibit sigmoidal binding behavior with a detection limit in the low nanomolar range. Time-resolved binding studies indicate rapid subsecond binding of the metal to the protein. The crystal structures obtained of mutant eGFP205C indicate a possible access route of the metal into the core of the protein. To our knowledge, this engineered protein is a first example of a biosensor that allows for noninvasive and real-time imaging of mercury uptake in a living cell. A major advantage is that its expression can be genetically controlled in many organisms to enable unprecedented studies of tissue specific mercury uptake.« less

Amyloid Plaques in PSAPP Mice Bind Less Metal than Plaques in Human Alzheimer's Disease

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

Leskovjan, A.; Lanzirotti, A; Miller, L

2009-01-01

Amyloid beta (A{Beta}) is the primary component of Alzheimer's disease (AD) plaques, a key pathological feature of the disease. Metal ions of zinc (Zn), copper (Cu), iron (Fe), and calcium (Ca) are elevated in human amyloid plaques and are thought to be involved in neurodegeneration. Transgenic mouse models of AD also exhibit amyloid plaques, but fail to exhibit the high degree of neurodegeneration observed in humans. In this study, we imaged the Zn, Cu, Fe, and Ca ion distribution in the PSAPP transgenic mouse model representing end-stage AD (N = 6) using synchrotron X-ray fluorescence (XRF) microprobe. In order tomore » account for differences in density in the plaques, the relative protein content was imaged with synchrotron Fourier transform infrared microspectroscopy (FTIRM) on the same samples. FTIRM results revealed a 61% increase in protein content in the plaques compared to the surrounding tissue. After normalizing to protein density, we found that the PSAPP plaques contained only a 29% increase in Zn and there was actually less Cu, Fe, and Ca in the plaque compared to the surrounding tissue. Since metal binding to A{beta} is thought to induce redox chemistry that is toxic to neurons, the reduced metal binding in PSAPP mice is consistent with the lack of neurodegeneration in these animals. These findings were in stark contrast to the high metal ion content observed in human AD plaques, further implicating the role of metal ions in human AD pathology.« less

Targeted Type 1 phototherapeutic agents using azido-peptide bioconjugates

NASA Astrophysics Data System (ADS)

Rajagopalan, Raghavan; Achilefu, Samuel I.; Jimenez, Hermo N.; Webb, Elizabeth G.; Schmidt, Michelle A.; Bugaj, Joseph E.; Dorshow, Richard B.

2001-07-01

Five peptides binding to somatostatin and bombesin receptors were conjugated to 4-azido-2,3,4,6-tetrafluorophenylbenzoic acid, a Type 1 photosensitizer, at the N-terminal position. The receptor affinities were determined by competition binding assay using two different pancreatic tumor cell lines, CA20948 and AR42-J, that expresses somatostatin-2 (SST-2) and bombesin receptors receptively. All compounds exhibited high receptor specificity, i.e., the IC50 values ranged between 1.0 to 64.0 nM. These conjugates may be useful for targeted Type 1 phototherapy via the generation of nitrenes at the cell surfaces expressing these receptors.

Biochemical characterization of a thermophilic β-mannanase from Talaromyces leycettanus JCM12802 with high specific activity.

PubMed

Wang, Caihong; Luo, Huiying; Niu, Canfang; Shi, Pengjun; Huang, Huoqing; Meng, Kun; Bai, Yingguo; Wang, Kun; Hua, Huifang; Yao, Bin

2015-02-01

Thermophilic β-mannanases are of increasing importance for wide industrial applications. In the current study, gene cloning, functional expression in Pichia pastoris, and characterization of a thermophilic β-mannanase (Man5A) from thermophilic Talaromyces leycettanus JCM12802 are reported. Deduced Man5A exhibits the highest identity with a putative β-mannanase from Talaromyces stipitatus ATCC10500 (70.3 %) and is composed of an N-terminal signal peptide, a fungal-type carbohydrate-binding module (CBM) of family 1, and a catalytic domain of glycosyl hydrolase (GH) family 5 at the C-terminus. Two recombinant proteins with different glycosylation levels, termed Man5A1 (72 kDa) and Man5A2 (60 kDa), were identified after purification. Both enzymes were thermophilic, exhibiting optimal activity at 85-90 °C, and were highly stable at 70 °C. Man5A1 and Man5A2 had a pH optimum of 4.5 and 4.0, respectively, and were highly stable over the broad pH range of 3.0-10.0. Most metal ions and sodium dodecyl sulfate (SDS) had no effect on the enzymatic activities. Man5A1 and Man5A2 exhibited high specific activity (2,160 and 1,800 U/mg, respectively) when using locust bean gum as the substrate. The CBM1 and two key residues D191 and R286 were found to affect Man5A thermostability. Man5A displays a classical four-site-binding mode, hydrolyzing mannooligosaccharides into smaller units, galactomannan into mannose and mannobiose, and glucomanman into mannose, mannobiose, and mannopentaose, respectively. All these properties make Man5A a good candidate for extensive applications in the bioconversion, pulp bleaching, textile, food, and feed industries.

Crystal structures of spleen tyrosine kinase in complex with novel inhibitors: structural insights for design of anticancer drugs.

PubMed

Lee, Sang Jae; Choi, Jang-Sik; Han, Byeong-Gu; Kim, Hyoun Sook; Song, Ho-Juhn; Lee, Jaekyoo; Nam, Seungyoon; Goh, Sung-Ho; Kim, Jung-Ho; Koh, Jong Sung; Lee, Byung Il

2016-10-01

Spleen tyrosine kinase (SYK) is a cytosolic nonreceptor protein tyrosine kinase that mediates key signal transduction pathways following the activation of immune cell receptors. SYK regulates cellular events induced by the B-cell receptor and Fc receptors with high intrinsic activity. Furthermore, SYK has been regarded as an attractive target for the treatment of autoimmune diseases and cancers. Here, we report the crystal structures of SYK in complex with seven newly developed inhibitors (G206, G207, O178, O194, O259, O272, and O282) to provide structural insights into which substituents of the inhibitors and binding regions of SYK are essential for lead compound optimization. Our kinase inhibitors exhibited high inhibitory activities against SYK, with half-maximal inhibitory concentrations (IC 50 ) of approximately 0.7-33 nm, but they showed dissimilar inhibitory activities against KDR, RET, JAK2, JAK3, and FLT3. Among the seven SYK inhibitors, O272 and O282 exhibited highly specific inhibitions against SYK, whereas O194 exhibited strong inhibition of both SYK and FLT3. Three inhibitors (G206, G207, and O178) more efficiently inhibited FLT3 while still substantially inhibiting SYK activity. The binding mode analysis suggested that a highly selective SYK inhibitor can be developed by optimizing the functional groups that facilitate direct interactions with Asn499. The atomic coordinates and structure factors for human SYK are in the Protein Data Bank under accession codes 4XG2 (inhibitor-free form), 4XG3 (G206), 4XG4 (G207), 5GHV (O178), 4XG6 (O194), 4XG7 (O259), 4XG8 (O272), and 4XG9 (O282). © 2016 Federation of European Biochemical Societies.

Critical evaluation of methods to incorporate entropy loss upon binding in high-throughput docking.

PubMed

Salaniwal, Sumeet; Manas, Eric S; Alvarez, Juan C; Unwalla, Rayomand J

2007-02-01

Proper accounting of the positional/orientational/conformational entropy loss associated with protein-ligand binding is important to obtain reliable predictions of binding affinity. Herein, we critically examine two simplified statistical mechanics-based approaches, namely a constant penalty per rotor method, and a more rigorous method, referred to here as the partition function-based scoring (PFS) method, to account for such entropy losses in high-throughput docking calculations. Our results on the estrogen receptor beta and dihydrofolate reductase proteins demonstrate that, while the constant penalty method over-penalizes molecules for their conformational flexibility, the PFS method behaves in a more "DeltaG-like" manner by penalizing different rotors differently depending on their residual entropy in the bound state. Furthermore, in contrast to no entropic penalty or the constant penalty approximation, the PFS method does not exhibit any bias towards either rigid or flexible molecules in the hit list. Preliminary enrichment studies using a lead-like random molecular database suggest that an accurate representation of the "true" energy landscape of the protein-ligand complex is critical for reliable predictions of relative binding affinities by the PFS method. Copyright 2006 Wiley-Liss, Inc.

Structural Determination of the Broadly Reactive Anti-IGHV1-69 Anti-idiotypic Antibody G6 and Its Idiotope.

PubMed

Avnir, Yuval; Prachanronarong, Kristina L; Zhang, Zhen; Hou, Shurong; Peterson, Eric C; Sui, Jianhua; Zayed, Hatem; Kurella, Vinodh B; McGuire, Andrew T; Stamatatos, Leonidas; Hilbert, Brendan J; Bohn, Markus-Frederik; Kowalik, Timothy F; Jensen, Jeffrey D; Finberg, Robert W; Wang, Jennifer P; Goodall, Margaret; Jefferis, Roy; Zhu, Quan; Kurt Yilmaz, Nese; Schiffer, Celia A; Marasco, Wayne A

2017-12-12

The heavy chain IGHV1-69 germline gene exhibits a high level of polymorphism and shows biased use in protective antibody (Ab) responses to infections and vaccines. It is also highly expressed in several B cell malignancies and autoimmune diseases. G6 is an anti-idiotypic monoclonal Ab that selectively binds to IGHV1-69 heavy chain germline gene 51p1 alleles that have been implicated in these Ab responses and disease processes. Here, we determine the co-crystal structure of humanized G6 (hG6.3) in complex with anti-influenza hemagglutinin stem-directed broadly neutralizing Ab D80. The core of the hG6.3 idiotope is a continuous string of CDR-H2 residues starting with M53 and ending with N58. G6 binding studies demonstrate the remarkable breadth of binding to 51p1 IGHV1-69 Abs with diverse CDR-H3, light chain, and antigen binding specificities. These studies detail the broad expression of the G6 cross-reactive idiotype (CRI) that further define its potential role in precision medicine. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Enhanced Human-Type Receptor Binding by Ferret-Transmissible H5N1 with a K193T Mutation.

PubMed

Peng, Wenjie; Bouwman, Kim M; McBride, Ryan; Grant, Oliver C; Woods, Robert J; Verheije, Monique H; Paulson, James C; de Vries, Robert P

2018-05-15

All human influenza pandemics have originated from avian influenza viruses. Although multiple changes are needed for an avian virus to be able to transmit between humans, binding to human-type receptors is essential. Several research groups have reported mutations in H5N1 viruses that exhibit specificity for human-type receptors and promote respiratory droplet transmission between ferrets. Upon detailed analysis, we have found that these mutants exhibit significant differences in fine receptor specificity compared to human H1N1 and H3N2 and retain avian-type receptor binding. We have recently shown that human influenza viruses preferentially bind to α2-6-sialylated branched N-linked glycans, where the sialic acids on each branch can bind to receptor sites on two protomers of the same hemagglutinin (HA) trimer. In this binding mode, the glycan projects over the 190 helix at the top of the receptor-binding pocket, which in H5N1 would create a stearic clash with lysine at position 193. Thus, we hypothesized that a K193T mutation would improve binding to branched N-linked receptors. Indeed, the addition of the K193T mutation to the H5 HA of a respiratory-droplet-transmissible virus dramatically improves both binding to human trachea epithelial cells and specificity for extended α2-6-sialylated N-linked glycans recognized by human influenza viruses. IMPORTANCE Infections by avian H5N1 viruses are associated with a high mortality rate in several species, including humans. Fortunately, H5N1 viruses do not transmit between humans because they do not bind to human-type receptors. In 2012, three seminal papers have shown how these viruses can be engineered to transmit between ferrets, the human model for influenza virus infection. Receptor binding, among others, was changed, and the viruses now bind to human-type receptors. Receptor specificity was still markedly different compared to that of human influenza viruses. Here we report an additional mutation in ferret-transmissible H5N1 that increases human-type receptor binding. K193T seems to be a common receptor specificity determinant, as it increases human-type receptor binding in multiple subtypes. The K193T mutation can now be used as a marker during surveillance of emerging viruses to assess potential pandemic risk. Copyright © 2018 American Society for Microbiology.

Cows with follicular fluid androgen excess exhibit anovulation and have altered circulating sex hormone binding globulin, gonadotropin secretion and plasma and follicular fluid composition

USDA-ARS?s Scientific Manuscript database

Our laboratory identified a group of cows with excess intrafollicular concentrations of androstenedione (A4; >30 fold), reduced calving rates, and theca gene expression profiles similar to women with Polycystic Ovary Syndrome (PCOS). Based on these previous studies, we hypothesized that High A4 cows...

Unfolding mechanism of thrombin-binding aptamer revealed by molecular dynamics simulation and Markov State Model

NASA Astrophysics Data System (ADS)

Zeng, Xiaojun; Zhang, Liyun; Xiao, Xiuchan; Jiang, Yuanyuan; Guo, Yanzhi; Yu, Xinyan; Pu, Xuemei; Li, Menglong

2016-04-01

Thrombin-binding aptamer (TBA) with the sequence 5‧GGTTGGTGTGGTTGG3‧ could fold into G-quadruplex, which correlates with functionally important genomic regionsis. However, unfolding mechanism involved in the structural stability of G-quadruplex has not been satisfactorily elucidated on experiments so far. Herein, we studied the unfolding pathway of TBA by a combination of molecular dynamics simulation (MD) and Markov State Model (MSM). Our results revealed that the unfolding of TBA is not a simple two-state process but proceeds along multiple pathways with multistate intermediates. One high flux confirms some observations from NMR experiment. Another high flux exhibits a different and simpler unfolding pathway with less intermediates. Two important intermediate states were identified. One is similar to the G-triplex reported in the folding of G-quadruplex, but lack of H-bonding between guanines in the upper plane. More importantly, another intermediate state acting as a connector to link the folding region and the unfolding one, was the first time identified, which exhibits higher population and stability than the G-triplex-like intermediate. These results will provide valuable information for extending our understanding the folding landscape of G-quadruplex formation.

Monoclonal Antibodies Against Fusicoccin with Binding Characteristics Similar to the Putative Fusicoccin Receptor of Higher Plants 1

PubMed Central

Feyerabend, Martin; Weiler, Elmar W.

1987-01-01

Monoclonal antibodies were raised against fusicoccin. The toxin, linked to bovine serum albumin through its t-pentenyl moiety, served as immunogen. Hybridomas secreting anti-fusicoccin antibodies were screened by radioimmunoassay employing a novel radioactive derivative, [3H]-nor-fusicoccin-alcohol of high specific activity (1.5 × 1014Bq/mole). The two monoclonal antibodies reported here are of high apparent affinity for fusicoccin (0.71 × 10−9 molar and 1.85 × 10−9 molar). This is comparable to the apparent affinity of rabbit antiserum raised against the same type of conjugate (9.3 × 10−9 molar). A method for the single step purification of the monoclonal antibodies from ascites fluid is reported. A solid-phase immunoassay, using alkaline phosphatase as enzyme, exhibits a measuring range from 0.1 to 1.5 picomoles (about 70 picograms to 1 nanogram) of fusicoccin. The displacement of [3H]-nor-fusicoccin-alcohol from the antibodies by compounds structurally related to fusicoccin exhibits similar selectivity as a microsomal binding assay with the same tracer as radiolabeled probe. Images Fig. 2 PMID:16665786

Discovery and evaluation of triple inhibitors of VEGFR-2, TIE-2 and EphB4 as anti-angiogenic and anti-cancer agents

PubMed Central

Zhang, Lin; Shan, Yuanyuan; Ji, Xingyue; Zhu, Mengyuan; Li, Chuansheng; Sun, Ying; Si, Ru; Pan, Xiaoyan; Wang, Jinfeng; Ma, Weina; Dai, Bingling; Wang, Binghe; Zhang, Jie

2017-01-01

Receptor tyrosine kinases (RTKs), especially VEGFR-2, TIE-2, and EphB4, play a crucial role in both angiogenesis and tumorigenesis. Moreover, complexity and heterogeneity of angiogenesis make it difficult to treat such pathological traits with single-target agents. Herein, we developed two classes of multi-target RTK inhibitors (RTKIs) based on the highly conserved ATP-binding pocket of VEGFR-2/TIE-2/EphB4, using previously reported BPS-7 as a lead compound. These multi-target RTKIs exhibited considerable potential as novel anti-angiogenic and anticancer agents. Among them, QDAU5 displayed the most promising potency and selectivity. It significantly suppressed viability of EA.hy926 and proliferation of several cancer cells. Further investigations indicated that QDAU5 showed high affinity to VEGFR-2 and reduced the phosphorylation of VEGFR-2. We identified QDAU5 as a potent multiple RTKs inhibitor exhibiting prominent anti-angiogenic and anticancer potency both in vitro and in vivo. Moreover, quinazolin-4(3H)-one has been identified as an excellent hinge binding moiety for multi-target inhibitors of angiogenic VEGFR-2, Tie-2, and EphB4. PMID:29285210

Direct binding to antigen-coated beads refines the specificity and cross-reactivity of four monoclonal antibodies that recognize polymorphic epitopes of HLA class I molecules.

PubMed

Hilton, H G; Parham, P

2013-04-01

Monoclonal antibodies with specificity for human leukocyte antigen (HLA) class I determinants of HLA were originally characterized using serological assays in which the targets were cells expressing three to six HLA class I variants. Because of this complexity, the specificities of the antibodies were defined indirectly by correlation. Here we use a direct binding assay, in which the targets are synthetic beads coated with 1 of 111 HLA class I variants, representing the full range of HLA-A, -B and -C variation. We studied one monoclonal antibody with monomorphic specificity (W6/32) and four with polymorphic specificity (MA2.1, PA2.1, BB7.2 and BB7.1) and compared the results with those obtained previously. W6/32 reacted with all HLA class I variants. MA2.1 not only exhibits high specificity for HLA-A*02, -B*57 and -B*58, but also exhibited cross-reactivity with HLA-A*11 and -B*15:16. At low concentration (1 µg/ml), PA2.1 and BB7.2 were both specific for HLA-A*02 and -A*69, and at high concentration (50 µg/ml) exhibited significant cross-reactions with HLA-A*68, -A*23 and -A*24. BB7.1 exhibits specificity for HLA-B*07 and -B*42, as previously described, but reacts equally well with HLA-B*81, a rare allotype defined some 16 years after the description of BB7.1. The results obtained with cell-based and bead-based assays are consistent and, in combination with amino acid sequence comparison, increase understanding of the polymorphic epitopes recognized by the MA2.1, PA2.1, BB7.2 and BB7.1 antibodies. Comparison of two overlapping but distinctive bead sets from two sources gave similar results, but the overall levels of binding were significantly different. Several weaker reactions were observed with only one of the bead sets. © 2013 John Wiley & Sons A/S.

Synthesis of isatin thiosemicarbazones derivatives: in vitro anti-cancer, DNA binding and cleavage activities.

PubMed

Ali, Amna Qasem; Teoh, Siang Guan; Salhin, Abdussalam; Eltayeb, Naser Eltaher; Khadeer Ahamed, Mohamed B; Abdul Majid, A M S

2014-05-05

New derivatives of thiosemicarbazone Schiff base with isatin moiety were synthesized L1-L6. The structures of these compounds were characterized based on the spectroscopic techniques. Compound L6 was further characterized by XRD single crystal. The interaction of these compounds with calf thymus (CT-DNA) exhibited high intrinsic binding constant (k(b)=5.03-33.00×10(5) M(-1)) for L1-L3 and L5 and (6.14-9.47×10(4) M(-1)) for L4 and L6 which reflect intercalative activity of these compounds toward CT-DNA. This result was also confirmed by the viscosity data. The electrophoresis studies reveal the higher cleavage activity of L1-L3 than L4-L6. The in vitro anti-proliferative activity of these compounds against human colon cancer cell line (HCT 116) revealed that the synthesized compounds (L3, L6 and L2) exhibited good anticancer potency. Copyright © 2014 Elsevier B.V. All rights reserved.

Molecular and functional characterization of single-box high-mobility group B (HMGB) chromosomal protein from Aedes aegypti.

PubMed

de Abreu da Silva, Isabel Caetano; Vicentino, Amanda Roberta Revoredo; Dos Santos, Renata Coutinho; da Fonseca, Rodrigo Nunes; de Mendonça Amarante, Anderson; Carneiro, Vitor Coutinho; de Amorim Pinto, Marcia; Aguilera, Estefania Anahi; Mohana-Borges, Ronaldo; Bisch, Paulo Mascarello; da Silva-Neto, Mario Alberto Cardoso; Fantappié, Marcelo Rosado

2018-05-30

High-mobility group B (HMGB) proteins have highly conserved, unique DNA-binding domains, HMG boxes, that can bind non-B-type DNA structures, such as bent, kinked and unwound structures, with high affinity. HMGB proteins also promote DNA bending, looping and unwinding. In this study, we determined the role of the Aedes aegypti single HMG-box domain protein AaHMGB; characterized its structure, spatiotemporal expression levels, subcellular localization, and nucleic acid binding activities; and compared these properties with those of its double-HMG-box counterpart protein, AaHMGB1. Via qRT-PCR, we showed that AaHMGB is expressed at much higher levels than AaHMGB1 throughout mosquito development. In situ hybridization results suggested a role for AaHMGB and AaHMGB1 during embryogenesis. Immunolocalization in the midgut revealed that AaHMGB is exclusively nuclear. Circular dichroism and fluorescence spectroscopy analyses showed that AaHMGB exhibits common features of α-helical structures and is more stably folded than AaHMGB1, likely due to the presence of one or two HMG boxes. Using several DNA substrates or single-stranded RNAs as probes, we observed significant differences between AaHMGB and AaHMGB1 in terms of their binding patterns, activity and/or specificity. Importantly, we showed that the phosphorylation of AaHMGB plays a critical role in its DNA-binding activity. Our study provides additional insight into the roles of single- versus double-HMG-box-containing proteins in nucleic acid interactions for better understanding of mosquito development, physiology and homeostasis. Copyright © 2017. Published by Elsevier B.V.

In vitro hypoglycemic and cholesterol lowering effects of dietary fiber prepared from cocoa (Theobroma cacao L.) shells.

PubMed

Nsor-Atindana, John; Zhong, Fang; Mothibe, Kebitsamang Joseph

2012-10-01

Three dietary fiber (DF) powders; soluble dietary fiber (SDF), insoluble dietary fiber (IDF) and total dietary fiber (TDF) were prepared from cocoa bean shells (CBS) by enzymatic treatment. These DFs were evaluated for their effects on glucose adsorption, glucose diffusion, starch hydrolysis, cholesterol binding, sodium cholate binding and oil binding capacities using in vitro model systems by simulating gastric intestinal conditions. The results showed that SDF generally exhibited significantly (p < 0.05) higher glucose adsorption capacity (GAC), α-amylase inhibition activity, cholesterol and sodium cholate binding capacity, but less significant (>0.05) glucose dialysis retardation index (GDRI) and oil binding capacity, when compared with IDF and TDF which both showed similar effects. Moreover, it was discovered that the three CBS dietary fiber powders contained intrinsic antioxidants (phenolic compounds). The study suggested that CBS could be an alternative cheap source of DF with additional benefits. Thus, CBS fibers could be incorporated as low calorie bulk ingredients in high-fiber diet to reduce calorie and cholesterol levels and control blood glucose level.

Autoinhibitory mechanisms of ERG studied by molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Lu, Yan; Salsbury, Freddie R.

2015-01-01

ERG, an ETS-family transcription factor, acts as a regulator of differentiation of early hematopoietic cells. It contains an autoinhibitory domain, which negatively regulates DNA-binding. The mechanism of autoinhibitory is still illusive. To understand the mechanism, we study the dynamical properties of ERG protein by molecular dynamics simulations. These simulations suggest that DNA binding autoinhibition associates with the internal dynamics of ERG. Specifically, we find that (1), The N-C terminal correlation in the inhibited ERG is larger than that in uninhibited ERG that contributes to the autoinhibition of DNA-binding. (2), DNA-binding changes the property of the N-C terminal correlation from being anti-correlated to correlated, that is, changing the relative direction of the correlated motions and (3), For the Ets-domain specifically, the inhibited and uninhibited forms exhibit essentially the same dynamics, but the binding of the DNA decreases the fluctuation of the Ets-domain. We also find from PCA analysis that the three systems, even with quite different dynamics, do have highly similar free energy surfaces, indicating that they share similar conformations.

Complex between α-bungarotoxin and an α7 nicotinic receptor ligand-binding domain chimaera

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

Huang, Sun; Li, Shu-Xing; Bren, Nina

2013-09-01

To identify high-affinity interactions between long-chain α-neurotoxins and nicotinic receptors, we determined the crystal structure of the complex between α-btx (α-bungarotoxin) and a pentameric ligand-binding domain constructed from the human α7 AChR (acetylcholine receptor) and AChBP (acetylcholine-binding protein). The complex buries ~2000 Å 2 (1 Å=0.1 nm) of surface area, within which Arg 36 and Phe 32 from finger II of α-btx form a π-cation stack that aligns edge-to-face with the conserved Tyr 184 from loop-C of α7, while Asp 30 of α-btx forms a hydrogen bond with the hydroxy group of Tyr 184. These inter-residue interactions diverge from thosemore » in a 4.2 Å structure of α-ctx (α-cobratoxin) bound to AChBP, but are similar to those in a 1.94 Å structure of α-btx bound to the monomeric α1 extracellular domain, although compared with the monomer-bound complex, the α-btx backbone exhibits a large shift relative to the protein surface. Mutational analyses show that replacing Tyr 184 with a threonine residue abolishes high-affinity α-btx binding, whereas replacing with a phenylalanine residue maintains high affinity. Comparison of the α-btx complex with that coupled to the agonist epibatidine reveals structural rearrangements within the binding pocket and throughout each subunit. The overall findings highlight structural principles by which α-neurotoxins interact with nicotinic receptors.« less

(3H)WB4101 labels the 5-HT1A serotonin receptor subtype in rat brain. Guanine nucleotide and divalent cation sensitivity

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

Norman, A.B.; Battaglia, G.; Creese, I.

1985-12-01

In the presence of a 30 nM prazosin mask, (/sup 3/H)-2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1,4-benzodioxane ((/sup 3/H)WB4101) can selectively label 5-HT1 serotonin receptors. Serotonin exhibits high affinity (Ki = 2.5 nM) and monophasic competition for (/sup 3/H) WB4101 binding in cerebral cortex. We have found a significant correlation (r = 0.96) between the affinities of a number of serotonergic and nonserotonergic compounds at (/sup 3/H)WB4101-binding sites in the presence of 30 nM prazosin and (/sup 3/H) lysergic acid diethylamide ((/sup 3/H)LSD)-labeled 5-HT1 serotonin receptors in homogenates of rat cerebral cortex. Despite similar pharmacological profiles, distribution studies indicate that, in the presence of 5more » mM MgSO4, the Bmax of (/sup 3/H)WB4101 is significantly lower than the Bmax of (/sup 3/H)LSD in various brain regions. WB4101 competition for (/sup 3/H) LSD-labeled 5-HT1 receptors fits best to a computer-derived model assuming two binding sites, with the KH for WB4101 being similar to the KD of (/sup 3/H)WB4101 binding derived from saturation experiments. This suggests that (/sup 3/H)WB4101 labels only one of the subtypes of the 5-HT1 serotonin receptors labeled by (/sup 3/H)LSD. The selective 5-HT1A serotonin receptor antagonist, spiperone, and the selective 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino) tetraline, exhibit high affinity and monophasic competition for (/sup 3/H)WB4101 but compete for multiple (/sup 3/H)LSD 5-HT1 binding sites. These data indicate that (/sup 3/H)WB4101 selectively labels the 5-HT1A serotonin receptor, whereas (/sup 3/H) LSD appears to label both the 5-HT1A and the 5-HT1B serotonin receptor subtypes. The divalent cations, Mn2+, Mg2+, and Ca2+ were found to markedly increase the affinity and Bmax of (/sup 3/H)WB4101 binding in cerebral cortex. Conversely, the guanine nucleotides guanylylimidodiphosphate and GTP, but not the adenosine nucleotide ATP, markedly reduce the Bmax of (/sup 3/H)WB4101 binding.« less

Development and cytotoxicity of Schiff base derivative as a fluorescence probe for the detection of L-Arginine

NASA Astrophysics Data System (ADS)

Shang, Xuefang; Li, Jie; Guo, Kerong; Ti, Tongyu; Wang, Tianyun; Zhang, Jinlian

2017-04-01

Inspired from biological counter parts, chemical modification of Schiff base derivatives with function groups may provide a highly efficient method to detect amino acids. Therefore, a fluorescent probe involving Schiff base and hydroxyl group has been designed and prepared, which showed high response and specificity for Arginine (Arg) among normal eighteen standard kinds of amino acids (Alanine, Valine, Leucine, Isoleucine, Methionine, Asparticacid, Glutamicacid, Arginine, Glycine, Serine, Threonine, Asparagine, Phenylalanine, Histidine, Tryptophan, Proline, Lysine, Glutamine, Tyrosine and Cysteine). Furthermore, theoretical investigation further illustrated the possible binding mode in the host-guest interaction and the roles of molecular frontier orbitals in molecular interplay. In addition, the synthesized fluorescent probe exhibited high binding ability for Arg and low cytotoxicity to MCF-7 cells over a concentration range of 0-200 μg mL-1 which can be also used as a biosensor for the Arg detection in vivo.

Mapping the Tail Fiber as the Receptor Binding Protein Responsible for Differential Host Specificity of Pseudomonas aeruginosa Bacteriophages PaP1 and JG004

PubMed Central

Le, Shuai; He, Xuesong; Tan, Yinling; Huang, Guangtao; Zhang, Lin; Lux, Renate; Shi, Wenyuan; Hu, Fuquan

2013-01-01

The first step in bacteriophage infection is recognition and binding to the host receptor, which is mediated by the phage receptor binding protein (RBP). Different RBPs can lead to differential host specificity. In many bacteriophages, such as Escherichia coli and Lactococcal phages, RBPs have been identified as the tail fiber or protruding baseplate proteins. However, the tail fiber-dependent host specificity in Pseudomonas aeruginosa phages has not been well studied. This study aimed to identify and investigate the binding specificity of the RBP of P. aeruginosa phages PaP1 and JG004. These two phages share high DNA sequence homology but exhibit different host specificities. A spontaneous mutant phage was isolated and exhibited broader host range compared with the parental phage JG004. Sequencing of its putative tail fiber and baseplate region indicated a single point mutation in ORF84 (a putative tail fiber gene), which resulted in the replacement of a positively charged lysine (K) by an uncharged asparagine (N). We further demonstrated that the replacement of the tail fiber gene (ORF69) of PaP1 with the corresponding gene from phage JG004 resulted in a recombinant phage that displayed altered host specificity. Our study revealed the tail fiber-dependent host specificity in P. aeruginosa phages and provided an effective tool for its alteration. These contributions may have potential value in phage therapy. PMID:23874674

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

Chen, S.-S.; IGE Therapeutics, Inc., Cellular and Cancer Immunology, 6370 Lusk Boulevard, F109, San Diego, CA 92121; Yang Yongmin

GFP-C{kappa} fusion protein was previously shown selectable on ribosome display platform with solid phase antibodies against GFP determinant [Y.-M. Yang, T.J. Barankiewicz, M. He, M. Taussig, S.-S. Chen, Selection of antigenic markers on a GFP-C{kappa} fusion scaffold with high sensitivity by eukaryotic ribosome display, Biochem. Biophys. Res. Commun. 359 (2007) 251-257]. Herein, we show that members of aptameric peptide library constructed within the site 6 and site 8/9 loops of GFP of the ribosome display construct are selectable upon binding to the solid phase IgE antigen. An input of 1.0 {mu}g of the dual site aptameric GFP library exhibiting amore » diversity of 7.5 x 10{sup 11} was transcribed, translated and incubated with solid phase IgE. RT-PCR products were amplified from mRNA of the aptamer-ribosome-mRNA (ARM) complex captured on the solid phase IgE. Clones of aptameric GFP were prepared from RT-PCR product of ARM complex following repetitive selection. Recombinant aptameric GFP proteins from the selected clones bind IgE coated on the 96-well plate, and the binding was abrogated by incubation with soluble human IgE but not human IgG. Selected aptameric GFP proteins also exhibit binding to three different sources of human IgE (IgE PS, BED, and JW8) but not irrelevant proteins. These observations indicate that appropriately selected aptameric GFP on a solid phase ligand by ribosome display may serve as an affinity reagent for blocking reactivity of a biological ligand.« less

Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

NASA Astrophysics Data System (ADS)

Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

1990-10-01

The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

Structural and immunologic characterization of bovine, horse, and rabbit serum albumins

PubMed Central

Majorek, Karolina A.; Porebski, Przemyslaw J.; Dayal, Arjun; Zimmerman, Matthew D.; Jablonska, Kamila; Stewart, Alan J.; Chruszcz, Maksymilian; Minor, Wladek

2012-01-01

Serum albumin (SA) is the most abundant plasma protein in mammals. SA is a multifunctional protein with extraordinary ligand binding capacity, making it a transporter molecule for a diverse range of metabolites, drugs, nutrients, metals and other molecules. Due to its ligand binding properties, albumins have wide clinical, pharmaceutical, and biochemical applications. Albumins are also allergenic, and exhibit a high degree of cross-reactivity due to significant sequence and structure similarity of SAs from different organisms. Here we present crystal structures of albumins from cattle (BSA), horse (ESA) and rabbit (RSA) serums. The structural data are correlated with the results of immunological studies of SAs. We also analyze the conservation or divergence of structures and sequences of SAs in the context of their potential allergenicity and cross-reactivity. In addition, we identified a previously uncharacterized ligand binding site in the structure of RSA, and calcium binding sites in the structure of BSA, which is the first serum albumin structure to contain metal ions. PMID:22677715

Mechanosensing Potentials Gate Fuel Consumption in a Bipedal DNA Nanowalker

NASA Astrophysics Data System (ADS)

Tee, Shern Ren; Hu, Xinpeng; Loh, Iong Ying; Wang, Zhisong

2018-03-01

A bipedal DNA nanowalker was recently reported to convert chemical energy into directional motion autonomously and efficiently. To elucidate its chemomechanical coupling mechanisms, three-dimensional molecular modeling is used to obtain coarse-grained foot-track binding potentials of the DNA nanowalker via unbiased and biased sampling techniques (for the potentials' basin and high-energy edges, respectively). The binding state that is protected against fuel-induced dissociation responds asymmetrically to forward versus backward forces, unlike the unprotected state, demonstrating a mechanosensing capability to gate fuel binding. Despite complex DNA mechanics, the foot-track potential exhibits a surprisingly neat three-part profile, offering some general guidelines to rationally design efficient nanowalkers. Subsequent modeling of the bipedal walker attached to the track gives estimates of the free energy for each bipedal state, showing how the mechanosensing foot-track binding breaks the symmetry between the rear and front feet, enabling the rear foot to be selectively dissociated by fuel and generating efficient chemomechanical coupling.

Discovery of a Novel General Anesthetic Chemotype Using High-throughput Screening

PubMed Central

McKinstry-Wu, Andrew R.; Bu, Weiming; Rai, Ganesha; Lea, Wendy A.; Weiser, Brian P.; Liang, David F.; Simeonov, Anton; Jadhav, Ajit; Maloney, David J.; Eckenhoff, Roderic G.

2014-01-01

Background The development of novel anesthetics has historically been a process of combined serendipity and empiricism, with most recent new anesthetics developed via modification of existing anesthetic structures. Methods Using a novel high-throughput screen employing the fluorescent anesthetic 1-aminoanthracene (1-AMA) and apoferritin as a surrogate for on-pathway anesthetic protein target(s), we screened a 350,000 compound library for competition with 1-AMA-apoferritin binding. Hit compounds meeting structural criteria had their binding affinities for apoferritin quantified with isothermal titration calorimetry and were tested for γ-aminobutyric acid type A-receptor binding using a flunitrazepam binding assay. Chemotypes with a strong presence in the top 700 and exhibiting activity via isothermal titration calorimetry were selected for medicinal chemistry optimization including testing for anesthetic potency and toxicity in an in vivo Xenopus laevis tadpole assay. Compounds with low toxicity and high potency were tested for anesthetic potency in mice. Results From an initial chemical library of over 350,000 compounds, we identified 2,600 compounds that potently inhibited 1-AMA binding to apoferritin. A subset of compounds chosen by structural criteria (700) was successfully reconfirmed using the initial assay. Based upon a strong presence in both the initial and secondary screens the 6-phenylpyridazin-3(2H)-one chemotype was assessed for anesthetic activity in tadpoles. Medicinal chemistry efforts identified four compounds with high potency and low toxicity in tadpoles, two were found to be effective novel anesthetics in mice. Conclusions We demonstrate the first use of a high-throughput screen to successfully identify a novel anesthetic chemotype and show mammalian anesthetic activity for members of that chemotype. PMID:25603205

Tactics for preclinical validation of receptor-binding radiotracers

PubMed Central

Lever, Susan Z.; Fan, Kuo-Hsien; Lever, John R.

2016-01-01

Introduction Aspects of radiopharmaceutical development are illustrated through preclinical studies of [125I]-(E)-1-(2-(2,3-dihydrobenzofuran-5-yl)ethyl)-4-(iodoallyl)piperazine ([125I]-E-IA- BF-PE-PIPZE), a radioligand for sigma-1 (σ1) receptors, coupled with examples from the recent literature. Findings are compared to those previously observed for [125I]-(E)-1-(2-(2,3-dimethoxy-5-yl)ethyl)-4-(iodoallyl)piperazine ([125I]-E-IA-DM-PE-PIPZE). Methods Syntheses of E-IA-BF-PE-PIPZE and [125I]-E-IA-BF-PE-PIPZE were accomplished by standard methods. In vitro receptor binding studies and autoradiography were performed, and binding potential was predicted. Measurements of lipophilicity and protein binding were obtained. In vivo studies were conducted in mice to evaluate radioligand stability, as well as specific binding to σ1 sites in brain, brain regions and peripheral organs in the presence and absence of potential blockers. Results E-IA-BF-PE-PIPZE exhibited high affinity and selectivity for σ1 receptors (Ki = 0.43 ± 0.03 nM, σ2 / σ1 = 173). [125I]-E-IA-BF-PE-PIPZE was prepared in good yield and purity, with high specific activity. Radioligand binding provided dissociation (koff) and association (kon) rate constants, along with a measured Kd of 0.24 ± 0.01 nM and Bmax of 472 ± 13 fmol / mg protein. The radioligand proved suitable for quantitative autoradiography in vitro using brain sections. Moderate lipophilicity, Log D7.4 2.69 ± 0.28, was determined, and protein binding was 71 ± 0.3%. In vivo, high initial whole brain uptake, > 6% injected dose / g, cleared slowly over 24 h. Specific binding represented 75% to 93% of total binding from 15 min to 24 h. Findings were confirmed and extended by regional brain biodistribution. Radiometabolites were not observed in brain (1%). Conclusions Substitution of dihydrobenzofuranylethyl for dimethoxyphenethyl increased radioligand affinity for σ1 receptors by 16-fold. While high specific binding to σ1 receptors was observed for both radioligands in vivo, [125I]-E-IA-BF-PE-PIPZE displayed much slower clearance kinetics than [125I]-E-IA-DM-PE-PIPZE. Thus, minor structural modifications of σ1 receptor radioligands lead to major differences in binding properties in vitro and in vivo. PMID:27755986

Stereoselective binding of doxazosin enantiomers to plasma proteins from rats, dogs and humans in vitro

PubMed Central

Sun, Jia-an; Kong, De-zhi; Zhen, Ya-qin; Li, Qing; Zhang, Wei; Zhang, Jiang-hua; Yin, Zhi-wei; Ren, Lei-ming

2013-01-01

Aim: (±)Doxazosin is a long-lasting inhibitor of α1-adrenoceptors that is widely used to treat benign prostatic hyperplasia and lower urinary tract symptoms. In this study we investigated the stereoselective binding of doxazosin enantiomers to the plasma proteins of rats, dogs and humans in vitro. Methods: Human, dog and rat plasma were prepared. Equilibrium dialysis was used to determine the plasma protein binding of each enantiomer in vitro. Chiral HPLC with fluorescence detection was used to measure the drug concentrations on each side of the dialysis membrane bag. Results: Both the enantiomers were highly bound to the plasma proteins of rats, dogs and humans [(−)doxazosin: 89.4%–94.3%; (+)doxazosin: 90.9%–95.4%]. (+)Doxazosin exhibited significantly higher protein binding capacities than (−)doxazosin in all the three species, and the difference in the bound concentration (Cb) between the two enantiomers was enhanced as their concentrations were increased. Although the percentage of the plasma protein binding in the dog plasma was significantly lower than that in the human plasma at 400 and 800 ng/mL, the corrected percentage of plasma protein binding was dog>human>rat. Conclusion: (−)Doxazosin and (+)doxazosin show stereoselective plasma protein binding with a significant species difference among rats, dogs and humans. PMID:24241343

No significant regulation of bicoid mRNA by Pumilio or Nanos in the early Drosophila embryo.

PubMed

Wharton, Tammy H; Nomie, Krystle J; Wharton, Robin P

2018-01-01

Drosophila Pumilio (Pum) is a founding member of the conserved Puf domain class of RNA-binding translational regulators. Pum binds with high specificity, contacting eight nucleotides, one with each of the repeats in its RNA-binding domain. In general, Pum is thought to block translation in collaboration with Nanos (Nos), which exhibits no binding specificity in isolation but is recruited jointly to regulatory sequences containing a Pum binding site in the 3'-UTRs of target mRNAs. Unlike Pum, which is ubiquitous in the early embryo, Nos is tightly restricted to the posterior, ensuring that repression of its best-characterized target, maternal hunchback (hb) mRNA, takes place exclusively in the posterior. An exceptional case of Nos-independent regulation by Pum has been described-repression of maternal bicoid (bcd) mRNA at the anterior pole of the early embryo, dependent on both Pum and conserved Pum binding sites in the 3'-UTR of the mRNA. We have re-investigated regulation of bcd in the early embryo; our experiments reveal no evidence of a role for Pum or its conserved binding sites in regulation of the perdurance of bcd mRNA or protein. Instead, we find that Pum and Nos control the accumulation of bcd mRNA in testes.

Architecture of the human regulatory network derived from ENCODE data.

PubMed

Gerstein, Mark B; Kundaje, Anshul; Hariharan, Manoj; Landt, Stephen G; Yan, Koon-Kiu; Cheng, Chao; Mu, Xinmeng Jasmine; Khurana, Ekta; Rozowsky, Joel; Alexander, Roger; Min, Renqiang; Alves, Pedro; Abyzov, Alexej; Addleman, Nick; Bhardwaj, Nitin; Boyle, Alan P; Cayting, Philip; Charos, Alexandra; Chen, David Z; Cheng, Yong; Clarke, Declan; Eastman, Catharine; Euskirchen, Ghia; Frietze, Seth; Fu, Yao; Gertz, Jason; Grubert, Fabian; Harmanci, Arif; Jain, Preti; Kasowski, Maya; Lacroute, Phil; Leng, Jing Jane; Lian, Jin; Monahan, Hannah; O'Geen, Henriette; Ouyang, Zhengqing; Partridge, E Christopher; Patacsil, Dorrelyn; Pauli, Florencia; Raha, Debasish; Ramirez, Lucia; Reddy, Timothy E; Reed, Brian; Shi, Minyi; Slifer, Teri; Wang, Jing; Wu, Linfeng; Yang, Xinqiong; Yip, Kevin Y; Zilberman-Schapira, Gili; Batzoglou, Serafim; Sidow, Arend; Farnham, Peggy J; Myers, Richard M; Weissman, Sherman M; Snyder, Michael

2012-09-06

Transcription factors bind in a combinatorial fashion to specify the on-and-off states of genes; the ensemble of these binding events forms a regulatory network, constituting the wiring diagram for a cell. To examine the principles of the human transcriptional regulatory network, we determined the genomic binding information of 119 transcription-related factors in over 450 distinct experiments. We found the combinatorial, co-association of transcription factors to be highly context specific: distinct combinations of factors bind at specific genomic locations. In particular, there are significant differences in the binding proximal and distal to genes. We organized all the transcription factor binding into a hierarchy and integrated it with other genomic information (for example, microRNA regulation), forming a dense meta-network. Factors at different levels have different properties; for instance, top-level transcription factors more strongly influence expression and middle-level ones co-regulate targets to mitigate information-flow bottlenecks. Moreover, these co-regulations give rise to many enriched network motifs (for example, noise-buffering feed-forward loops). Finally, more connected network components are under stronger selection and exhibit a greater degree of allele-specific activity (that is, differential binding to the two parental alleles). The regulatory information obtained in this study will be crucial for interpreting personal genome sequences and understanding basic principles of human biology and disease.

Structural characterization of metal binding to a cold-adapted frataxin.

PubMed

Noguera, Martín E; Roman, Ernesto A; Rigal, Juan B; Cousido-Siah, Alexandra; Mitschler, André; Podjarny, Alberto; Santos, Javier

2015-06-01

Frataxin is an evolutionary conserved protein that participates in iron metabolism. Deficiency of this small protein in humans causes a severe neurodegenerative disease known as Friedreich's ataxia. A number of studies indicate that frataxin binds iron and regulates Fe-S cluster biosynthesis. Previous structural studies showed that metal binding occurs mainly in a region of high density of negative charge. However, a comprehensive characterization of the binding sites is required to gain further insights into the mechanistic details of frataxin function. In this work, we have solved the X-ray crystal structures of a cold-adapted frataxin from a psychrophilic bacterium in the presence of cobalt or europium ions. We have identified a number of metal-binding sites, mainly solvent exposed, several of which had not been observed in previous studies on mesophilic homologues. No major structural changes were detected upon metal binding, although the structures exhibit significant changes in crystallographic B-factors. The analysis of these B-factors, in combination with crystal packing and RMSD among structures, suggests the existence of localized changes in the internal motions. Based on these results, we propose that bacterial frataxins possess binding sites of moderate affinity for a quick capture and transfer of iron to other proteins and for the regulation of Fe-S cluster biosynthesis, modulating interactions with partner proteins.

Crystal structure of RlmAI: Implications for understanding the 23S rRNA G745/G748-methylation at the macrolide antibiotic-binding site

PubMed Central

Das, Kalyan; Acton, Thomas; Chiang, Yiwen; Shih, Lydia; Arnold, Eddy; Montelione, Gaetano T.

2004-01-01

The RlmA class of enzymes (RlmAI and RlmAII) catalyzes N1-methylation of a guanine base (G745 in Gram-negative and G748 in Gram-positive bacteria) of hairpin 35 of 23S rRNA. We have determined the crystal structure of Escherichia coli RlmAI at 2.8-Å resolution, providing 3D structure information for the RlmA class of RNA methyltransferases. The dimeric protein structure exhibits features that provide new insights into its molecular function. Each RlmAI molecule has a Zn-binding domain, responsible for specific recognition and binding of its rRNA substrate, and a methyltransferase domain. The asymmetric RlmAI dimer observed in the crystal structure has a well defined W-shaped RNA-binding cleft. Two S-adenosyl-l-methionine substrate molecules are located at the two valleys of the W-shaped RNA-binding cleft. The unique shape of the RNA-binding cleft, different from that of known RNA-binding proteins, is highly specific and structurally complements the 3D structure of hairpin 35 of bacterial 23S rRNA. Apart from the hairpin 35, parts of hairpins 33 and 34 also interact with the RlmAI dimer. PMID:14999102

Composite Reinforcement using Boron Nitride Nanotubes

DTIC Science & Technology

2016-11-15

atomistic environment, which often results in notable changes of its binding to the system. By contrast, Cu and Ti exhibits quite uniform behaviour ...contrast, Cu and Ti exhibits quite uniform behaviour and hardly change their binding energy with tube size for the systems investigated. Studies on...reinforced Cu is rather brittle and therefore comparison of the behaviour of Al, Ti and Cu is important, and also because Cu is used in alloys to produce

Lessons learned about [F-18]-AV-1451 off-target binding from an autopsy-confirmed Parkinson's case.

PubMed

Marquié, Marta; Verwer, Eline E; Meltzer, Avery C; Kim, Sally Ji Who; Agüero, Cinthya; Gonzalez, Jose; Makaretz, Sara J; Siao Tick Chong, Michael; Ramanan, Prianca; Amaral, Ana C; Normandin, Marc D; Vanderburg, Charles R; Gomperts, Stephen N; Johnson, Keith A; Frosch, Matthew P; Gómez-Isla, Teresa

2017-10-19

[F-18]-AV-1451 is a novel positron emission tomography (PET) tracer with high affinity to neurofibrillary tau pathology in Alzheimer's disease (AD). PET studies have shown increased tracer retention in patients clinically diagnosed with dementia of AD type and mild cognitive impairment in regions that are known to contain tau lesions. In vivo uptake has also consistently been observed in midbrain, basal ganglia and choroid plexus in elderly individuals regardless of their clinical diagnosis, including clinically normal whose brains are not expected to harbor tau pathology in those areas. We and others have shown that [F-18]-AV-1451 exhibits off-target binding to neuromelanin, melanin and blood products on postmortem material; and this is important for the correct interpretation of PET images. In the present study, we further investigated [F-18]-AV-1451 off-target binding in the first autopsy-confirmed Parkinson's disease (PD) subject who underwent antemortem PET imaging. The PET scan showed elevated [F-18]-AV-1451 retention predominantly in inferior temporal cortex, basal ganglia, midbrain and choroid plexus. Neuropathologic examination confirmed the PD diagnosis. Phosphor screen and high resolution autoradiography failed to show detectable [F-18]-AV-1451 binding in multiple brain regions examined with the exception of neuromelanin-containing neurons in the substantia nigra, leptomeningeal melanocytes adjacent to ventricles and midbrain, and microhemorrhages in the occipital cortex (all reflecting off-target binding), in addition to incidental age-related neurofibrillary tangles in the entorhinal cortex. Additional legacy postmortem brain samples containing basal ganglia, choroid plexus, and parenchymal hemorrhages from 20 subjects with various neuropathologic diagnoses were also included in the autoradiography experiments to better understand what [F-18]-AV-1451 in vivo positivity in those regions means. No detectable [F-18]-AV-1451 autoradiographic binding was present in the basal ganglia of the PD case or any of the other subjects. Off-target binding in postmortem choroid plexus samples was only observed in subjects harboring leptomeningeal melanocytes within the choroidal stroma. Off-target binding to parenchymal hemorrhages was noticed in postmortem material from subjects with cerebral amyloid angiopathy. The imaging-postmortem correlation analysis in this PD case reinforces the notion that [F-18]-AV-1451 has strong affinity for neurofibrillary tau pathology but also exhibits off-target binding to neuromelanin, melanin and blood components. The robust off-target in vivo retention in basal ganglia and choroid plexus, in the absence of tau deposits, meningeal melanocytes or any other identifiable binding substrate by autoradiography in the PD case reported here, also suggests that the PET signal in those regions may be influenced, at least in part, by biological or technical factors that occur in vivo and are not captured by autoradiography.

pH-induced conformational change of the rotavirus VP4 spike: implications for cell entry and antibody neutralization.

PubMed

Pesavento, Joseph B; Crawford, Sue E; Roberts, Ed; Estes, Mary K; Prasad, B V Venkataram

2005-07-01

The rotavirus spike protein, VP4, is a major determinant of infectivity and neutralization. Previously, we have shown that trypsin-enhanced infectivity of rotavirus involves a transformation of the VP4 spike from a flexible to a rigid bilobed structure. Here we show that at elevated pH the spike undergoes a drastic, irreversible conformational change and becomes stunted, with a pronounced trilobed appearance. These particles with altered spikes, at a normal pH of 7.5, despite the loss of infectivity and the ability to hemagglutinate, surprisingly exhibit sialic acid (SA)-independent cell binding in contrast to the SA-dependent cell binding exhibited by native virions. Remarkably, a neutralizing monoclonal antibody that remains bound to spikes throughout the pH changes (pH 7 to 11 and back to pH 7) completely prevents this conformational change, preserving the SA-dependent cell binding and hemagglutinating functions of the virion. A hypothesis that emerges from the present study is that high-pH treatment triggers a conformational change that mimics a post-SA-attachment step to expose an epitope recognized by a downstream receptor in the rotavirus cell entry process. This process involves sequential interactions with multiple receptors, and the mechanism by which the antibody neutralizes is by preventing this conformational change.

Biochemical Regulatory Features of Activation-Induced Cytidine Deaminase Remain Conserved from Lampreys to Humans

PubMed Central

King, Justin J.; Amemiya, Chris T.; Hsu, Ellen

2017-01-01

ABSTRACT Activation-induced cytidine deaminase (AID) is a genome-mutating enzyme that initiates class switch recombination and somatic hypermutation of antibodies in jawed vertebrates. We previously described the biochemical properties of human AID and found that it is an unusual enzyme in that it exhibits binding affinities for its substrate DNA and catalytic rates several orders of magnitude higher and lower, respectively, than a typical enzyme. Recently, we solved the functional structure of AID and demonstrated that these properties are due to nonspecific DNA binding on its surface, along with a catalytic pocket that predominantly assumes a closed conformation. Here we investigated the biochemical properties of AID from a sea lamprey, nurse shark, tetraodon, and coelacanth: representative species chosen because their lineages diverged at the earliest critical junctures in evolution of adaptive immunity. We found that these earliest-diverged AID orthologs are active cytidine deaminases that exhibit unique substrate specificities and thermosensitivities. Significant amino acid sequence divergence among these AID orthologs is predicted to manifest as notable structural differences. However, despite major differences in sequence specificities, thermosensitivities, and structural features, all orthologs share the unusually high DNA binding affinities and low catalytic rates. This absolute conservation is evidence for biological significance of these unique biochemical properties. PMID:28716949

In vitro high throughput phage display selection of ovarian cancer avid phage clones for near-infrared optical imaging.

PubMed

Soendergaard, Mette; Newton-Northup, Jessica R; Deutscher, Susan L

2014-01-01

Ovarian cancer is among the leading causes of cancer deaths in women, and is the most fatal gynecological malignancy. Poor outcomes of the disease are a direct result of inadequate detection and diagnostic methods, which may be overcome by the development of novel efficacious screening modalities. However, the advancement of such technologies is often time-consuming and costly. To overcome this hurdle, our laboratory has established a time and cost effective method of selecting and identifying ovarian carcinoma avid bacteriophage (phage) clones using high throughput phage display technology. These phage clones were selected from a filamentous phage fusion vector (fUSE5) 15-amino acid peptide library against human ovarian carcinoma (SKOV-3) cells, and identified by DNA sequencing. Two phage clones, pM6 and pM9, were shown to exhibit high binding affinity and specificity for SKOV-3 cells using micropanning, cell binding and fluorescent microscopy studies. To validate that the binding was mediated by the phage-displayed peptides, biotinylated peptides (M6 and M9) were synthesized and the specificity for ovarian carcinoma cells was analyzed. These results showed that M6 and M9 bound to SKOV-3 cells in a dose-response manner and exhibited EC50 values of 22.9 ± 2.0 μM and 12.2 ± 2.1μM (mean ± STD), respectively. Based on this, phage clones pM6 and pM9 were labeled with the near-infrared fluorophore AF680, and examined for their pharmacokinetic properties and tumor imaging abilities in vivo. Both phage successfully targeted and imaged SKOV-3 tumors in xenografted nude mice, demonstrating the ability of this method to quickly and cost effectively develop novel ovarian carcinoma avid phage.

Hydrophobic Peptides Affect Binding of Calmodulin and Ca2+ as Explored by H/D Amide Exchange and Mass Spectrometry

PubMed Central

Sperry, Justin B.; Huang, Richard Y-C.; Zhu, Mei M.; Rempel, Don L.; Gross, Michael L.

2010-01-01

Calmodulin (CaM), a ubiquitous intracellular sensor protein, binds Ca2+ and interacts with various targets as part of signal transduction. Using hydrogen/deuterium exchange (H/DX) and a high resolution PLIMSTEX (Protein-Ligand Interactions by Mass Spectrometry, Titration, and H/D Exchange) protocol, we examined five different states of calmodulin: calcium-free, calcium-loaded, and three states of calcium-loaded in the presence of either melittin, mastoparan, or skeletal myosin light-chain kinase (MLCK). When CaM binds Ca2+, the extent of HDX decreased, consistent with the protein becoming stabilized upon binding. Furthermore, Ca2+-saturated calmodulin exhibits increased protection when bound to the peptides, forming high affinity complexes. The protocol reveals significant changes in EF hands 1, 3, and 4 with saturating levels of Ca2+. Titration of the protein using PLIMSTEX provides the binding affinity of Ca2+ to calmodulin within previously reported values. The affinities of calmodulin to Ca2+ increase by factors of 300 and 1000 in the presence of melittin and mastoparan, respectively. A modified PLIMSTEX protocol whereby the protein is digested to component peptides gives a region-specific titration. The titration data taken in this way show a decrease in the root mean square fit of the residuals, indicating a better fit of the data. The global H/D exchange results and those obtained in a region-specific way provide new insight into the Ca2+-binding properties of this well-studied protein. PMID:21765646

A Conformational Investigation of Propeptide Binding to the Integral Membrane Protein γ-Glutamyl Carboxylase Using Nanodisc Hydrogen Exchange Mass Spectrometry

PubMed Central

2015-01-01

Gamma (γ)-glutamyl carboxylase (GGCX) is an integral membrane protein responsible for the post-translational catalytic conversion of select glutamic acid (Glu) residues to γ-carboxy glutamic acid (Gla) in vitamin K-dependent (VKD) proteins. Understanding the mechanism of carboxylation and the role of GGCX in the vitamin K cycle is of biological interest in the development of therapeutics for blood coagulation disorders. Historically, biophysical investigations and structural characterizations of GGCX have been limited due to complexities involving the availability of an appropriate model membrane system. In previous work, a hydrogen exchange mass spectrometry (HX MS) platform was developed to study the structural configuration of GGCX in a near-native nanodisc phospholipid environment. Here we have applied the nanodisc–HX MS approach to characterize specific domains of GGCX that exhibit structural rearrangements upon binding the high-affinity consensus propeptide (pCon; AVFLSREQANQVLQRRRR). pCon binding was shown to be specific for monomeric GGCX-nanodiscs and promoted enhanced structural stability to the nanodisc-integrated complex while maintaining catalytic activity in the presence of carboxylation co-substrates. Noteworthy modifications in HX of GGCX were prominently observed in GGCX peptides 491–507 and 395–401 upon pCon association, consistent with regions previously identified as sites for propeptide and glutamate binding. Several additional protein regions exhibited minor gains in solvent protection upon propeptide incorporation, providing evidence for a structural reorientation of the GGCX complex in association with VKD carboxylation. The results herein demonstrate that nanodisc–HX MS can be utilized to study molecular interactions of membrane-bound enzymes in the absence of a complete three-dimensional structure and to map dynamic rearrangements induced upon ligand binding. PMID:24512177

Identification of cross-reactive proteins amongst different Curvularia species.

PubMed

Gupta, Ratna; Singh, Bhanu P; Sridhara, Susheela; Gaur, Shailendra N; Kumar, Raj; Chaudhary, Vijay K; Arora, Naveen

2002-01-01

Curvularia lunata is an important inhalant allergen. The present study was undertaken to investigate the shared IgG- and IgE-binding components among seven Curvularia species prevalent in the aerospora. Seven different Curvularia species were grown in a semisynthetic medium for 13 days. The extracts were analyzed by SDS-PAGE, immunoblot and ELISA/immunoblot inhibition using sera from C. lunata-positive patients and anti-C. lunata rabbit serum. Different Curvularia species showed 11-19 protein bands on SDS-PAGE. Proteins of 12, 20, 31, 45, 53, 78 and 97 kD were present in all the species. Eight out of 98 nasobronchial patients exhibited positive skin tests to C. lunata and to at least five Curvularia species. ELISA using these sera showed IgE binding with Curvularia species. Immunoblot using pooled anti-C. lunata sera from patients showed 5-12 allergenic proteins. Proteins of 12, 31, 45, 53 and 78 kD showed IgE binding in Curvularia species. Antibodies against C. lunata detected 6-14 antigenic proteins on immunoblot. Proteins of 31, 45 and 53 kD showed IgG binding in all the species. Proteins of 31 and 53 kD showed complete IgE/IgG binding inhibition. IgE/IgG ELISA inhibition showed dose-dependent inhibition in Curvularia species. C. lunata extract required 0.17 and 0.11 microg of protein for 50% IgE and IgG inhibition, respectively. C. clavata and C. pallescens required 10 times more protein to exhibit the same inhibition and other species required similar protein levels as those required by C. lunata. A high degree of cross-reactivity was observed between C. lunata and the six other Curvularia species tested. C. lunata and C. senegalensis shared maximum allergenic and antigenic components.

Loop III region of platelet-derived growth factor (PDGF) B-chain mediates binding to PDGF receptors and heparin.

PubMed Central

Schilling, D; Reid IV, J D; Hujer, A; Morgan, D; Demoll, E; Bummer, P; Fenstermaker, R A; Kaetzel, D M

1998-01-01

Site-directed mutagenesis of the platelet-derived growth factor (PDGF) B-chain was conducted to determine the importance of cationic amino acid residues (Arg160-Lys161-Lys162; RKK) located within the loop III region in mediating the biological and cell-association properties of the molecule. Binding to both PDGF alpha-and beta-receptors was inhibited by the conversion of all three cationic residues into anionic glutamates (RKK-->EEE), whereas an RKK-->SSS mutant also exhibited a modest loss in affinity for beta-receptors. Replacements with serine at either Arg160 (RKK-->SKK) or at all three positions (RKK-->SSS) had little effect on binding to alpha-receptors. Replacements with either glutamic or serine residues at any of the three positions also resulted in significant inhibition of heparin-binding activity. Furthermore, the RKK-->EEE mutant exhibited decreased association with the cell surface and accumulated in the culture medium as 29-32 kDa forms. Stable transfection of U87 astrocytoma cells with RKK-->EEE mutants of either the A-chain or the B-chain inhibited malignant growth in athymic nude mice. Despite altered receptor-binding activities, each of the loop III mutants retained full mitogenic activity when applied to cultured Swiss 3T3 cells. CD spectrophotometric analysis of the RKK-->EEE mutant revealed a secondary structure indistinguishable from the wild type, with a high degree of beta-sheet structure and random coil content (50% and 43% respectively). These findings indicate an important role of the Arg160-Lys161-Lys162 sequence in mediating the biological and cell-associative activities of the PDGF-BB homodimer, and reveal that the mitogenic activity of PDGF-BB is insufficient to mediate its full oncogenic properties. PMID:9677323

Binding of [3H]MSX-2 (3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine) to rat striatal membranes--a new, selective antagonist radioligand for A(2A) adenosine receptors.

PubMed

Müller, C E; Maurinsh, J; Sauer, R

2000-01-01

The present study describes the preparation and binding properties of a new, potent, and selective A(2A) adenosine receptor (AR) antagonist radioligand, [3H]3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargy lxanth ine ([3H]MSX-2). [3H]MSX-2 binding to rat striatal membranes was saturable and reversible. Saturation experiments showed that [3H]MSX-2 labeled a single class of binding sites with high affinity (K(d)=8.0 nM) and limited capacity (B(max)=1.16 fmol.mg(-1) of protein). The presence of 100 microM GTP, or 10 mM magnesium chloride, respectively, had no effect on [3H]MSX-2 binding. AR agonists competed with the binding of 1 nM [3H]MSX-2 with the following order of potency: 5'-N-ethylcarboxamidoadenosine (NECA)>2-[4-(carboxyethyl)phenylethylamino]-5'-N-ethylcarboxami doaden osine (CGS-21680)>2-chloroadenosine (2-CADO)>N(6)-cyclopentyladenosine (CPA). AR antagonists showed the following order of potency: 8-(m-bromostyryl)-3, 7-dimethyl-1-propargylxanthine (BS-DMPX)>1, 3-dipropyl-8-cyclopentylxanthine (DPCPX)>(R)-5, 6-dimethyl-7-(1-phenylethyl)-2-(4-pyridyl)-7H-pyrrolo[2, 3-d]pyrimidine-4-amine (SH-128)>3,7-dimethyl-1-propargylxanthine (DMPX)>caffeine. The K(i) values for antagonists were in accordance with data from binding studies with the agonist radioligand [3H]CGS21680, while agonist affinities were 3-7-fold lower. [3H]MSX-2 is a highly selective A(2A) AR antagonist radioligand exhibiting a selectivity of at least two orders of magnitude versus all other AR subtypes. The new radioligand shows high specific radioactivity (85 Ci/mmol, 3150 GBq/mmol) and acceptable nonspecific binding at rat striatal membranes of 20-30%, at 1 nM.

Novobiocin and additional inhibitors of the Hsp90 C-terminal nucleotide-binding pocket.

PubMed

Donnelly, Alison; Blagg, Brian S J

2008-01-01

The 90 kDa heat shock proteins (Hsp90), which are integrally involved in cell signaling, proliferation, and survival, are ubiquitously expressed in cells. Many proteins in tumor cells are dependent upon the Hsp90 protein folding machinery for their stability, refolding, and maturation. Inhibition of Hsp90 uniquely targets client proteins associated with all six hallmarks of cancer. Thus, Hsp90 has emerged as a promising target for the treatment of cancer. Hsp90 exists as a homodimer, which contains three domains. The N-terminal domain contains an ATP-binding site that binds the natural products geldanamycin and radicicol. The middle domain is highly charged and has high affinity for co-chaperones and client proteins. Initial studies by Csermely and co-workers suggested a second ATP-binding site in the C-terminus of Hsp90. This C-terminal nucleotide binding pocket has been shown to not only bind ATP, but cisplatin, novobiocin, epilgallocatechin-3-gallate (EGCG) and taxol. The coumarin antibiotics novobiocin, clorobiocin, and coumermycin A1 were isolated from several streptomyces strains and exhibit potent activity against Gram-positive bacteria. These compounds bind type II topoisomerases, including DNA gyrase, and inhibit the enzyme-catalyzed hydrolysis of ATP. As a result, novobiocin analogues have garnered the attention of numerous researchers as an attractive agent for the treatment of bacterial infection. Novobiocin was reported to bind weakly to the newly discovered Hsp90 C-terminal ATP binding site ( approximately 700 M in SkBr3 cells) and induce degradation of Hsp90 client proteins. Structural modification of this compound has led to an increase of 1000-fold in activity in anti-proliferative assays. Recent studies of structure-activity relationship (SAR) by Renoir and co-workers highlighted the crucial role of the C-4 and/or C-7 positions of the coumarin and removal of the noviose moiety, which appeared to be essential for degradation of Hsp90 client proteins. Unlike the N-terminal ATP binding site, there is no reported co-crystal structure of Hsp90 C-terminus bound to any inhibitor. The Hsp90 C-terminal domain, however, is known to contain a conserved pentapeptide sequence (MEEVD) which is recognized by co-chaperones. Cisplatin is a platinum-containing chemotherapeutic used to treat various types of cancers, including testicular, ovarian, bladder, and small cell lung cancer. Most notably, cisplatin coordinates to DNA bases, resulting in cross-linked DNA, which prohibits rapidly dividing cells from duplicating DNA for mitosis. Itoh and co-workers reported that cisplatin decreases the chaperone activity of Hsp90. This group applied bovine brain cytosol to a cisplatin affinity column, eluted with cisplatin and detected Hsp90 in the eluent. Subsequent experiments indicated that cisplatin exhibits high affinity for Hsp90. Moreover Csermely and co-workers determined that the cisplatin binding site is located proximal to the C-terminal ATP binding site. EGCG is one of the active ingredients found in green tea. EGCG is known to inhibit the activity of many Hsp90-dependent client proteins, including telomerase, several kinases, and the aryl hydrocarbon receptor (AhR). Recently Gasiewicz and co-workers reported that EGCG manifests its antagonistic activity against AhR through binding Hsp90. Similar to novobiocin, EGCG was shown to bind the C-terminus of Hsp90. Unlike previously identified N-terminal Hsp90 inhibitors, EGCG does not appear to prevent Hsp90 from forming multiprotein complexes. Studies are currently underway to determine whether EGCG competes with novobiocin or cisplatin binding. Taxol, a well-known drug for the treatment of cancer, is responsible for the stabilization of microtubules and the inhibition of mitosis. Previous studies have shown that taxol induces the activation of kinases and transcription factors, and mimics the effect of bacterial lipopolysaccharide (LPS), an attribute unrelated to its tubulin-binding properties. Rosen and co-workers prepared a biotinylated taxol derivative and performed affinity chromatography experiments with lysates from both mouse brain and macrophage cell lines. These studies led to identification of two chaperones, Hsp70 and Hsp90, by mass spectrometry. In contrast to typical Hsp90-binding drugs, taxol exhibits a stimulatory response. Recently it was reported that the geldanamycin derivative 17-AAG behaves synergistically with taxol-induced apoptosis. This review describes the different C-terminal inhibitors of Hsp90, with specific emphasis on structure-activity relationship studies of novobiocin and their effects on anti-proliferative activity.

Novobiocin and Additional Inhibitors of the Hsp90 C-Terminal Nucleotide-binding Pocket

PubMed Central

Donnelly, Alison; Blagg, Brian S. J.

2009-01-01

The 90 kDa heal shock proteins (Hsp90), which are integrally involved in cell signaling, proliferation, and survival, are ubiquitously expressed in cells. Many proteins in tumor cells are dependent upon the Hsp90 protein folding machinery for their stability, refolding, and maturation. Inhibition of Hsp90 uniquely targets client proteins associated with all six hallmarks of cancer. Thus, Hsp90 has emerged as a promising target for the treatment of cancer. Hsp90 exists as a homodimer, which contains three domains. The N-terminal domain contains an ATP-binding site that binds the natural products geldanamycin and radicicol. The middle domain is highly charged and has high affinity for co-chaperones and client proteins. Initial studies by Csermely and co-workers suggested a second ATP-binding site in the C-terminus of Hsp90. This C-terminal nucleotide binding pocket has been shown to not only bind ATP, but cisplatin, novobiocin, epilgallocatechin-3-gallate (EGCG) and taxol. The coumarin antibiotics novobiocin, clorobiocin, and coumermycin A1 were isolated from several streptomyces strains and exhibit potent activity against Gram-positive bacteria. These compounds bind type II topoisomerases, including DNA gyrase, and inhibit the enzyme-catalyzed hydrolysis of ATP. As a result, novobiocin analogues have garnered the attention of numerous researchers as an attractive agent for the treatment of bacterial infection. Novobiocin was reported to bind weakly to the newly discovered Hsp90 C-terminal ATP binding site (~700 M in SkBr3 cells) and induce degradation of Hsp90 client proteins. Structural modification of this compound has led to an increase of 1000-fold in activity in anti-proliferative assays. Recent studies of structure-activity relationship (SAR) by Renoir and co-workers highlighted the crucial role of the C-4 and/or C-7 positions of the coumarin and removal of the noviose moiety, which appeared to be essential for degradation of Hsp90 client proteins. Unlike the N-terminal ATP binding site, there is no reported co-crystal structure of Hsp90 C-terminus bound to any inhibitor. The Hsp90 C-terminal domain, however, is known to contain a conserved pentapeptide sequence (MEEVD) which is recognized by co-chaperones. Cisplatin is a platinum-containing chemotherapeutic used to treat various types of cancers, including testicular, ovarian, bladder, and small cell lung cancer. Most notably, cisplatin coordinates to DNA bases, resulting in cross-linked DNA, which prohibits rapidly dividing cells from duplicating DNA for mitosis. Itoh and co-workers reported that cisplatin decreases the chaperone activity of Hsp90. This group applied bovine brain cytosol to a cisplatin affinity column, eluted with cisplatin and detected Hsp90 in the eluent. Subsequent experiments indicated that cisplatin exhibits high affinity for Hsp90. Moreover Csermely and co-workers determined that the cisplatin binding site is located proximal to the C-terminal ATP binding site. EGCG is one of the active ingredients found in green tea EGCG is known to inhibit the activity of many Hsp90-dependent client proteins, including telomerase, several kinases, and the aryl hydrocarbon receptor (AhR). Recently Gasiewicz and co-workers reported that EGCG manifests its antagonistic activity against AhR through binding Hsp90. Similar to novobiocin, EGCG was shown to bind the C-terminus of Hsp90. Unlike previously identified N-terminal Hsp90 inhibitors, EGCG does not appear to prevent Hsp90 from forming multiprotein complexes. Studies are currently underway to determine whether EGCG competes with novobiocin or cisplatin binding. Taxol, a well-known drug for the treatment of cancer, is responsible for the stabilization of microtubules and the inhibition of mitosis. Previous studies have shown that taxol induces the activation of kinases and transcription factors, and mimies the effect of bacterial lipopolysaccharide (LPS), an attribute unrelated to its tubulin-binding properties. Rosen and co-workers prepared a biotinylated taxol derivative and performed affinity chromatography experiments with lysates from both mouse brain and macrophage cell lines. These studies led to identification of two chaperones. Hsp70 and Hsp90, by mass spectrometry. In contrast to typical Hsp90-binding drugs, taxol exhibits a stimulatory response. Recently it was reported that the geldanamycin derivative 17-AAG behaves synergistically with taxol-induced apoptosis. This review describes the different C-terminal inhibitors of Hsp90, with specific emphasis on structure-activity relationship studies of novobiocin and their effects on anti-proliferative activity. PMID:18991631

Luminescent macrocyclic lanthanide complexes

DOEpatents

Raymond, Kenneth N; Corneillie, Todd M; Xu, Jide

2014-05-20

The present invention provides a novel class of macrocyclic compounds as well as complexes formed between a metal (e.g., lanthanide) ion and the compounds of the invention. Preferred complexes exhibit high stability as well as high quantum yields of lanthanide ion luminescence in aqueous media without the need for secondary activating agents. Preferred compounds incorporate hydroxy-isophthalamide moieties within their macrocyclic structure and are characterized by surprisingly low, non-specific binding to a variety of polypeptides such as antibodies and proteins as well as high kinetic stability. These characteristics distinguish them from known, open-structured ligands.

Occurrence of tetrodotoxin-binding high molecular weight substances in the body fluid of shore crab (Hemigrapsus sanguineus).

PubMed

Shiomi, K; Yamaguchi, S; Kikuchi, T; Yamamori, K; Matsui, T

1992-12-01

The shore crab (Hemigrapsus sanguineus) is highly resistant to tetrodotoxin (TTX) although it contains no detectable amount of TTX (less than 5 MU/g, where 1 MU is defined as the amount of TTX killing a 20 g mouse in 30 min). Its body fluid was examined for neutralizing effects against the lethal activity of TTX. When the mixture of the body fluid and TTX was injected i.p. into mice, the lethal activity of TTX was significantly reduced; 1 ml of the body fluid was evaluated to neutralize 3.6-4.0 MU of TTX. Higher neutralizing activity (7.2-12.5 MU/ml of the body fluid) was exhibited by i.v. administration of the body fluid into mice before or after i.p. challenge of TTX. The lethal effect of paralytic shellfish poisons was not counteracted by the body fluid. Analysis by gel filtration on Sepharose 6B revealed that the body fluid contained TTX-binding high mol. wt substances (> 2,000,000) responsible for the neutralizing activity of the body fluid against TTX, which accounts for the high resistibility of the crab to TTX. When the crude toxin extracted from the liver of puffer (Takifugu niphobles) was mixed with the body fluid and chromatographed on Sepharose 6B, almost pure TTX was obtained from the fractions containing the TTX-binding high mol. wt substances, suggesting that the TTX-binding high mol. wt substances could be useful in purification of TTX from biological samples.

TAF(II)170 interacts with the concave surface of TATA-binding protein to inhibit its DNA binding activity.

PubMed

Pereira, L A; van der Knaap, J A; van den Boom, V; van den Heuvel, F A; Timmers, H T

2001-11-01

The human RNA polymerase II transcription factor B-TFIID consists of TATA-binding protein (TBP) and the TBP-associated factor (TAF) TAF(II)170 and can rapidly redistribute over promoter DNA. Here we report the identification of human TBP-binding regions in human TAF(II)170. We have defined the TBP interaction domain of TAF(II)170 within three amino-terminal regions: residues 2 to 137, 290 to 381, and 380 to 460. Each region contains a pair of Huntington-elongation-A subunit-Tor repeats and exhibits species-specific interactions with TBP family members. Remarkably, the altered-specificity TBP mutant (TBP(AS)) containing a triple mutation in the concave surface is defective for binding the TAF(II)170 amino-terminal region of residues 1 to 504. Furthermore, within this region the TAF(II)170 residues 290 to 381 can inhibit the interaction between Drosophila TAF(II)230 (residues 2 to 81) and TBP through competition for the concave surface of TBP. Biochemical analyses of TBP binding to the TATA box indicated that TAF(II)170 region 290-381 inhibits TBP-DNA complex formation. Importantly, the TBP(AS) mutant is less sensitive to TAF(II)170 inhibition. Collectively, our results support a mechanism in which TAF(II)170 induces high-mobility DNA binding by TBP through reversible interactions with its concave DNA binding surface.

Dual-mode fluorophore-doped nickel nitrilotriacetic acid-modified silica nanoparticles combine histidine-tagged protein purification with site-specific fluorophore labeling.

PubMed

Kim, Sung Hoon; Jeyakumar, M; Katzenellenbogen, John A

2007-10-31

We present the first example of a fluorophore-doped nickel chelate surface-modified silica nanoparticle that functions in a dual mode, combining histidine-tagged protein purification with site-specific fluorophore labeling. Tetramethylrhodamine (TMR)-doped silica nanoparticles, estimated to contain 700-900 TMRs per ca. 23 nm particle, were surface modified with nitrilotriacetic acid (NTA), producing TMR-SiO2-NTA-Ni2+. Silica-embedded TMR retains very high quantum yield, is resistant to quenching by buffer components, and is modestly quenched and only to a certain depth (ca. 2 nm) by surface-attached Ni2+. When exposed to a bacterial lysate containing estrogen receptor alpha ligand binding domain (ERalpha) as a minor component, these beads showed very high specificity binding, enabling protein purification in one step. The capacity and specificity of these beads for binding a his-tagged protein were characterized by electrophoresis, radiometric counting, and MALDI-TOF MS. ERalpha, bound to TMR-SiO2-NTA-Ni++ beads in a site-specific manner, exhibited good activity for ligand binding and for ligand-induced binding to coactivators in solution FRET experiments and protein microarray fluorometric and FRET assays. This dual-mode type TMR-SiO2-NTA-Ni2+ system represents a powerful combination of one-step histidine-tagged protein purification and site-specific labeling with multiple fluorophore species.

Pb-binding to various dissolved organic matter in urban aquatic systems: Key role of the most hydrophilic fraction

NASA Astrophysics Data System (ADS)

Pernet-Coudrier, Benoît; Companys, Encarnació; Galceran, Josep; Morey, Margalida; Mouchel, Jean-Marie; Puy, Jaume; Ruiz, Núria; Varrault, Gilles

2011-07-01

Dissolved organic matter (DOM) from the treated effluent of a wastewater treatment plant and from the river Seine under high human pressure has been separated into three fractions: hydrophobic (containing humic and fulvic substances), transphilic and hydrophilic using a two column array of XAD-8 and XAD-4 resins. The acid base properties and the binding characteristics with respect to Pb ions (using the new electroanalytical technique AGNES, Absence of Gradients and Nernstian Equilibrium Stripping) have been studied and fitted to NICA (Non-Ideal Competitive Isotherm). We evaluated the binding potential of each DOM fraction in order to better predict the speciation of Pb and, later, its bioavailability in the river. The total binding capacity of the different fractions to Pb, as well as the total titratable charge, reaches its maximum value at the most hydrophilic fraction from the treated effluent. Specific properties of the distribution of the complexing sites within each DOM fraction have been exposed by plotting the conditional affinity spectrum (CAS). The addition of these distributions, weighted according to the respective abundance of each organic fraction, allows for a full description of the Pb binding properties of the whole DOM of a sampling site. Despite its weak aromaticity, the hydrophilic fraction from the wastewater treatment plant effluent exhibits a high lead binding affinity, so that at typical environmental pH and free Pb levels (0.1 μg L -1), Pb is mainly bound to the most hydrophilic fraction of the treated effluent (49% of bound Pb at pH 7). This feature may greatly enhance the transport of Pb and highlights that Pb speciation should also consider other fractions apart from humic and/or fulvic acids when studying surface waters under high human pressure.

A novel astaxanthin-binding photooxidative stress-inducible aqueous carotenoprotein from a eukaryotic microalga isolated from asphalt in midsummer.

PubMed

Kawasaki, Shinji; Mizuguchi, Keisuke; Sato, Masaru; Kono, Tetsuya; Shimizu, Hirofumi

2013-07-01

Water-soluble orange carotenoid proteins (OCPs) that bind 3'-hydroxyechinenone are found in cyanobacteria, and are thought to play a key role in photoprotection. The distribution of OCPs in eukaryotes remains largely unknown. In this study, we identified a novel OCP that predominantly binds astaxanthin from a eukaryotic microalga, strain Ki-4, isolated from a dry surface of heated asphalt in midsummer. A purified astaxanthin-binding OCP, named AstaP, shows high solubility in water with an absorption peak at 484 nm, and possesses a heat-stable activity that quenches singlet oxygen. The deduced amino acid sequence of AstaP comprises an N-terminal hydrophobic signal peptide, fasciclin domains found in secreted and cell surface proteins, and N-linked glycosylation sites, the first example of a carotenoprotein among fasciclin family proteins. AstaP homologs of unknown function are distributed mainly in organisms from the hydrosphere, such as marine bacteria, cyanobacteria, sea anemone and eukaryotic microalgae; however, AstaP exhibits a unique extraordinarily high isoelectric point (pI) value among homologs. The gene encoding AstaP, as well as the AstaP peptide, is expressed abundantly under conditions of dehydration and salt stress in conjunction with high light exposure. As a unique aqueous carotenoprotein, AstaP will provide a novel function of OCPs in protection against extreme photooxidative stresses.

Fast Kinetics of Calcium Signaling and Sensor Design

PubMed Central

Tang, Shen; Reddish, Florence; Zhuo, You; Yang, Jenny J.

2015-01-01

Fast calcium signaling is regulated by numerous calcium channels exhibiting high spatiotemporal profiles which are currently measured by fluorescent calcium sensors. There is still a strong need to improve the kinetics of genetically encoded calcium indicators (sensors) to capture calcium dynamics in the millisecond time frame. In this review, we summarize several major fast calcium signaling pathways and discuss the recent developments and application of genetically encoded calcium indicators to detect these pathways. A new class of genetically encoded calcium indicators designed with site-directed mutagenesis on the surface of beta-barrel fluorescent proteins to form a pentagonal bipyramidal-like calcium binding domain dramatically accelerates calcium binding kinetics. Furthermore, novel genetically encoded calcium indicators with significantly increased fluorescent lifetime change are advantageous in deep-field imaging with high light-scattering and notable morphology change. PMID:26151819

A High Throughput Protein Microarray Approach to Classify HIV Monoclonal Antibodies and Variant Antigens

PubMed Central

Dotsey, Emmanuel Y.; Gorlani, Andrea; Ingale, Sampat; Achenbach, Chad J.; Forthal, Donald N.; Felgner, Philip L.; Gach, Johannes S.

2015-01-01

In recent years, high throughput discovery of human recombinant monoclonal antibodies (mAbs) has been applied to greatly advance our understanding of the specificity, and functional activity of antibodies against HIV. Thousands of antibodies have been generated and screened in functional neutralization assays, and antibodies associated with cross-strain neutralization and passive protection in primates, have been identified. To facilitate this type of discovery, a high throughput-screening tool is needed to accurately classify mAbs, and their antigen targets. In this study, we analyzed and evaluated a prototype microarray chip comprised of the HIV-1 recombinant proteins gp140, gp120, gp41, and several membrane proximal external region peptides. The protein microarray analysis of 11 HIV-1 envelope-specific mAbs revealed diverse binding affinities and specificities across clades. Half maximal effective concentrations, generated by our chip analysis, correlated significantly (P<0.0001) with concentrations from ELISA binding measurements. Polyclonal immune responses in plasma samples from HIV-1 infected subjects exhibited different binding patterns, and reactivity against printed proteins. Examining the totality of the specificity of the humoral response in this way reveals the exquisite diversity, and specificity of the humoral response to HIV. PMID:25938510

Proton and metal ion binding to natural organic polyelectrolytes-II. Preliminary investigation with a peat and a humic acid

USGS Publications Warehouse

Marinsky, J.A.; Reddy, M.M.

1984-01-01

We summarize here experimental studies of proton and metal ion binding to a peat and a humic acid. Data analysis is based on a unified physico-chemical model for reaction of simple ions with polyelectrolytes employing a modified Henderson-Hasselbalch equation. Peat exhibited an apparent intrinsic acid dissociation constant of 10-4.05, and an apparent intrinsic metal ion binding constant of: 400 for cadmium ion; 600 for zinc ion; 4000 for copper ion; 20000 for lead ion. A humic acid was found to have an apparent intrinsic proton binding constant of 10-2.6. Copper ion binding to this humic acid sample occurred at two types of sites. The first site exhibited reaction characteristics which were independent of solution pH and required the interaction of two ligands on the humic acid matrix to simultaneously complex with each copper ion. The second complex species is assumed to be a simple monodentate copper ion-carboxylate species with a stability constant of 18. ?? 1984.

Targeting Matrix Metalloproteinases in Cancer: Bringing New Life to Old Ideas.

PubMed

Cathcart, Jillian; Pulkoski-Gross, Ashleigh; Cao, Jian

2015-03-01

Since the identification of matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases, as being a driving factor for cancer progression and patient prognosis, MMPs have been studied extensively. Although early programs targeting MMPs were largely unsuccessful in clinical trials, they remain a viable and highly desirable therapeutic target based on preclinical studies and their role in disease progression. As information regarding the structure and function of these proteinases is compiled and biotechnology evolves, tools to develop better inhibitors is within our grasp. Improved methods for high throughput screening and in silico drug design programs have identified compounds which are highly potent, have high binding affinities, and exhibit favorable pharmacokinetic profiles. More recently, advances in drug delivery methods or compounds which bind outside the active site have brought new light to the field. In this review, we highlight the role of MMPs in cancer, clinical trials for MMP inhibitors, and novel approaches to targeting MMPs in cancer.

DOTA-derivatives of octreotide dicarba-analogues with high affinity for somatostatin sst2,5 receptors

NASA Astrophysics Data System (ADS)

Pratesi, Alessandro; Ginanneschi, Mauro; Lumini, Marco; Papini, Anna M.; Novellino, Ettore; Brancaccio, Diego; Carotenuto, Alfonso

2017-02-01

In vivo somatostatin receptor scintigraphy is a valuable method for the visualization of human endocrine tumours and their metastases. In fact, peptide ligands of somatostatin receptors (sst’s) conjugated with chelating agents are in clinical use. We have recently developed octreotide dicarba-analogues, which show interesting binding profiles at sst’s. In this context, it was mandatory to explore the possibility that our analogues could maintain their activity also upon conjugation with DOTA. In this paper, we report and discuss the synthesis, binding affinity and conformational preferences of three DOTA-conjugated dicarba-analogues of octreotide. Interestingly, two conjugated analogues exhibited nanomolar affinities on sst2 and sst5 somatostatin receptor subtypes.

Effect of Various Material Properties on the Adhesive Stage of Fretting

NASA Technical Reports Server (NTRS)

Buckley, D. H.

1974-01-01

Various properties of metals and alloys were studied with respect to their effect on the initial stage of the fretting process, namely adhesion. Crystallographic orientation, crystal structure, interfacial binding energies of dissimiliar metal, segregation of alloy constituents and the nature and structure of surface films were found to influence adhesion. High atomic density, low surface energy grain orientations exhibited lower adhesion than other orientations. Knowledge of interfacial surface binding energies assists in predicting adhesive transfer and wear. Selective surface segregation of alloy constituents accomplishes both a reduction in adhesion and improved surface oxidation characteristics. Equivalent surface coverages of various adsorbed species indicate that some are markedly more effective in inhibiting adhesion than others.

DOTA-Derivatives of Octreotide Dicarba-Analogs with High Affinity for Somatostatin sst2,5 Receptors.

PubMed

Pratesi, Alessandro; Ginanneschi, Mauro; Lumini, Marco; Papini, Anna M; Novellino, Ettore; Brancaccio, Diego; Carotenuto, Alfonso

2017-01-01

In vivo somatostatin receptor scintigraphy is a valuable method for the visualization of human endocrine tumors and their metastases. In fact, peptide ligands of somatostatin receptors (sst's) conjugated with chelating agents are in clinical use. We have recently developed octreotide dicarba-analogs, which show interesting binding profiles at sst's. In this context, it was mandatory to explore the possibility that our analogs could maintain their activity also upon conjugation with DOTA. In this paper, we report and discuss the synthesis, binding affinity and conformational preferences of three DOTA-conjugated dicarba-analogs of octreotide. Interestingly, two conjugated analogs exhibited nanomolar affinities on sst 2 and sst 5 somatostatin receptor subtypes.

A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis.

PubMed

Duggin, Iain G; Matthews, Jacqueline M; Dixon, Nicholas E; Wake, R Gerry; Mackay, Joel P

2005-04-01

Two dimers of the replication terminator protein (RTP) of Bacillus subtilis bind to a chromosomal DNA terminator site to effect polar replication fork arrest. Cooperative binding of the dimers to overlapping half-sites within the terminator is essential for arrest. It was suggested previously that polarity of fork arrest is the result of the RTP dimer at the blocking (proximal) side within the complex binding very tightly and the permissive-side RTP dimer binding relatively weakly. In order to investigate this "differential binding affinity" model, we have constructed a series of mutant terminators that contain half-sites of widely different RTP binding affinities in various combinations. Although there appeared to be a correlation between binding affinity at the proximal half-site and fork arrest efficiency in vivo for some terminators, several deviated significantly from this correlation. Some terminators exhibited greatly reduced binding cooperativity (and therefore have reduced affinity at each half-site) but were highly efficient in fork arrest, whereas one terminator had normal affinity over the proximal half-site, yet had low fork arrest efficiency. The results show clearly that there is no direct correlation between the RTP binding affinity (either within the full complex or at the proximal half-site within the full complex) and the efficiency of replication fork arrest in vivo. Thus, the differential binding affinity over the proximal and distal half-sites cannot be solely responsible for functional polarity of fork arrest. Furthermore, efficient fork arrest relies on features in addition to the tight binding of RTP to terminator DNA.

A calmodulin-binding/CGCG box DNA-binding protein family involved in multiple signaling pathways in plants

NASA Technical Reports Server (NTRS)

Yang, Tianbao; Poovaiah, B. W.

2002-01-01

We reported earlier that the tobacco early ethylene-responsive gene NtER1 encodes a calmodulin-binding protein (Yang, T., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 38467-38473). Here we demonstrate that there is one NtER1 homolog as well as five related genes in Arabidopsis. These six genes are rapidly and differentially induced by environmental signals such as temperature extremes, UVB, salt, and wounding; hormones such as ethylene and abscisic acid; and signal molecules such as methyl jasmonate, H(2)O(2), and salicylic acid. Hence, they were designated as AtSR1-6 (Arabidopsis thaliana signal-responsive genes). Ca(2+)/calmodulin binds to all AtSRs, and their calmodulin-binding regions are located on a conserved basic amphiphilic alpha-helical motif in the C terminus. AtSR1 targets the nucleus and specifically recognizes a novel 6-bp CGCG box (A/C/G)CGCG(G/T/C). The multiple CGCG cis-elements are found in promoters of genes such as those involved in ethylene signaling, abscisic acid signaling, and light signal perception. The DNA-binding domain in AtSR1 is located on the N-terminal 146 bp where all AtSR1-related proteins share high similarity but have no similarity to other known DNA-binding proteins. The calmodulin-binding nuclear proteins isolated from wounded leaves exhibit specific CGCG box DNA binding activities. These results suggest that the AtSR gene family encodes a family of calmodulin-binding/DNA-binding proteins involved in multiple signal transduction pathways in plants.

Agrobacterium uses a unique ligand-binding mode for trapping opines and acquiring a competitive advantage in the niche construction on plant host.

PubMed

Lang, Julien; Vigouroux, Armelle; Planamente, Sara; El Sahili, Abbas; Blin, Pauline; Aumont-Nicaise, Magali; Dessaux, Yves; Moréra, Solange; Faure, Denis

2014-10-01

By modifying the nuclear genome of its host, the plant pathogen Agrobacterium tumefaciens induces the development of plant tumours in which it proliferates. The transformed plant tissues accumulate uncommon low molecular weight compounds called opines that are growth substrates for A. tumefaciens. In the pathogen-induced niche (the plant tumour), a selective advantage conferred by opine assimilation has been hypothesized, but not experimentally demonstrated. Here, using genetics and structural biology, we deciphered how the pathogen is able to bind opines and use them to efficiently compete in the plant tumour. We report high resolution X-ray structures of the periplasmic binding protein (PBP) NocT unliganded and liganded with the opine nopaline (a condensation product of arginine and α-ketoglurate) and its lactam derivative pyronopaline. NocT exhibited an affinity for pyronopaline (K(D) of 0.6 µM) greater than that for nopaline (KD of 3.7 µM). Although the binding-mode of the arginine part of nopaline/pyronopaline in NocT resembled that of arginine in other PBPs, affinity measurement by two different techniques showed that NocT did not bind arginine. In contrast, NocT presented specific residues such as M117 to stabilize the bound opines. NocT relatives that exhibit the nopaline/pyronopaline-binding mode were only found in genomes of the genus Agrobacterium. Transcriptomics and reverse genetics revealed that A. tumefaciens uses the same pathway for assimilating nopaline and pyronopaline. Fitness measurements showed that NocT is required for a competitive colonization of the plant tumour by A. tumefaciens. Moreover, even though the Ti-plasmid conjugal transfer was not regulated by nopaline, the competitive advantage gained by the nopaline-assimilating Ti-plasmid donors led to a preferential horizontal propagation of this Ti-plasmid amongst the agrobacteria colonizing the plant-tumour niche. This work provided structural and genetic evidences to support the niche construction paradigm in bacterial pathogens.

/sup 32/P-postlabeling assay in mice of transplacental DNA damage induced by the environmental carcinogens safrole, 4-aminobiphenyl, and benzo(a)pyrene

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

Lu, L.J.; Disher, R.M.; Reddy, M.V.

1986-06-01

Transplacental exposure of fetuses to carcinogens is known to induce tumors in the offspring, often with a high incidence and short latency. While covalent adduction of DNA appears to be essential for tumor initiation, little is known about the binding of carcinogens to the DNA of fetal tissues. A sensitive /sup 32/P-postlabeling method enabled us to study the binding of the environmental carcinogens safrole (600 mumol/kg p.o.), 4-aminobiphenyl (800 mumol/kg), and benzo(a)pyrene (200 mumol/kg) to the DNA of various maternal and fetal tissues after administration of test carcinogens to pregnant ICR mice on day 18 of gestation. The results showmore » that these carcinogens bound to the DNA of maternal and fetal liver, lung, kidney, heart, brain, intestine, skin, maternal uterus, and placenta, with organ-specific quantitative and qualitative differences. It was possible for the first time to analyze DNA adduct patterns in minute amounts of tissue, for example those available from fetal heart. The covalent binding index 24 h after safrole treatment was estimated for the different organs and ranged from 0.1 to 247 and 0.1 to 5.8 for maternal and fetal DNA, respectively. Covalent binding index values of 0.2 to 13 and 0.1 to 0.3 for maternal and fetal DNA, respectively, were found for 4-aminobiphenyl. Benzo(a)pyrene treatment yielded covalent binding index values of 0.6 to 6.5 and 0.3 to 0.7 for maternal and fetal DNA, respectively. In both maternal and fetal tissues, safrole exhibited preferential binding to liver DNA. 4-Aminobiphenyl bound preferentially to DNA of maternal liver and kidney but showed no preference among fetal tissues. Benzo(a)pyrene exhibited weak tissue preference in both maternal and fetal organs.« less

Agrobacterium Uses a Unique Ligand-Binding Mode for Trapping Opines and Acquiring A Competitive Advantage in the Niche Construction on Plant Host

PubMed Central

Planamente, Sara; El Sahili, Abbas; Blin, Pauline; Aumont-Nicaise, Magali; Dessaux, Yves; Moréra, Solange; Faure, Denis

2014-01-01

By modifying the nuclear genome of its host, the plant pathogen Agrobacterium tumefaciens induces the development of plant tumours in which it proliferates. The transformed plant tissues accumulate uncommon low molecular weight compounds called opines that are growth substrates for A. tumefaciens. In the pathogen-induced niche (the plant tumour), a selective advantage conferred by opine assimilation has been hypothesized, but not experimentally demonstrated. Here, using genetics and structural biology, we deciphered how the pathogen is able to bind opines and use them to efficiently compete in the plant tumour. We report high resolution X-ray structures of the periplasmic binding protein (PBP) NocT unliganded and liganded with the opine nopaline (a condensation product of arginine and α-ketoglurate) and its lactam derivative pyronopaline. NocT exhibited an affinity for pyronopaline (KD of 0.6 µM) greater than that for nopaline (KD of 3.7 µM). Although the binding-mode of the arginine part of nopaline/pyronopaline in NocT resembled that of arginine in other PBPs, affinity measurement by two different techniques showed that NocT did not bind arginine. In contrast, NocT presented specific residues such as M117 to stabilize the bound opines. NocT relatives that exhibit the nopaline/pyronopaline-binding mode were only found in genomes of the genus Agrobacterium. Transcriptomics and reverse genetics revealed that A. tumefaciens uses the same pathway for assimilating nopaline and pyronopaline. Fitness measurements showed that NocT is required for a competitive colonization of the plant tumour by A. tumefaciens. Moreover, even though the Ti-plasmid conjugal transfer was not regulated by nopaline, the competitive advantage gained by the nopaline-assimilating Ti-plasmid donors led to a preferential horizontal propagation of this Ti-plasmid amongst the agrobacteria colonizing the plant-tumour niche. This work provided structural and genetic evidences to support the niche construction paradigm in bacterial pathogens. PMID:25299655

Identification, characterization and leucocyte expression of Siglec-10, a novel human sialic acid-binding receptor.

PubMed Central

Munday, J; Kerr, S; Ni, J; Cornish, A L; Zhang, J Q; Nicoll, G; Floyd, H; Mattei, M G; Moore, P; Liu, D; Crocker, P R

2001-01-01

Here we characterize Siglec-10 as a new member of the Siglec family of sialic acid-binding Ig-like lectins. A full-length cDNA was isolated from a human spleen library and the corresponding gene identified. Siglec-10 is predicted to contain five extracellular Ig-like domains and a cytoplasmic tail containing three putative tyrosine-based signalling motifs. Siglec-10 exhibited a high degree of sequence similarity to CD33-related Siglecs and mapped to the same region, on chromosome 19q13.3. The expressed protein was able to mediate sialic acid-dependent binding to human erythrocytes and soluble sialoglycoconjugates. Using specific antibodies, Siglec-10 was detected on subsets of human leucocytes including eosinophils, monocytes and a minor population of natural killer-like cells. The molecular properties and expression pattern suggest that Siglec-10 may function as an inhibitory receptor within the innate immune system. PMID:11284738

Strong positive cooperativity in binding to the A3T3 repeat by Hoechst 33258 derivatives attaching the quinoline units at the end of a branched linker.

PubMed

Koda, Hironori; Brazier, John Alan; Onishi, Ippei; Sasaki, Shigeki

2015-08-01

Hoechst 33258 derivatives with additional interacting moieties attached at the ends of branched linkers were synthesized, and their DNA binding properties were investigated with regard to the A3T3 repeat by measuring fluorescence spectra. The binding property of the ligand was investigated by fluorescence titration, and the titration data were analyzed using the McGhee-von Hippel method. Ligand 6Q with the quinolin-6-yloxyacetyl group and Ligand IQ with isoquinolin-6-yloxyacetyl group at the ends of the branched linkers exhibit highly positive cooperativity for the DNA having 5 A3T3 sites with 3 base-insertions between them with sequence selectivity. The strategy developed in this study may be generally applicable for designing ligands for repetitive DNA sequences. Copyright © 2015 Elsevier Ltd. All rights reserved.

Bacteria as living patchy colloids: Phenotypic heterogeneity in surface adhesion

PubMed Central

Hermes, Michiel; Schwarz-Linek, Jana; Poon, Wilson C. K.

2018-01-01

Understanding and controlling the surface adhesion of pathogenic bacteria is of urgent biomedical importance. However, many aspects of this process remain unclear (for example, microscopic details of the initial adhesion and possible variations between individual cells). Using a new high-throughput method, we identify and follow many single cells within a clonal population of Escherichia coli near a glass surface. We find strong phenotypic heterogeneities: A fraction of the cells remain in the free (planktonic) state, whereas others adhere with an adhesion strength that itself exhibits phenotypic heterogeneity. We explain our observations using a patchy colloid model; cells bind with localized, adhesive patches, and the strength of adhesion is determined by the number of patches: Nonadherers have no patches, weak adherers bind with a single patch only, and strong adherers bind via a single or multiple patches. We discuss possible implications of our results for controlling bacterial adhesion in biomedical and other applications. PMID:29719861

A label-free fluorescent aptamer sensor based on regulation of malachite green fluorescence

PubMed Central

Xu, Weichen; Lu, Yi

2009-01-01

We report a label-free fluorescent aptamer sensor for adenosine based on the regulation of malachite green (MG) fluorescence, with comparable sensitivity and selectivity to other labeled adenosine aptamer-based sensors. The sensor consists of free MG, an aptamer strand containing an adenosine aptamer next to an MG aptamer, and a bridging strand that partially hybridizes to the aptamer strand. Such a hybridization prevents MG from binding to MG aptamer, resulting in low fluorescence of MG in the absence of adenosine. Addition of adenosine causes the adenosine aptamer to bind adenosine, weakening the hybridization of the aptamer strand with the bridging strand, making it possible for MG to bind to the aptamer strand and exhibits high fluorescence intensity. Since this design is based purely on nucleic acid hybridization, it can be generally applied to other aptamers for the label-free detection of a broad range of analytes. PMID:20017558

IgGs are made for walking on bacterial and viral surfaces

NASA Astrophysics Data System (ADS)

Preiner, Johannes; Kodera, Noriyuki; Tang, Jilin; Ebner, Andreas; Brameshuber, Mario; Blaas, Dieter; Gelbmann, Nicola; Gruber, Hermann J.; Ando, Toshio; Hinterdorfer, Peter

2014-07-01

Binding of antibodies to their cognate antigens is fundamental for adaptive immunity. Molecular engineering of antibodies for therapeutic and diagnostic purposes emerges to be one of the major technologies in combating many human diseases. Despite its importance, a detailed description of the nanomechanical process of antibody-antigen binding and dissociation on the molecular level is lacking. Here we utilize high-speed atomic force microscopy to examine the dynamics of antibody recognition and uncover a principle; antibodies do not remain stationary on surfaces of regularly spaced epitopes; they rather exhibit ‘bipedal’ stochastic walking. As monovalent Fab fragments do not move, steric strain is identified as the origin of short-lived bivalent binding. Walking antibodies gather in transient clusters that might serve as docking sites for the complement system and/or phagocytes. Our findings could inspire the rational design of antibodies and multivalent receptors to exploit/inhibit steric strain-induced dynamic effects.

Structural and biochemical characterization of the inhibitor complexes of XMRV protease

PubMed Central

Li, Mi; Gustchina, Alla; Matúz, Krisztina; Tözsér, Jozsef; Namwong, Sirilak; Goldfarb, Nathan E.; Dunn, Ben M.; Wlodawer, Alexander

2012-01-01

Summary Interactions between the protease (PR) encoded by the xenotropic murine leukemia virus-related virus (XMRV) and a number of potential inhibitors have been investigated by biochemical and structural techniques. It was observed that several inhibitors used clinically against HIV PR exhibit nanomolar or even subnanomolar values of Ki, depending on exact experimental conditions. TL-3, a universal inhibitor of retroviral proteases, as well as some inhibitors originally shown to inhibit plasmepsins were also quite potent, whereas inhibition by pepstatin A was considerably weaker. Crystal structures of the complexes of XMRV PR with TL-3, amprenavir, and pepstatin A were solved at high resolution and compared to the structures of these inhibitors complexed with other retropepsins. Whereas TL-3 and amprenavir bind in a predictable manner spanning the substrate-binding site of the enzyme, two molecules of pepstatin A bind simultaneously in an unprecedented manner, leaving the catalytic water molecule in place. PMID:21951660

4-Aminoquinoline-pyrimidine hybrids: synthesis, antimalarial activity, heme binding and docking studies.

PubMed

Kumar, Deepak; Khan, Shabana I; Tekwani, Babu L; Ponnan, Prija; Rawat, Diwan S

2015-01-07

A series of novel 4-aminoquinoline-pyrimidine hybrids has been synthesized and evaluated for their antimalarial activity. Several compounds showed promising in vitro antimalarial activity against both CQ-sensitive and CQ-resistant strains with high selectivity index. All the compounds were found to be non-toxic to the mammalian cell lines. Selected compound 7g exhibited significant suppression of parasitemia in the in vivo assay. The heme binding studies were conducted to determine the mode of action of these hybrid molecules. These compounds form a stable 1:1 complex with hematin suggesting that heme may be one of the possible targets of these hybrids. The interaction of these conjugate hybrids was also investigated by the molecular docking studies in the binding site of PfDHFR. The pharmacokinetic property analysis of best active compounds was also studied using ADMET prediction. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Combined Virtual Screening and Substructure Search for Discovery of Novel FABP4 Inhibitors.

PubMed

Cai, Haiyan; Wang, Ting; Yang, Zhuo; Xu, Zhijian; Wang, Guimin; Wang, He-Yao; Zhu, Weiliang; Chen, Kaixian

2017-09-25

Fatty acid-binding protein 4 (FABP4, AFABP) is a potential drug target for diabetes and atherosclerosis. In this study, a series of novel FABP4 inhibitors were discovered through combining virtual screening and substructure search. Seventeen compounds exhibited FABP4 inhibitory activities with IC 50 < 10 μM, among which 11 compounds showed high selectivity against FABP3. The best compound 36b displayed an IC 50 value of 1.5 μM. Molecular docking and point mutation studies revealed that Gln95, Arg126, and Tyr128 play key roles for these compounds binding with FABP4. Interestingly, Gln95 seems to be essential for conformation stability of FABP4. The new scaffolds of these compounds and their interaction mechanisms binding with FABP4 should provide an important clue for the further development of novel FABP4 inhibitors.

Synergistic activation of human pregnane X receptor by binary cocktails of pharmaceutical and environmental compounds.

PubMed

Delfosse, Vanessa; Dendele, Béatrice; Huet, Tiphaine; Grimaldi, Marina; Boulahtouf, Abdelhay; Gerbal-Chaloin, Sabine; Beucher, Bertrand; Roecklin, Dominique; Muller, Christina; Rahmani, Roger; Cavaillès, Vincent; Daujat-Chavanieu, Martine; Vivat, Valérie; Pascussi, Jean-Marc; Balaguer, Patrick; Bourguet, William

2015-09-03

Humans are chronically exposed to multiple exogenous substances, including environmental pollutants, drugs and dietary components. Many of these compounds are suspected to impact human health, and their combination in complex mixtures could exacerbate their harmful effects. Here we demonstrate that a pharmaceutical oestrogen and a persistent organochlorine pesticide, both exhibiting low efficacy when studied separately, cooperatively bind to the pregnane X receptor, leading to synergistic activation. Biophysical analysis shows that each ligand enhances the binding affinity of the other, so the binary mixture induces a substantial biological response at doses at which each chemical individually is inactive. High-resolution crystal structures reveal the structural basis for the observed cooperativity. Our results suggest that the formation of 'supramolecular ligands' within the ligand-binding pocket of nuclear receptors contributes to the synergistic toxic effect of chemical mixtures, which may have broad implications for the fields of endocrine disruption, toxicology and chemical risk assessment.

Changes in conformational dynamics of basic side chains upon protein–DNA association

PubMed Central

Esadze, Alexandre; Chen, Chuanying; Zandarashvili, Levani; Roy, Sourav; Pettitt, B. Montgometry; Iwahara, Junji

2016-01-01

Basic side chains play major roles in recognition of nucleic acids by proteins. However, dynamic properties of these positively charged side chains are not well understood. In this work, we studied changes in conformational dynamics of basic side chains upon protein–DNA association for the zinc-finger protein Egr-1. By nuclear magnetic resonance (NMR) spectroscopy, we characterized the dynamics of all side-chain cationic groups in the free protein and in the complex with target DNA. Our NMR order parameters indicate that the arginine guanidino groups interacting with DNA bases are strongly immobilized, forming rigid interfaces. Despite the strong short-range electrostatic interactions, the majority of the basic side chains interacting with the DNA phosphates exhibited high mobility, forming dynamic interfaces. In particular, the lysine side-chain amino groups exhibited only small changes in the order parameters upon DNA-binding. We found a similar trend in the molecular dynamics (MD) simulations for the free Egr-1 and the Egr-1–DNA complex. Using the MD trajectories, we also analyzed side-chain conformational entropy. The interfacial arginine side chains exhibited substantial entropic loss upon binding to DNA, whereas the interfacial lysine side chains showed relatively small changes in conformational entropy. These data illustrate different dynamic characteristics of the interfacial arginine and lysine side chains. PMID:27288446

How actin binds and assembles onto plasma membranes from Dictyostelium discoideum

PubMed Central

1988-01-01

We have shown previously (Schwartz, M. A., and E. J. Luna. 1986. J. Cell Biol. 102: 2067-2075) that actin binds with positive cooperativity to plasma membranes from Dictyostelium discoideum. Actin is polymerized at the membrane surface even at concentrations well below the critical concentration for polymerization in solution. Low salt buffer that blocks actin polymerization in solution also prevents actin binding to membranes. To further explore the relationship between actin polymerization and binding to membranes, we prepared four chemically modified actins that appear to be incapable of polymerizing in solution. Three of these derivatives also lost their ability to bind to membranes. The fourth derivative (EF actin), in which histidine-40 is labeled with ethoxyformic anhydride, binds to membranes with reduced affinity. Binding curves exhibit positive cooperativity, and cross- linking experiments show that membrane-bound actin is multimeric. Thus, binding and polymerization are tightly coupled, and the ability of these membranes to polymerize actin is dramatically demonstrated. EF actin coassembles weakly with untreated actin in solution, but coassembles well on membranes. Binding by untreated actin and EF actin are mutually competitive, indicating that they bind to the same membrane sites. Hill plots indicate that an actin trimer is the minimum assembly state required for tight binding to membranes. The best explanation for our data is a model in which actin oligomers assemble by binding to clustered membrane sites with successive monomers on one side of the actin filament bound to the membrane. Individual binding affinities are expected to be low, but the overall actin-membrane avidity is high, due to multivalency. Our results imply that extracellular factors that cluster membrane proteins may create sites for the formation of actin nuclei and thus trigger actin polymerization in the cell. PMID:3392099

Development, synthesis and structure-activity-relationships of inhibitors of the macrophage infectivity potentiator (Mip) proteins of Legionella pneumophila and Burkholderia pseudomallei.

PubMed

Seufert, Florian; Kuhn, Maximilian; Hein, Michael; Weiwad, Matthias; Vivoli, Mirella; Norville, Isobel H; Sarkar-Tyson, Mitali; Marshall, Laura E; Schweimer, Kristian; Bruhn, Heike; Rösch, Paul; Harmer, Nicholas J; Sotriffer, Christoph A; Holzgrabe, Ulrike

2016-11-01

The bacteria Burkholderia pseudomallei and Legionella pneumophila cause severe diseases like melioidosis and Legionnaire's disease with high mortality rates despite antibiotic treatment. Due to increasing antibiotic resistances against these and other Gram-negative bacteria, alternative therapeutical strategies are in urgent demand. As a virulence factor, the macrophage infectivity potentiator (Mip) protein constitutes an attractive target. The Mip proteins of B. pseudomallei and L. pneumophila exhibit peptidyl-prolyl cis/trans isomerase (PPIase) activity and belong to the PPIase superfamily. In previous studies, the pipecolic acid moiety proved to be a valuable scaffold for inhibiting this PPIase activity. Thus, a library of pipecolic acid derivatives was established guided by structural information and computational analyses of the binding site and possible binding modes. Stability and toxicity considerations were taken into account in iterative extensions of the library. Synthesis and evaluation of the compounds in PPIase assays resulted in highly active inhibitors. The activities can be interpreted in terms of a common binding mode obtained by docking calculations. Copyright © 2016 Elsevier Ltd. All rights reserved.

Multiple Targets of Salicylic Acid and Its Derivatives in Plants and Animals

PubMed Central

Klessig, Daniel F.; Tian, Miaoying; Choi, Hyong Woo

2016-01-01

Salicylic acid (SA) is a critical plant hormone that is involved in many processes, including seed germination, root initiation, stomatal closure, floral induction, thermogenesis, and response to abiotic and biotic stresses. Its central role in plant immunity, although extensively studied, is still only partially understood. Classical biochemical approaches and, more recently, genome-wide high-throughput screens have identified more than two dozen plant SA-binding proteins (SABPs), as well as multiple candidates that have yet to be characterized. Some of these proteins bind SA with high affinity, while the affinity of others exhibit is low. Given that SA levels vary greatly even within a particular plant species depending on subcellular location, tissue type, developmental stage, and with respect to both time and location after an environmental stimulus such as infection, the presence of SABPs exhibiting a wide range of affinities for SA may provide great flexibility and multiple mechanisms through which SA can act. SA and its derivatives, both natural and synthetic, also have multiple targets in animals/humans. Interestingly, many of these proteins, like their plant counterparts, are associated with immunity or disease development. Two recently identified SABPs, high mobility group box protein and glyceraldehyde 3-phosphate dehydrogenase, are critical proteins that not only serve key structural or metabolic functions but also play prominent roles in disease responses in both kingdoms. PMID:27303403

Tumor targeting profiling of hyaluronan-coated lipid based-nanoparticles

NASA Astrophysics Data System (ADS)

Mizrahy, Shoshy; Goldsmith, Meir; Leviatan-Ben-Arye, Shani; Kisin-Finfer, Einat; Redy, Orit; Srinivasan, Srimeenakshi; Shabat, Doron; Godin, Biana; Peer, Dan

2014-03-01

Hyaluronan (HA), a naturally occurring high Mw (HMw) glycosaminoglycan, has been shown to play crucial roles in cell growth, embryonic development, healing processes, inflammation, and tumor development and progression. Low Mw (LMw, <10 kDa) HA has been reported to provoke inflammatory responses, such as induction of cytokines, chemokines, reactive nitrogen species and growth factors. Herein, we prepared and characterized two types of HA coated (LMw and HMw) lipid-based targeted and stabilized nanoparticles (tsNPs) and tested their binding to tumor cells expressing the HA receptor (CD44), systemic immunotoxicity, and biodistribution in tumor bearing mice. In vitro, the Mw of the surface anchored HA had a significant influence on the affinity towards CD44 on B16F10 murine melanoma cells. LMw HA-tsNPs exhibited weak binding, while binding of tsNPs coated with HMw HA was characterized by high binding. Both types of tsNPs had no measured effect on cytokine induction in vivo following intravenous administration to healthy C57BL/6 mice suggesting no immune activation. HMw HA-tsNPs showed enhanced circulation time and tumor targeting specificity, mainly by accumulating in the tumor and its vicinity compared with LMw HA-tsNPs. Finally, we show that methotrexate (MTX), a drug commonly used in cancer chemotherapy, entrapped in HMw HA-tsNPs slowly diffused from the particles with a half-life of 13.75 days, and improved the therapeutic outcome in a murine B16F10 melanoma model compared with NPs suggesting an active cellular targeting beyond the Enhanced Permeability and Retention (EPR) effect. Taken together, these findings have major implications for the use of high molecular weight HA in nanomedicine as a selective and safe active cellular targeting moiety.Hyaluronan (HA), a naturally occurring high Mw (HMw) glycosaminoglycan, has been shown to play crucial roles in cell growth, embryonic development, healing processes, inflammation, and tumor development and progression. Low Mw (LMw, <10 kDa) HA has been reported to provoke inflammatory responses, such as induction of cytokines, chemokines, reactive nitrogen species and growth factors. Herein, we prepared and characterized two types of HA coated (LMw and HMw) lipid-based targeted and stabilized nanoparticles (tsNPs) and tested their binding to tumor cells expressing the HA receptor (CD44), systemic immunotoxicity, and biodistribution in tumor bearing mice. In vitro, the Mw of the surface anchored HA had a significant influence on the affinity towards CD44 on B16F10 murine melanoma cells. LMw HA-tsNPs exhibited weak binding, while binding of tsNPs coated with HMw HA was characterized by high binding. Both types of tsNPs had no measured effect on cytokine induction in vivo following intravenous administration to healthy C57BL/6 mice suggesting no immune activation. HMw HA-tsNPs showed enhanced circulation time and tumor targeting specificity, mainly by accumulating in the tumor and its vicinity compared with LMw HA-tsNPs. Finally, we show that methotrexate (MTX), a drug commonly used in cancer chemotherapy, entrapped in HMw HA-tsNPs slowly diffused from the particles with a half-life of 13.75 days, and improved the therapeutic outcome in a murine B16F10 melanoma model compared with NPs suggesting an active cellular targeting beyond the Enhanced Permeability and Retention (EPR) effect. Taken together, these findings have major implications for the use of high molecular weight HA in nanomedicine as a selective and safe active cellular targeting moiety. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr06102g

Synthesis and evaluation of ¹²³/¹³¹I-Iochlonicotinamide as a novel SPECT probe for malignant melanoma.

PubMed

Chang, Chih-Chao; Chang, Chih-Hsien; Shen, Chih-Chieh; Chen, Chuan-Lin; Liu, Ren-Shyan; Lin, Ming-Hsien; Wang, Hsin-Ell

2015-05-01

Malignant melanoma expresses a highly aggressive metastasis. Early diagnosis of malignant melanoma is important for patient survival. Radiolabeled benzamides and nicotinamides have been reported to be attractive candidates for malignant melanoma diagnosis as they bind to melanin, a characteristic substance that displays in malignant melanoma, and show high tumor accumulation and retention. Herein, we designed and synthesized a novel (123/131)I-labeled nicotinamide derivative that specifically binds to melanin. (123/131)I-Iochlonicotinamide was prepared with good radiochemical yield (50-70%, decay corrected) and high specific radioactivity (50-80 GBq/μmol). (131)I-Iochlonicotinamide exhibited good in vitro stability (radiochemical purity >95% after a 24-h incubation) in human serum. High uptake of (123/131)I-Iochlonicotinamide in B16F0 melanoma cells compared to that in A375 amelanotic cells demonstrated its selective binding to melanin. Intravenous administration of (123/131)I-Iochlonicotinamide in a melanoma-bearing mouse model revealed high uptake in melanotic melanoma and high tumor-to-muscle ratio. MicroSPECT scan of (123/131)I-Iochlonicotinamide injected mice also displayed high contrast tumor imaging as compared with normal organs. The radiation-absorbed dose projection for the administration of (131)I-Iochlonicotinamide to human was based on the results of biodistribution study. The effective dose appears to be approximately 0.44 mSv/MBq(-1). The specific binding of (123/131)I-Iochlonicotinamide to melanin along with a prolonged tumor retention and acceptable projected human dosimetry suggest that it may be a promising theranostic agent for treating malignant melanoma. Copyright © 2015 Elsevier Ltd. All rights reserved.

Targeting diverse protein–protein interaction interfaces with α/β-peptides derived from the Z-domain scaffold

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

Checco, James W.; Kreitler, Dale F.; Thomas, Nicole C.

Peptide-based agents derived from well-defined scaffolds offer an alternative to antibodies for selective and high-affinity recognition of large and topologically complex protein surfaces. In this paper, we describe a strategy for designing oligomers containing both α- and β-amino acid residues (“α/β-peptides”) that mimic several peptides derived from the three-helix bundle “Z-domain” scaffold. We show that α/β-peptides derived from a Z-domain peptide targeting vascular endothelial growth factor (VEGF) can structurally and functionally mimic the binding surface of the parent peptide while exhibiting significantly decreased susceptibility to proteolysis. The tightest VEGF-binding α/β-peptide inhibits the VEGF 165-induced proliferation of human umbilical vein endothelialmore » cells. We demonstrate the versatility of this strategy by showing how principles underlying VEGF signaling inhibitors can be rapidly extended to produce Z-domain–mimetic α/β-peptides that bind to two other protein partners, IgG and tumor necrosis factor-α. Because well-established selection techniques can identify high-affinity Z-domain derivatives from large DNA-encoded libraries, our findings should enable the design of biostable α/β-peptides that bind tightly and specifically to diverse targets of biomedical interest. Finally, such reagents would be useful for diagnostic and therapeutic applications.« less

A New Family of Nuclear Receptor Coregulators That Integrate Nuclear Receptor Signaling through CREB-Binding Protein

PubMed Central

Mahajan, Muktar A.; Samuels, Herbert H.

2000-01-01

We describe the cloning and characterization of a new family of nuclear receptor coregulators (NRCs) which modulate the function of nuclear hormone receptors in a ligand-dependent manner. NRCs are expressed as alternatively spliced isoforms which may exhibit different intrinsic activities and receptor specificities. The NRCs are organized into several modular structures and contain a single functional LXXLL motif which associates with members of the steroid hormone and thyroid hormone/retinoid receptor subfamilies with high affinity. Human NRC (hNRC) harbors a potent N-terminal activation domain (AD1), which is as active as the herpesvirus VP16 activation domain, and a second activation domain (AD2) which overlaps with the receptor-interacting LXXLL region. The C-terminal region of hNRC appears to function as an inhibitory domain which influences the overall transcriptional activity of the protein. Our results suggest that NRC binds to liganded receptors as a dimer and this association leads to a structural change in NRC resulting in activation. hNRC binds CREB-binding protein (CBP) with high affinity in vivo, suggesting that hNRC may be an important functional component of a CBP complex involved in mediating the transcriptional effects of nuclear hormone receptors. PMID:10866662

Improved purification of immunoglobulin G from plasma by mixed-mode chromatography.

PubMed

Chai, Dong-Sheng; Sun, Yan; Wang, Xiao-Ning; Shi, Qing-Hong

2014-12-01

Efficient loading of immunoglobulin G in mixed-mode chromatography is often a serious bottleneck in the chromatographic purification of immunoglobulin G. In this work, a mixed-mode ligand, 4-(1H-imidazol-1-yl) aniline, was coupled to Sepharose Fast Flow to fabricate AN SepFF adsorbents with ligand densities of 15-64 mmol/L, and the chromatographic performances of these adsorbents were thoroughly investigated to identify a feasible approach to improve immunoglobulin G purification. The results indicate that a critical ligand density exists for immunoglobulin G on the AN SepFF adsorbents. Above the critical ligand density, the adsorbents showed superior selectivity to immunoglobulin G at high salt concentrations, and also exhibited much higher dynamic binding capacities. For immunoglobulin G purification, both the yield and binding capacity increased with adsorbent ligand density along with a decrease in purity. It is difficult to improve the binding capacity, purity, and yield of immunoglobulin G simultaneously in AN SepFF chromatography. By using tandem AN SepFF chromatography, a threefold increase in binding capacity as well as high purity and yield of immunoglobulin G were achieved. Therefore, the tandem chromatography demonstrates that AN SepFF adsorbent is a practical and feasible alternative to MEP HyperCel adsorbents for immunoglobulin G purification. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Targeting diverse protein–protein interaction interfaces with α/β-peptides derived from the Z-domain scaffold

DOE PAGES

Checco, James W.; Kreitler, Dale F.; Thomas, Nicole C.; ...

2015-03-30

Peptide-based agents derived from well-defined scaffolds offer an alternative to antibodies for selective and high-affinity recognition of large and topologically complex protein surfaces. In this paper, we describe a strategy for designing oligomers containing both α- and β-amino acid residues (“α/β-peptides”) that mimic several peptides derived from the three-helix bundle “Z-domain” scaffold. We show that α/β-peptides derived from a Z-domain peptide targeting vascular endothelial growth factor (VEGF) can structurally and functionally mimic the binding surface of the parent peptide while exhibiting significantly decreased susceptibility to proteolysis. The tightest VEGF-binding α/β-peptide inhibits the VEGF 165-induced proliferation of human umbilical vein endothelialmore » cells. We demonstrate the versatility of this strategy by showing how principles underlying VEGF signaling inhibitors can be rapidly extended to produce Z-domain–mimetic α/β-peptides that bind to two other protein partners, IgG and tumor necrosis factor-α. Because well-established selection techniques can identify high-affinity Z-domain derivatives from large DNA-encoded libraries, our findings should enable the design of biostable α/β-peptides that bind tightly and specifically to diverse targets of biomedical interest. Finally, such reagents would be useful for diagnostic and therapeutic applications.« less

Conformational selection in protein binding and function

PubMed Central

Weikl, Thomas R; Paul, Fabian

2014-01-01

Protein binding and function often involves conformational changes. Advanced nuclear magnetic resonance (NMR) experiments indicate that these conformational changes can occur in the absence of ligand molecules (or with bound ligands), and that the ligands may “select” protein conformations for binding (or unbinding). In this review, we argue that this conformational selection requires transition times for ligand binding and unbinding that are small compared to the dwell times of proteins in different conformations, which is plausible for small ligand molecules. Such a separation of timescales leads to a decoupling and temporal ordering of binding/unbinding events and conformational changes. We propose that conformational-selection and induced-change processes (such as induced fit) are two sides of the same coin, because the temporal ordering is reversed in binding and unbinding direction. Conformational-selection processes can be characterized by a conformational excitation that occurs prior to a binding or unbinding event, while induced-change processes exhibit a characteristic conformational relaxation that occurs after a binding or unbinding event. We discuss how the ordering of events can be determined from relaxation rates and effective on- and off-rates determined in mixing experiments, and from the conformational exchange rates measured in advanced NMR or single-molecule fluorescence resonance energy transfer experiments. For larger ligand molecules such as peptides, conformational changes and binding events can be intricately coupled and exhibit aspects of conformational-selection and induced-change processes in both binding and unbinding direction. PMID:25155241

Fibrous hybrid of graphene and sulfur nanocrystals for high-performance lithium-sulfur batteries.

PubMed

Zhou, Guangmin; Yin, Li-Chang; Wang, Da-Wei; Li, Lu; Pei, Songfeng; Gentle, Ian Ross; Li, Feng; Cheng, Hui-Ming

2013-06-25

Graphene-sulfur (G-S) hybrid materials with sulfur nanocrystals anchored on interconnected fibrous graphene are obtained by a facile one-pot strategy using a sulfur/carbon disulfide/alcohol mixed solution. The reduction of graphene oxide and the formation/binding of sulfur nanocrystals were integrated. The G-S hybrids exhibit a highly porous network structure constructed by fibrous graphene, many electrically conducting pathways, and easily tunable sulfur content, which can be cut and pressed into pellets to be directly used as lithium-sulfur battery cathodes without using a metal current-collector, binder, and conductive additive. The porous network and sulfur nanocrystals enable rapid ion transport and short Li(+) diffusion distance, the interconnected fibrous graphene provides highly conductive electron transport pathways, and the oxygen-containing (mainly hydroxyl/epoxide) groups show strong binding with polysulfides, preventing their dissolution into the electrolyte based on first-principles calculations. As a result, the G-S hybrids show a high capacity, an excellent high-rate performance, and a long life over 100 cycles. These results demonstrate the great potential of this unique hybrid structure as cathodes for high-performance lithium-sulfur batteries.

Formation of crystalline nanoparticles by iron binding to pentapeptide (Asp-His-Thr-Lys-Glu) from egg white hydrolysates.

PubMed

Sun, Na; Cui, Pengbo; Li, Dongmei; Jin, Ziqi; Zhang, Shuyu; Lin, Songyi

2017-09-20

A novel peptide from egg white, Asp-His-Thr-Lys-Glu (DHTKE), contains specific amino acids associated with iron binding. The present study aims to better understand the molecular basis of interactions between the DHTKE peptide and iron ions. The ultraviolet-visible and fluorescence spectra indicate an interaction between the DHTKE peptide and iron ions, which leads to the formation of a DHTKE-iron complex. Notably, Asp, Glu, His, and Lys in the DHTKE peptide play crucial roles in the formation of the DHTKE-iron complex, and the iron-binding site of the DHTKE peptide corresponds primarily to the amide and carboxyl groups. The DHTKE peptide can bind iron ions in a 1 : 2 ratio with a binding constant of 1.312 × 10 5 M -1 . Moreover, the DHTKE-iron complex belongs to thermodynamically stable nanoparticles that are present in the crystalline structure, which might be attributed to peptide folding induced by iron binding. Meanwhile, the DHTKE-iron complex exhibits a relatively high iron-releasing percentage and exerts excellent solubility in the human gastrointestinal tract in vitro. This suggests a potential application of peptides containing Asp, Glu, His, or Lys residues as potential iron supplements.

Psychotomimetic opiate receptors labeled and visualized with (+)-(/sup 3/H)3-(3-hydroxyphenyl)-N-(1-propyl)piperidine

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

Largent, B.L.; Gundlach, A.L.; Snyder, S.H.

1984-08-01

3-(3-Hydroxyphenyl)-N-(1-propyl)piperidine (3-PPP) has been proposed as a selective dopamine autoreceptor agonist in the central nervous system. This report describes the pharmacology and localization of specific high-affinity binding sites for (+)-(/sup 3/H)3-PPP in brain. The drug specificity of (+)-(/sup 3/H)3-PPP binding is identical to that of sigma receptors, which may mediate psychotomimetic effects of some opiates. Haloperidol and the opioid derivatives, pentazocine, cyclazocine, and SKF 10,047 are potent inhibitors of (+)-(/sup 3/H)3-PPP binding. Stereoselectivity is exhibited for the (+) isomers of cyclazocine and SKF 10.047 at the sigma site, opposite to the stereoselectivity seen at ..mu.., sigma, and k opiate receptors.more » (+)-(/sup 3/H)3-PPP does not label dopamine receptors, as potent dopamine agonists and antagonists are weak inhibitors of binding and the localization of specific (+)-(/sup 3/H)3-PPP binding sites does not parallel that of dopamine neurons. Discrete localizations of (+)-(/sup 3/H)3-PPP binding sites in many brain areas including limbic, midbrain, brainstem, and cerebellar regions may explain psychotomimetic actions of opiates and behavior effects of 3-PPP. 41 references, 2 figures, 1 table.« less

Magnetic, core-shell structured and surface molecularly imprinted polymers for the rapid and selective recognition of salicylic acid from aqueous solutions

NASA Astrophysics Data System (ADS)

Zhang, Zulei; Niu, Dechao; Li, Yongsheng; Shi, Jianlin

2018-03-01

In this work, a novel kind of magnetic, core-shell structured and surface molecularly imprinted polymers (MMIPs) for the recognition of salicylic acid (SA) was facilely synthesized through a surface imprinting and sol-gel polymerization approach. The as-synthesized MMIPs exhibit uniform core-shell structure and favorable magnetic properties with a saturation magnetization of 22.8 emu g-1. The binding experiments demonstrated that MMIPs possessed high binding and specific recognition capacity, as well as fast binding kinetics and phase separation rate. The maximum binding capacity of MMIPs is around 36.8 mg g-1, nearly 6 times that of the magnetic non-imprinted polymers (MNIPs). Moreover, the selectivity experiments show that all the relative selectivity coefficients towards SA over its structure analogs are higher than 18, further indicating the markedly enhanced binding selectivity of MMIPs. Furthermore, the MMIPs were successfully applied for the determination of SA in environmental water samples with the recovery rates ranging from 94.0 to 108.0 %. This strategy may provide a versatile approach for the fabrication of well-defined molecularly imprinted polymers on nanomaterials for the analysis of complicated matrixes.

Identification of Lectins from Metastatic Cancer Cells through Magnetic Glyconanoparticles

PubMed Central

Kavunja, Herbert W.; Voss, Patricia G.

2016-01-01

Cancer cells can have characteristic carbohydrate binding properties. Previously, it was shown that a highly metastatic melanoma cell line B16F10 bound to galacto-side-functionalized nanoparticles much stronger than the corresponding less metastatic B16F1 cells. To better understand the carbohydrate binding properties of cancer cells, herein, we report the isolation and characterization of endogenous galactose binding proteins from B16F10 cells using magnetic glyconanoparticles. The galactose-coated magnetic glyconanoparticles could bind with lectins present in the cells and be isolated through magnet-mediated separation. Through Western blot and mass spectrometry, the arginine/serine rich splicing factor Sfrs1 was identified as a galactose-selective endogenous lectin, overexpressed in B16F10 cells, compared with B16F1 cells. In addition, galactin-3 was found in higher amounts in B16F10 cells. Finally, the glyconanoparticles exhibited a superior efficiency in lectin isolation, from both protein mixtures and live cells, than the corresponding more traditional microparticles functionalized with carbohydrates. Thus, the magnetic glyconanoparticles present a useful tool for discovery of endogenous lectins, as well as binding partners of lectins, without prior knowledge of protein identities. PMID:27110035

High-affinity, noninhibitory pathogenic C1 domain antibodies are present in patients with hemophilia A and inhibitors

PubMed Central

Batsuli, Glaivy; Deng, Wei; Healey, John F.; Parker, Ernest T.; Baldwin, W. Hunter; Cox, Courtney; Nguyen, Brenda; Kahle, Joerg; Königs, Christoph; Li, Renhao; Lollar, Pete

2016-01-01

Inhibitor formation in hemophilia A is the most feared treatment-related complication of factor VIII (fVIII) therapy. Most inhibitor patients with hemophilia A develop antibodies against the fVIII A2 and C2 domains. Recent evidence demonstrates that the C1 domain contributes to the inhibitor response. Inhibitory anti-C1 monoclonal antibodies (mAbs) have been identified that bind to putative phospholipid and von Willebrand factor (VWF) binding epitopes and block endocytosis of fVIII by antigen presenting cells. We now demonstrate by competitive enzyme-linked immunosorbent assay and hydrogen-deuterium exchange mass spectrometry that 7 of 9 anti-human C1 mAbs tested recognize an epitope distinct from the C1 phospholipid binding site. These mAbs, designated group A, display high binding affinities for fVIII, weakly inhibit fVIII procoagulant activity, poorly inhibit fVIII binding to phospholipid, and exhibit heterogeneity with respect to blocking fVIII binding to VWF. Another mAb, designated group B, inhibits fVIII procoagulant activity, fVIII binding to VWF and phospholipid, fVIIIa incorporation into the intrinsic Xase complex, thrombin generation in plasma, and fVIII uptake by dendritic cells. Group A and B epitopes are distinct from the epitope recognized by the canonical, human-derived inhibitory anti-C1 mAb, KM33, whose epitope overlaps both groups A and B. Antibodies recognizing group A and B epitopes are present in inhibitor plasmas from patients with hemophilia A. Additionally, group A and B mAbs increase fVIII clearance and are pathogenic in a hemophilia A mouse tail snip bleeding model. Group A anti-C1 mAbs represent the first identification of pathogenic, weakly inhibitory antibodies that increase fVIII clearance. PMID:27381905

Interaction of antithrombin with sulfated, low molecular weight lignins: opportunities for potent, selective modulation of antithrombin function.

PubMed

Henry, Brian L; Connell, Justin; Liang, Aiye; Krishnasamy, Chandravel; Desai, Umesh R

2009-07-31

Antithrombin, a major regulator of coagulation and angiogenesis, is known to interact with several natural sulfated polysaccharides. Previously, we prepared sulfated low molecular weight variants of natural lignins, called sulfated dehydrogenation polymers (DHPs) (Henry, B. L., Monien, B. H., Bock, P. E., and Desai, U. R. (2007) J. Biol. Chem. 282, 31891-31899), which have now been found to exhibit interesting antithrombin binding properties. Sulfated DHPs represent a library of diverse noncarbohydrate aromatic scaffolds that possess structures completely different from heparin and heparan sulfate. Fluorescence binding studies indicate that sulfated DHPs bind to antithrombin with micromolar affinity under physiological conditions. Salt dependence of binding affinity indicates that the antithrombin-sulfated DHP interaction involves a massive 80-87% non-ionic component to the free energy of binding. Competitive binding studies with heparin pentasaccharide, epicatechin sulfate, and full-length heparin indicate that sulfated DHPs bind to both the pentasaccharide-binding site and extended heparin-binding site of antithrombin. Affinity capillary electrophoresis resolves a limited number of peaks of antithrombin co-complexes suggesting preferential binding of selected DHP structures to the serpin. Computational genetic algorithm-based virtual screening study shows that only one sulfated DHP structure, out of the 11 present in a library of plausible sequences, bound in the heparin-binding site with a high calculated score supporting selectivity of recognition. Enzyme inhibition studies indicate that only one of the three sulfated DHPs studied is a potent inhibitor of free factor VIIa in the presence of antithrombin. Overall, the chemo-enzymatic origin and antithrombin binding properties of sulfated DHPs present novel opportunities for potent and selective modulation of the serpin function, especially for inhibiting the initiation phase of hemostasis.

Direct observation of a single nanoparticle-ubiquitin corona formation

NASA Astrophysics Data System (ADS)

Ding, Feng; Radic, Slaven; Chen, Ran; Chen, Pengyu; Geitner, Nicholas K.; Brown, Jared M.; Ke, Pu Chun

2013-09-01

The advancement of nanomedicine and the increasing applications of nanoparticles in consumer products have led to administered biological exposure and unintentional environmental accumulation of nanoparticles, causing concerns over the biocompatibility and sustainability of nanotechnology. Upon entering physiological environments, nanoparticles readily assume the form of a nanoparticle-protein corona that dictates their biological identity. Consequently, understanding the structure and dynamics of a nanoparticle-protein corona is essential for predicting the fate, transport, and toxicity of nanomaterials in living systems and for enabling the vast applications of nanomedicine. Here we combined multiscale molecular dynamics simulations and complementary experiments to characterize the silver nanoparticle-ubiquitin corona formation. Notably, ubiquitins competed with citrates for the nanoparticle surface, governed by specific electrostatic interactions. Under a high protein/nanoparticle stoichiometry, ubiquitins formed a multi-layer corona on the particle surface. The binding exhibited an unusual stretched-exponential behavior, suggesting a rich binding kinetics. Furthermore, the binding destabilized the α-helices while increasing the β-sheet content of the proteins. This study revealed the atomic and molecular details of the structural and dynamic characteristics of nanoparticle-protein corona formation.The advancement of nanomedicine and the increasing applications of nanoparticles in consumer products have led to administered biological exposure and unintentional environmental accumulation of nanoparticles, causing concerns over the biocompatibility and sustainability of nanotechnology. Upon entering physiological environments, nanoparticles readily assume the form of a nanoparticle-protein corona that dictates their biological identity. Consequently, understanding the structure and dynamics of a nanoparticle-protein corona is essential for predicting the fate, transport, and toxicity of nanomaterials in living systems and for enabling the vast applications of nanomedicine. Here we combined multiscale molecular dynamics simulations and complementary experiments to characterize the silver nanoparticle-ubiquitin corona formation. Notably, ubiquitins competed with citrates for the nanoparticle surface, governed by specific electrostatic interactions. Under a high protein/nanoparticle stoichiometry, ubiquitins formed a multi-layer corona on the particle surface. The binding exhibited an unusual stretched-exponential behavior, suggesting a rich binding kinetics. Furthermore, the binding destabilized the α-helices while increasing the β-sheet content of the proteins. This study revealed the atomic and molecular details of the structural and dynamic characteristics of nanoparticle-protein corona formation. Electronic supplementary information (ESI) available: Experimental and computational methods as well as supporting figures. See DOI: 10.1039/c3nr02147e

A versatile method for fabricating ion-exchange hydrogel nanofibrous membranes with superb biomolecule adsorption and separation properties.

PubMed

Lv, Huan; Wang, Xueqin; Fu, Qiuxia; Si, Yang; Yin, Xia; Li, Xiaoran; Sun, Gang; Yu, Jianyong; Ding, Bin

2017-11-15

Construction ion-exchange membranes with superb biomolecules adsorption and purification performance plays a greatly important role in the fields of biotechnological and biopharmaceutical industry, yet still remains an extremely challenging. Herein, we in situ synthesized the cis-butenedioic anhydride grafted poly(vinyl alcohol) hydrogel nanofibrous membranes (CBA-g-PVA HNFM) by combining electrospinning technique with the grafting-copolymerization crosslinking. Taking advantages of the large specific surface area which could provide numerous sites available for functional groups and biomolecules binding, highly tortuous and interconnected porous channel for biomolecules transfer, and enhanced mechanical strength, the resultant CBA-g-PVA HNFM exhibited relatively high binding amount of 170mgg -1 , rapid equilibrium time of 8h towards the biomolecule template of lysozyme, and the performance could be tailored by regulating the buffer properties and protein concentrations. Additionally, the resultant functional HNFM also possessed superior acid resistance property, excellent reversibility and regeneration performance. More importantly, the obtained CBA-g-PVA HNFM could directly extract lysozyme from fresh chicken eggs with capacity of 125mgg -1 , exhibiting excellent practical application properties. The fabrication of proposed CBA-g-PVA HNFM offers a feasible alternative for construction of ion-exchange chromatograph column for bio-separation and purification engineering. Copyright © 2017 Elsevier Inc. All rights reserved.

Entropy as a Driver of Selectivity for Inhibitor Binding to Histone Deacetylase 6.

PubMed

Porter, Nicholas J; Wagner, Florence F; Christianson, David W

2018-05-18

Among the metal-dependent histone deacetylases, the class IIb isozyme HDAC6 is remarkable because of its role in the regulation of microtubule dynamics in the cytosol. Selective inhibition of HDAC6 results in microtubule hyperacetylation, leading to cell cycle arrest and apoptosis, which is a validated strategy for cancer chemotherapy and the treatment of other disorders. HDAC6 inhibitors generally consist of a Zn 2+ -binding group such as a hydroxamate, a linker, and a capping group; the capping group is a critical determinant of isozyme selectivity. Surprisingly, however, even "capless" inhibitors exhibit appreciable HDAC6 selectivity. To probe the chemical basis for this selectivity, we now report high-resolution crystal structures of HDAC6 complexed with capless cycloalkyl hydroxamate inhibitors 1-4. Each inhibitor hydroxamate group coordinates to the catalytic Zn 2+ ion with canonical bidentate geometry. Additionally, the olefin moieties of compounds 2 and 4 bind in an aromatic crevice between the side chains of F583 and F643. Reasoning that similar binding could be achieved in the representative class I isozyme HDAC8, we employed isothermal titration calorimetry to study the thermodynamics of inhibitor binding. These measurements indicate that the entropy of inhibitor binding is generally positive for binding to HDAC6 and negative for binding to HDAC8, resulting in ≤313-fold selectivity for binding to HDAC6 relative to HDAC8. Thus, favorable binding entropy contributes to HDAC6 selectivity. Notably, cyclohexenyl hydroxamate 2 represents a promising lead for derivatization with capping groups that may further enhance its impressive 313-fold thermodynamic selectivity for HDAC6 inhibition.

Optimization of reverse chemical ecology method: false positive binding of Aenasius bambawalei odorant binding protein 1 caused by uncertain binding mechanism.

PubMed

Li, Q L; Yi, S C; Li, D Z; Nie, X P; Li, S Q; Wang, M-Q; Zhou, A M

2018-06-01

Odorant binding proteins (OBPs) are considered as the core molecular targets in reverse chemical ecology, which is a convenient and efficient method by which to screen potential semiochemicals. Herein, we identified a classic OBP, AbamOBP1 from Aenasius bambawalei, which showed high mRNA expression in male antennae. Fluorescence competitive binding assay (FCBA) results demonstrated that AbamOBP1 has higher binding affinity with ligands at acid pH, suggesting the physiologically inconsistent binding affinity of this protein. Amongst the four compounds with the highest binding affinities at acid pH, 2, 4, 4-trimethyl-2-pentene and 1-octen-3-one were shown to have attractant activity for male adults, whereas (-)-limonene and an analogue of 1-octen-3-ol exhibited nonbehavioural activity. Further homology modelling and fluorescence quenching experiments demonstrated that the stoichiometry of the binding of this protein to these ligands was not 1: 1, suggesting that the results of FCBA were false. In contrast, the apparent association constants (Ka) of fluorescence quenching experiments seemed to be more reliable, because 2, 4, 4-trimethyl-2-pentene and 1-octen-3-one had observably higher Ka than (-)-limonene and 1-octen-3-ol at neutral pH. Based on the characteristics of different OBPs, various approaches should be applied to study their binding affinities with ligands, which could modify and complement the results of FCBA and contribute to the application of reverse chemical ecology. © 2018 The Royal Entomological Society.

Dimethyltryptamine and other hallucinogenic tryptamines exhibit substrate behavior at the serotonin uptake transporter and the vesicle monoamine transporter.

PubMed

Cozzi, Nicholas V; Gopalakrishnan, Anupama; Anderson, Lyndsey L; Feih, Joel T; Shulgin, Alexander T; Daley, Paul F; Ruoho, Arnold E

2009-12-01

N,N-dimethyltryptamine (DMT) is a potent plant hallucinogen that has also been found in human tissues. When ingested, DMT and related N,N-dialkyltryptamines produce an intense hallucinogenic state. Behavioral effects are mediated through various neurochemical mechanisms including activity at sigma-1 and serotonin receptors, modification of monoamine uptake and release, and competition for metabolic enzymes. To further clarify the pharmacology of hallucinogenic tryptamines, we synthesized DMT, N-methyl-N-isopropyltryptamine (MIPT), N,N-dipropyltryptamine (DPT), and N,N-diisopropyltryptamine. We then tested the abilities of these N,N-dialkyltryptamines to inhibit [(3)H]5-HT uptake via the plasma membrane serotonin transporter (SERT) in human platelets and via the vesicle monoamine transporter (VMAT2) in Sf9 cells expressing the rat VMAT2. The tryptamines were also tested as inhibitors of [(3)H]paroxetine binding to the SERT and [(3)H]dihydrotetrabenazine binding to VMAT2. Our results show that DMT, MIPT, DPT, and DIPT inhibit [(3)H]5-HT transport at the SERT with K ( I ) values of 4.00 +/- 0.70, 8.88 +/- 4.7, 0.594 +/- 0.12, and 2.32 +/- 0.46 microM, respectively. At VMAT2, the tryptamines inhibited [(3)H]5-HT transport with K ( I ) values of 93 +/- 6.8, 20 +/- 4.3, 19 +/- 2.3, and 19 +/- 3.1 muM, respectively. On the other hand, the tryptamines were very poor inhibitors of [(3)H]paroxetine binding to SERT and of [(3)H]dihydrotetrabenazine binding to VMAT2, resulting in high binding-to-uptake ratios. High binding-to-uptake ratios support the hypothesis that the tryptamines are transporter substrates, not uptake blockers, at both SERT and VMAT2, and also indicate that there are separate substrate and inhibitor binding sites within these transporters. The transporters may allow the accumulation of tryptamines within neurons to reach relatively high levels for sigma-1 receptor activation and to function as releasable transmitters.

Sequence specificity of mutagen-nucleic acid complexes in solution: intercalation and mutagen-base pair overlap geometries for proflavine binding to dC-dC-dG-dG and dG-dG-dC-dC self-complementary duplexes.

PubMed

Patel, D J; Canuel, L L

1977-07-01

The complex formed between the mutagen proflavine and the dC-dC-dG-dG and dG-dG-dC-dC self-complementary tetranucleotide duplexes has been monitored by proton high resolution nuclear magnetic resonance spectroscopy in 0.1 M phosphate solution at high nucleotide/drug ratios. The large upfield shifts (0.5 to 0.85 ppm) observed at all the proflavine ring nonexchangeable protons on complex formation are consistent with intercalation of the mutagen between base pairs of the tetranucleotide duplex. We have proposed an approximate overlap geometry between the proflavine ring and nearest neighbor base pairs at the intercalation site from a comparison between experimental shifts and those calculated for various stacking orientations. We have compared the binding of actinomycin D, propidium diiodide, and proflavine to self-complementary tetranucleotide sequences dC-dC-dG-dG and dG-dG-dC-dC by UV absorbance changes in the drug bands between 400 and 500 nm. Actinomycin D exhibits a pronounced specificity for sequences with dG-dC sites (dG-dG-dC-dC), while propidium diiodide and proflavine exhibit a specificity for sequences with dC-dG sites (dC-dC-dG-dG). Actinomycin D binds more strongly than propidium diiodide and proflavine to dC-dG-dC-dG (contains dC-dG and dG-dC binding sites), indicative of the additional stabilization from hydrogen bonding and hydrophobic interactions between the pentapeptide lactone rings of actinomycin D and the base pair edges and sugar-phosphate backbone of the tetranucleotide duplex.

Sequence specificity of mutagen-nucleic acid complexes in solution: Intercalation and mutagen-base pair overlap geometries for proflavine binding to dC-dC-dG-dG and dG-dG-dC-dC self-complementary duplexes

PubMed Central

Patel, Dinshaw J.; Canuel, Lita L.

1977-01-01

The complex formed between the mutagen proflavine and the dC-dC-dG-dG and dG-dG-dC-dC self-complementary tetranucleotide duplexes has been monitored by proton high resolution nuclear magnetic resonance spectroscopy in 0.1 M phosphate solution at high nucleotide/drug ratios. The large upfield shifts (0.5 to 0.85 ppm) observed at all the proflavine ring nonexchangeable protons on complex formation are consistent with intercalation of the mutagen between base pairs of the tetranucleotide duplex. We have proposed an approximate overlap geometry between the proflavine ring and nearest neighbor base pairs at the intercalation site from a comparison between experimental shifts and those calculated for various stacking orientations. We have compared the binding of actinomycin D, propidium diiodide, and proflavine to self-complementary tetranucleotide sequences dC-dC-dG-dG and dG-dG-dC-dC by UV absorbance changes in the drug bands between 400 and 500 nm. Actinomycin D exhibits a pronounced specificity for sequences with dG-dC sites (dG-dG-dC-dC), while propidium diiodide and proflavine exhibit a specificity for sequences with dC-dG sites (dC-dC-dG-dG). Actinomycin D binds more strongly than propidium diiodide and proflavine to dC-dG-dC-dG (contains dC-dG and dG-dC binding sites), indicative of the additional stabilization from hydrogen bonding and hydrophobic interactions between the pentapeptide lactone rings of actinomycin D and the base pair edges and sugar-phosphate backbone of the tetranucleotide duplex. PMID:268613

Interaction and inhibitory influence of the azo dye carmoisine on lysozyme amyloid fibrillogenesis.

PubMed

Basu, Anirban; Suresh Kumar, Gopinatha

2017-07-25

The binding of the common food colorant carmoisine and its inhibitory effect on amyloid fibrillation in lysozyme have been investigated. Since humans are increasingly exposed to various food colorants like carmoisine, such studies are highly relevant. In the presence of lysozyme, the carmoisine absorption spectrum exhibited hypochromic changes. The intrinsic fluorescence of lysozyme was also quenched on interaction. Time-resolved fluorescence results suggested that the binding mechanism involved ground state complexation. The binding was predominantly dominated by non-polyelectrolytic forces. The molecular distance between the donor (lysozyme) and the acceptor (carmoisine), calculated from FRET theory, was found to be 3.37 nm, indicating that carmoisine binds close to Trp-62/63 residues in the β-domain of the protein. Information on alterations in the microenvironment surrounding the Trp-residues was also obtained from synchronous fluorescence data. Carmoisine binding induced significant loss in the alpha helical organization of lysozyme. The binding, nevertheless, did not influence the thermal stability of lysozyme significantly. The binding reaction was exothermic and driven by large negative enthalpy and small but favourable entropic contributions. Thioflavin T assay, far-UV circular dichroism studies and AFM imaging profiles testified that carmoisine had a significant inhibitory effect on amyloid fibrillogenesis in lysozyme. Carmoisine also had a definitive defibrillating effect on existing fibrils. The results may provide new insights for designing new small molecule inhibitors for amyloid related diseases.

Multi-specificity of a Psathyrella velutina mushroom lectin: heparin/pectin binding occurs at a site different from the N-acetylglucosamine/N-acetylneuraminic acid-specific site.

PubMed

Ueda, H; Saitoh, T; Kojima, K; Ogawa, H

1999-09-01

An N-acetylglucosamine (GlcNAc)/N-acetylneuraminic acid-specific lectin from the fruiting body of Psathyrella velutina (PVL) is a useful probe for the detection and fractionation of specific carbohydrates. In this study, PVL was found to exhibit multispecificity to acidic polysaccharides and sulfatides. Purified PVL and a counterpart lectin to PVL in the mycelium interact with heparin neoproteoglycans, as detected by both membrane analysis and solid phase assay. The pH-dependencies of the binding to heparin and GlcNAc5-6 differ. The heparin binding of PVL is inhibited best by pectin, polygalacturonic acid, and highly sulfated polysaccharides, but not by GlcNAc, colominic acid, or other glycosaminoglycans. Sandwich affinity chromatography indicated that PVL can simultaneously interact with heparin- and GlcNAc-containing macromolecules. Extensive biotinylation was found to suppress the binding activity to heparin while the GlcNAc binding activity is retained. On the other hand, biotinyl PVL binds to sulfatide and the binding is not inhibited by GlcNAc, N-acetylneuraminic acid, or heparin. These results indicate that PVL is a multi-ligand adhesive lectin that can interact with various glycoconjugates. This multispecificity needs to be recognized when using PVL as a sugar-specific probe to avoid misleading information about the nature of glycoforms.

Human metabolites of synthetic cannabinoids JWH-018 and JWH-073 bind with high affinity and act as potent agonists at cannabinoid type-2 receptors

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

Rajasekaran, Maheswari; Brents, Lisa K.; Franks, Lirit N.

K2 or Spice is an emerging drug of abuse that contains synthetic cannabinoids, including JWH-018 and JWH-073. Recent reports indicate that monohydroxylated metabolites of JWH-018 and JWH-073 retain high affinity and activity at cannabinoid type-1 receptors (CB{sub 1}Rs), potentially contributing to the enhanced toxicity of K2 compared to marijuana. Since the parent compounds also bind to cannabinoid type-2 receptors (CB{sub 2}Rs), this study investigated the affinity and intrinsic activity of JWH-018, JWH-073 and several monohydroxylated metabolites at human CB{sub 2}Rs (hCB{sub 2}Rs). The affinity of cannabinoids for hCB{sub 2}Rs was determined by competition binding studies employing CHO-hCB{sub 2} membranes. Intrinsicmore » activity of compounds was assessed by G-protein activation and adenylyl cyclase (AC)-inhibition in CHO-hCB{sub 2} cells. JWH-073, JWH-018 and several of their human metabolites exhibit nanomolar affinity and act as potent agonists at hCB{sub 2}Rs. Furthermore, a major omega hydroxyl metabolite of JWH-073 (JWH-073-M5) binds to CB{sub 2}Rs with 10-fold less affinity than the parent molecule, but unexpectedly, is equipotent in regulating AC-activity when compared to the parent molecule. Finally, when compared to CP-55,940 and Δ{sup 9}-tetrahydrocannabinol (Δ{sup 9}-THC), JWH-018, JWH-018-M5 and JWH-073-M5 require significantly less CB{sub 2}R occupancy to produce similar levels of AC-inhibition, indicating that these compounds may more efficiently couple CB{sub 2}Rs to AC than the well characterized cannabinoid agonists examined. These results indicate that JWH-018, JWH-073 and several major human metabolites of these compounds exhibit high affinity and demonstrate distinctive signaling properties at CB{sub 2}Rs. Therefore, future studies examining pharmacological and toxicological properties of synthetic cannabinoids present in K2 products should consider potential actions of these drugs at both CB{sub 1} and CB{sub 2}Rs. - Highlights: • JWH-018 and JWH-073 are synthetic cannabinoids present in abused K2 products. • JWH-018, JWH-073 and their human metabolites have high affinity for CB{sub 2} receptors. • JWH-018, JWH-073 and their human metabolites are potent agonists at CB{sub 2} receptors. • JWH-018, JWH-073 and their metabolites exhibit distinct CB{sub 2} signaling properties. • Studies of JWH-018 and JWH-073 should consider actions at CB{sub 1} and CB{sub 2} receptors.« less

Kinetic study of the effects of calcium ions on cationic artichoke (Cynara scolymus L.) peroxidase: calcium binding, steady-state kinetics and reactions with hydrogen peroxide.

PubMed

Hiner, Alexander N P; Sidrach, Lara; Chazarra, Soledad; Varón, Ramón; Tudela, José; García-Cánovas, Francisco; Rodríguez-López, José Neptuno

2004-01-01

The apparent catalytic constant (k(cat)) of artichoke (Cynara scolymus L.) peroxidase (AKPC) with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) increased 130-fold in the presence of calcium ions (Ca2+) but the affinity (K(m)) of the enzyme for ABTS was 500 times lower than for Ca2+-free AKPC. AKPC is known to exhibit an equilibrium between 6-aquo hexa-coordinate and penta-coordinate forms of the haem iron that is modulated by Ca2+ and affects compound I formation. Measurements of the Ca2+ dissociation constant (K(D)) were complicated by the water-association/dissociation equilibrium yielding a global value more than 1000 times too high. The value for the Ca2+ binding step alone has now been determined to be K(D) approximately 10 nM. AKPC-Ca2+ was more resistant to inactivation by hydrogen peroxide (H(2)O(2)) and exhibited increased catalase activity. An analysis of the complex H(2)O(2) concentration dependent kinetics of Ca2+-free AKPC is presented.

The “gating” residues Ile199 and Tyr326 in human monoamine oxidase B function in substrate and inhibitor recognition

PubMed Central

Milczek, Erika M.; Binda, Claudia; Rovida, Stefano; Mattevi, Andrea; Edmondson, Dale E.

2011-01-01

Summary The major structural difference between human monoamine oxidases A (MAO A) and B (MAO B) is that MAO A has a monopartite substrate cavity of ~550 Å3 volume and MAO B contains a dipartite cavity structure with volumes of ~290 Å3 (entrance cavity) and ~400 Å3 (substrate cavity). Ile199 and Tyr326 side chains separate these two cavities in MAO B. To probe the function of these gating residues, Ile199Ala and Ile199Ala Tyr326Ala mutant forms of MAO B were investigated. Structural data on the Ile199Ala MAO B mutant show no alterations in active site geometries compared to WT enzyme while the Ile199Ala-Tyr326Ala MAO B mutant exhibits alterations in residues 100–103 which are part of the loop gating the entrance to the active site. Both mutant enzymes exhibit catalytic properties with increased amine KM but unaltered kcat values. The altered KM values on mutation are attributed to the influence of the cavity structure in the binding and subsequent deprotonation of the amine substrate. Both mutant enzymes exhibit weaker binding affinities relative to WT enzyme for small reversible inhibitors. Ile199Ala MAO B exhibits an increase in binding affinity for reversible MAO B specific inhibitors which bridge both cavities. The Ile199Ala-Tyr326Ala double mutant exhibits inhibitor binding properties more similar to those of MAO A than to MAO B. These results demonstrate the bipartite cavity structure in MAO B plays an important role in substrate and inhibitor recognition to distinguish its specificities from those of MAO A and provides insights into specific reversible inhibitor design for these membrane-bound enzymes. PMID:21978362

Cell wall reactivity of acidophilic and alkaliphilic bacteria determined by potentiometric titrations and Cd adsorption experiments.

PubMed

Kenney, Janice P L; Fein, Jeremy B

2011-05-15

In this study, we used potentiometric titrations and Cd adsorption experiments to determine the binding capacities of two acidophilic (A. cryptum and A. acidophilum) and two alkaliphilic (B. pseudofirmus and B. circulans) bacterial species in order to determine if any consistent trends could be observed relating bacterial growth environment to proton and Cd binding properties and to compare those binding behaviors to those of neutrophilic bacteria. All of the bacterial species studied exhibited significant proton buffering over the pH range in this study, with the alkaliphiles exhibiting significantly higher acidity constants than the acidophiles as well as the neutrophilic bacterial consortia. The calculated average site concentrations for each of the bacteria in this study are within 2σ experimental error of each other, with the exception of A. cryptum, which has a significantly higher Site 2 concentration than the other species. Despite differing acidity constants between the acidophiles and alkaliphiles, all bacteria except A. cryptum exhibited remarkably similar Cd adsorption behavior to each other, and the observed extent of adsorption was also similar to that predicted from a generalized model derived using neutrophilic bacterial consortia. This study demonstrates that bacteria that grow under extreme conditions exhibit similar proton and metal adsorption behavior to that of previously studied neutrophilic species and that a single set of proton and metal binding constants can be used to model the behavior of bacterial adsorption under a wide range of environmental conditions.

Human Blue Cone Opsin Regeneration Involves Secondary Retinal Binding with Analog Specificity.

PubMed

Srinivasan, Sundaramoorthy; Fernández-Sampedro, Miguel A; Morillo, Margarita; Ramon, Eva; Jiménez-Rosés, Mireia; Cordomí, Arnau; Garriga, Pere

2018-03-27

Human color vision is mediated by the red, green, and blue cone visual pigments. Cone opsins are G-protein-coupled receptors consisting of an opsin apoprotein covalently linked to the 11-cis-retinal chromophore. All visual pigments share a common evolutionary origin, and red and green cone opsins exhibit a higher homology, whereas blue cone opsin shows more resemblance to the dim light receptor rhodopsin. Here we show that chromophore regeneration in photoactivated blue cone opsin exhibits intermediate transient conformations and a secondary retinoid binding event with slower binding kinetics. We also detected a fine-tuning of the conformational change in the photoactivated blue cone opsin binding site that alters the retinal isomer binding specificity. Furthermore, the molecular models of active and inactive blue cone opsins show specific molecular interactions in the retinal binding site that are not present in other opsins. These findings highlight the differential conformational versatility of human cone opsin pigments in the chromophore regeneration process, particularly compared to rhodopsin, and point to relevant functional, unexpected roles other than spectral tuning for the cone visual pigments. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Modulation of the binding of basic fibroblast growth factor and heparanase activity by purified λ-carrageenan oligosaccharides.

PubMed

Niu, Ting-Ting; Zhang, Dong-Sheng; Chen, Hai-Min; Yan, Xiao-Jun

2015-07-10

Inhibitors of angiogenesis and tumor metastasis are increasingly emerging as promising agents for cancer therapy. Here, we report λ-carrageenan oligosaccharides (λ-COs), highly-sulfated oligosaccharides acting as a basic fibroblast growth factor (bFGF) antagonist and heparanase inhibitor. λ-COs with degree of polymerization (DP) from 2 to 8 degraded by λ-carrageenase were separated and purified. The structures were identified by mass spectrometry. The activities of λ-COs are closely related with DP. λ-COs showed no cytotoxicity, but inactivated bFGF-induced cell proliferation; among them, λ-carraheptaose showed highest capability. Only λ-carraheptaose can effectively bind to bFGF. Binding kinetics showed that λ-carraheptaose and suramin had different binding modes, i.e., suramin displayed a fast association and fast dissociation, but λ-carraheptaose exhibited a slow association and slow dissociation. In addition, λ-COs showed the highest heparanase inhibitory ability and abolished the endothelial cell invasion. Thus, λ-COs may provide a tool to develop of new carbohydrate-based therapeutics against cancer and angiogenesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Calmodulin fishing with a structurally disordered bait triggers CyaA catalysis

PubMed Central

O’Brien, Darragh P.; Durand, Dominique; Voegele, Alexis; Hourdel, Véronique; Davi, Marilyne; Chamot-Rooke, Julia; Vachette, Patrice; Brier, Sébastien; Ladant, Daniel

2017-01-01

Once translocated into the cytosol of target cells, the catalytic domain (AC) of the adenylate cyclase toxin (CyaA), a major virulence factor of Bordetella pertussis, is potently activated by binding calmodulin (CaM) to produce supraphysiological levels of cAMP, inducing cell death. Using a combination of small-angle X-ray scattering (SAXS), hydrogen/deuterium exchange mass spectrometry (HDX-MS), and synchrotron radiation circular dichroism (SR-CD), we show that, in the absence of CaM, AC exhibits significant structural disorder, and a 75-residue-long stretch within AC undergoes a disorder-to-order transition upon CaM binding. Beyond this local folding, CaM binding induces long-range allosteric effects that stabilize the distant catalytic site, whilst preserving catalytic loop flexibility. We propose that the high enzymatic activity of AC is due to a tight balance between the CaM-induced decrease of structural flexibility around the catalytic site and the preservation of catalytic loop flexibility, allowing for fast substrate binding and product release. The CaM-induced dampening of AC conformational disorder is likely relevant to other CaM-activated enzymes. PMID:29287065

Self-Consistent Charge Density Functional Tight-Binding Study of Poly(3,4-ethylenedioxythiophene): Poly(styrenesulfonate) Ammonia Gas Sensor.

PubMed

Marutaphan, Ampaiwan; Seekaew, Yotsarayuth; Wongchoosuk, Chatchawal

2017-12-01

Geometric and electronic properties of 3,4-ethylenedioxythiophene (EDOT), styrene sulfonate (SS), and EDOT: SS oligomers up to 10 repeating units were studied by the self-consistent charge density functional tight-binding (SCC-DFTB) method. An application of PEDOT:PSS for ammonia (NH 3 ) detection was highlighted and investigated both experimentally and theoretically. The results showed an important role of H-bonds in EDOT:SS oligomers complex conformation. Electrical conductivity of EDOT increased with increasing oligomers and doping SS due to enhancement of π conjugation. Printed PEDOT:PSS gas sensor exhibited relatively high response and selectivity to NH 3 . The SCC-DFTB calculation suggested domination of direct charge transfer process in changing of PEDOT:PSS conductivity upon NH 3 exposure at room temperature. The NH 3 molecules preferred to bind with PEDOT:PSS via physisorption. The most favorable adsorption site for PEDOT:PSS-NH 3 interaction was found to be at the nitrogen atom of NH 3 and hydrogen atoms of SS with an average optimal binding distance of 2.00 Å.

The solvatochromic effects of side chain substitution on the binding interaction of novel tricarbocyanine dyes with human serum albumin.

PubMed

Beckford, Garfield; Owens, Eric; Henary, Maged; Patonay, Gabor

2012-04-15

The effects of solvatochromism on protein-ligand interactions have been studied by absorbance and near-infrared laser induced fluorescence (NIR-LIF) spectroscopy. The utility of three novel classes of cyanine dyes designed for this purpose illustrates that the affinity interactions of ligands at the hydrophobic binding pockets of Human Serum Albumin (HSA) are not only dependent on the overall hydrophobic characteristics of the molecules but are highly influenced by the size of the ligands as well. Whereas changes to the chromophore moiety exhibited slight to moderate changes to the hydrophobic nature of these molecules, substitution at the alkyl indolium side chain has enabled us to vary the binding affinity towards serum albumin. Substitution at the indolium side chain among an ethyl to butyl group results in improved binding characteristics and an almost three-fold increase in affinity constant. In addition, replacement of the ethyl side chain with a phenylpropyl group also yielded unique solvotachromic patterns such as increased hydrophobicity and subsequent biocompatibility with the HSA binding regions. Ligand interaction was however inhibited by steric hindrance associated with the bulky phenyl ring system thus affecting the increased binding that could be realized from the improved hydrophobic nature of the molecules. This characteristic change in binding affinity is of potential interest to developing a methodology which reveals information on the hydrophobic character and steric specificity of the binding cavities. Copyright © 2012 Elsevier B.V. All rights reserved.

Facile Synthesis of Molecularly Imprinted Graphene Quantum Dots for the Determination of Dopamine with Affinity-Adjustable.

PubMed

Zhou, Xi; Wang, Anqi; Yu, Chenfei; Wu, Shishan; Shen, Jian

2015-06-10

A facilely prepared fluorescence sensor was developed for dopamine (DA) determination based on polyindole/graphene quantum dots molecularly imprinted polymers (PIn/GQDs@MIPs). The proposed sensor exhibits a high sensitivity with a linear range of 5 × 10(-10) to 1.2 × 10(-6) M and the limit of detection as low as 1 × 10(-10) M in the determination of DA, which is probably due to the tailor-made imprinted cavities for binding DA thought hydrogen bonds between amine groups of DA and oxygen-containing groups of the novel composite. Furthermore, the prepared sensor can rebind DA in dual-type: a low affinity type (noncovalent interaction is off) and a high affinity type (noncovalent interaction is on), and the rebinding interaction can be adjusted by tuning the pH, which shows a unique potential for adjusting the binding interaction while keeping the specificity, allowing for wider applications.

Computational Optimization and Characterization of Molecularly Imprinted Polymers

NASA Astrophysics Data System (ADS)

Terracina, Jacob J.

Molecularly imprinted polymers (MIPs) are a class of materials containing sites capable of selectively binding to the imprinted target molecule. Computational chemistry techniques were used to study the effect of different fabrication parameters (the monomer-to-target ratios, pre-polymerization solvent, temperature, and pH) on the formation of the MIP binding sites. Imprinted binding sites were built in silico for the purposes of better characterizing the receptor - ligand interactions. Chiefly, the sites were characterized with respect to their selectivities and the heterogeneity between sites. First, a series of two-step molecular mechanics (MM) and quantum mechanics (QM) computational optimizations of monomer -- target systems was used to determine optimal monomer-to-target ratios for the MIPs. Imidazole- and xanthine-derived target molecules were studied. The investigation included both small-scale models (one-target) and larger scale models (five-targets). The optimal ratios differed between the small and larger scales. For the larger models containing multiple targets, binding-site surface area analysis was used to evaluate the heterogeneity of the sites. The more fully surrounded sites had greater binding energies. Molecular docking was then used to measure the selectivities of the QM-optimized binding sites by comparing the binding energies of the imprinted target to that of a structural analogue. Selectivity was also shown to improve as binding sites become more fully encased by the monomers. For internal sites, docking consistently showed selectivity favoring the molecules that had been imprinted via QM geometry optimizations. The computationally imprinted sites were shown to exhibit size-, shape-, and polarity-based selectivity. This represented a novel approach to investigate the selectivity and heterogeneity of imprinted polymer binding sites, by applying the rapid orientation screening of MM docking to the highly accurate QM-optimized geometries. Next, we sought to computationally construct and investigate binding sites for their enantioselectivity. Again, a two-step MM [special characters removed] QM optimization scheme was used to "computationally imprint" chiral molecules. Using docking techniques, the imprinted binding sites were shown to exhibit an enantioselective preference for the imprinted molecule over its enantiomer. Docking of structurally similar chiral molecules showed that the sites computationally imprinted with R- or S-tBOC-tyrosine were able to differentiate between R- and S-forms of other tyrosine derivatives. The cross-enantioselectivity did not hold for chiral molecules that did not share the tyrosine H-bonding functional group orientations. Further analysis of the individual monomer - target interactions within the binding site led us to conclude that H-bonding functional groups that are located immediately next to the target's chiral center, and therefore spatially fixed relative to the chiral center, will have a stronger contribution to the enantioselectivity of the site than those groups separated from the chiral center by two or more rotatable bonds. These models were the first computationally imprinted binding sites to exhibit this enantioselective preference for the imprinted target molecules. Finally, molecular dynamics (MD) was used to quantify H-bonding interactions between target molecules, monomers, and solvents representative of the pre-polymerization matrix. It was found that both target dimerization and solvent interference decrease the number of monomer - target H-bonds present. Systems were optimized via simulated annealing to create binding sites that were then subjected to molecular docking analysis. Docking showed that the presence of solvent had a detrimental effect on the sensitivity and selectivity of the sites, and that solvents with more H-bonding capabilities were more disruptive to the binding properties of the site. Dynamic simulations also showed that increasing the temperature of the solution can significantly decrease the number of H-bonds formed between the targets and monomers. It is believed that the monomer - target complexes formed within the pre-polymerization matrix are translated into the selective binding cavities formed during polymerization. Elucidating the nature of these interactions in silico improves our understanding of MIPs, ultimately allowing for more optimized sensing materials.

Mechanism of pathogen recognition by human dectin-2.

PubMed

Feinberg, Hadar; Jégouzo, Sabine A F; Rex, Maximus J; Drickamer, Kurt; Weis, William I; Taylor, Maureen E

2017-08-11

Dectin-2, a C-type lectin on macrophages and other cells of the innate immune system, functions in response to pathogens, particularly fungi. The carbohydrate-recognition domain (CRD) in dectin-2 is linked to a transmembrane sequence that interacts with the common Fc receptor γ subunit to initiate immune signaling. The molecular mechanism by which dectin-2 selectively binds to pathogens has been investigated by characterizing the CRD expressed in a bacterial system. Competition binding studies indicated that the CRD binds to monosaccharides with modest affinity and that affinity was greatly enhanced for mannose-linked α1-2 or α1-4 to a second mannose residue. Glycan array analysis confirmed selective binding of the CRD to glycans that contain Manα1-2Man epitopes. Crystals of the CRD in complex with a mammalian-type high-mannose Man 9 GlcNAc 2 oligosaccharide exhibited interaction with Manα1-2Man on two different termini of the glycan, with the reducing-end mannose residue ligated to Ca 2+ in a primary binding site and the nonreducing terminal mannose residue occupying an adjacent secondary site. Comparison of the binding sites in DC-SIGN and langerin, two other pathogen-binding receptors of the innate immune system, revealed why these two binding sites accommodate only terminal Manα1-2Man structures, whereas dectin-2 can bind Manα1-2Man in internal positions in mannans and other polysaccharides. The specificity and geometry of the dectin-2-binding site provide the molecular mechanism for binding of dectin-2 to fungal mannans and also to bacterial lipopolysaccharides, capsular polysaccharides, and lipoarabinomannans that contain the Manα1-2Man disaccharide unit. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Chalcone Based Homodimeric PET Agent, 11C-(Chal)2DEA-Me, for Beta Amyloid Imaging: Synthesis and Bioevaluation.

PubMed

Chauhan, Kanchan; Tiwari, Anjani K; Chadha, Nidhi; Kaul, Ankur; Singh, Ajai Kumar; Datta, Anupama

2018-04-02

Homodimeric chalcone based 11 C-PET radiotracer, 11 C-(Chal) 2 DEA-Me, was synthesized, and binding affinity toward beta amyloid (Aβ) was evaluated. The computational studies revealed multiple binding of the tracer at the recognition sites of Aβ fibrils. The bivalent ligand 11 C-(Chal) 2 DEA-Me displayed higher binding affinity compared to the corresponding monomer, 11 C-Chal-Me, and classical Aβ agents. The radiolabeling yield with carbon-11 was 40-55% (decay corrected) with specific activity of 65-90 GBq/μmol. A significant ( p < 0.0001) improvement in the binding affinity of 11 C-(Chal) 2 DEA-Me with synthetic Aβ42 aggregates over the monomer, 11 C-Chal-Me, demonstrates the utility of the bivalent approach. The PET imaging and biodistribution data displayed suitable brain pharmacokinetics of both ligands with higher brain uptake in the case of the bivalent ligand. Metabolite analysis of healthy ddY mouse brain homogenates exhibited high stability of the radiotracers in the brain with >93% intact tracer at 30 min post injection. Both chalcone derivatives were fluorescent in nature and demonstrated significant changes in the emission properties after binding with Aβ42. The preliminary analysis indicates high potential of 11 C-(Chal) 2 DEA-Me as in vivo Aβ42 imaging tracer and highlights the significance of the bivalent approach to achieve a higher biological response for detection of early stages of amyloidosis.

Vsx2 Controls Eye Organogenesis and Retinal Progenitor Identity Via Homeodomain and Non-Homeodomain Residues Required for High Affinity DNA Binding

PubMed Central

Zou, Changjiang; Levine, Edward M.

2012-01-01

The homeodomain and adjacent CVC domain in the visual system homeobox (VSX) proteins are conserved from nematodes to humans. Humans with missense mutations in these regions of VSX2 have microphthalmia, suggesting both regions are critical for function. To assess this, we generated the corresponding mutations in mouse Vsx2. The homeodomain mutant protein lacked DNA binding activity and the knock-in mutant phenocopied the null mutant, ocular retardation J. The CVC mutant protein exhibited weakened DNA binding; and, although the corresponding knock-in allele was recessive, it unexpectedly caused the strongest phenotype, as indicated by severe microphthalmia and hyperpigmentation of the neural retina. This occurred through a cryptic transcriptional feedback loop involving the transcription factors Mitf and Otx1 and the Cdk inhibitor p27Kip1. Our data suggest that the phenotypic severity of the CVC mutant depends on the weakened DNA binding activity elicited by the CVC mutation and a previously unknown protein interaction between Vsx2 and its regulatory target Mitf. Our data also suggest that an essential function of the CVC domain is to assist the homeodomain in high-affinity DNA binding, which is required for eye organogenesis and unhindered execution of the retinal progenitor program in mammals. Finally, the genetic and phenotypic behaviors of the CVC mutation suggest it has the characteristics of a recessive neomorph, a rare type of genetic allele. PMID:23028343

Investigation of the interaction between berberine and nucleosomes in solution: Spectroscopic and equilibrium dialysis approach

NASA Astrophysics Data System (ADS)

Rabbani-Chadegani, Azra; Mollaei, Hossein; Sargolzaei, Javad

2017-02-01

Berberine is a natural plant alkaloid with high pharmacological potential. Although its interaction with free DNA has been the subject of several reports, to date there is no work concerning the effect of berberine on nucleoprotein structure of DNA, the nucleosomes. The present study focuses on the binding affinity of berberine to nucleosomes and histone H1 employing various spectroscopic techniques, fluorescence, circular dichroism, thermal denaturation as well as equilibrium dialysis. The results showed that the binding of berberine to nucleosomes is positive cooperative with Ka = 5.57 × 103 M- 1. Berberine quenched with the chromophores of protein moiety of nucleosomes and reduced fluorescence emission intensity at 335 nm with Ksv value of 0.135. Binding of berberine to nucleosomes decreased the absorbance at 210 and 260 nm, produced hypochromicity in thermal denaturation profiles and its affinity to nucleoprotein structure of nucleosomes was much higher than to free DNA. Berberine also exhibited high affinity to histone H1 in solution and the binding was positive cooperative with. Ka = 3.61 × 103 M- 1. Moreover berberine decreased fluorescence emission intensity of H1 by quenching with tyrosine residue in its globular core domain. The circular dichroism profiles demonstrated that the binding of drug induced secondary structural changes in both DNA stacking and histone H1. It is concluded that berberine is genotoxic drug, interacts with nucleosomes and in this process histone H1 is involved to exert its anticancer activity.

Entry Inhibition of Influenza Viruses with High Mannose Binding Lectin ESA-2 from the Red Alga Eucheuma serra through the Recognition of Viral Hemagglutinin

PubMed Central

Sato, Yuichiro; Morimoto, Kinjiro; Kubo, Takanori; Sakaguchi, Takemasa; Nishizono, Akira; Hirayama, Makoto; Hori, Kanji

2015-01-01

Lectin sensitivity of the recent pandemic influenza A virus (H1N1-2009) was screened for 12 lectins with various carbohydrate specificity by a neutral red dye uptake assay with MDCK cells. Among them, a high mannose (HM)-binding anti-HIV lectin, ESA-2 from the red alga Eucheuma serra, showed the highest inhibition against infection with an EC50 of 12.4 nM. Moreover, ESA-2 exhibited a wide range of antiviral spectrum against various influenza strains with EC50s of pico molar to low nanomolar levels. Besides ESA-2, HM-binding plant lectin ConA, fucose-binding lectins such as fungal AOL from Aspergillus oryzae and AAL from Aleuria aurantia were active against H1N1-2009, but the potency of inhibition was of less magnitude compared with ESA-2. Direct interaction between ESA-2 and a viral envelope glycoprotein, hemagglutinin (HA), was demonstrated by ELISA assay. This interaction was effectively suppressed by glycoproteins bearing HM-glycans, indicating that ESA-2 binds to the HA of influenza virus through HM-glycans. Upon treatment with ESA-2, no viral antigens were detected in the host cells, indicating that ESA-2 inhibited the initial steps of virus entry into the cells. ESA-2 would thus be useful as a novel microbicide to prevent penetration of viruses such as HIV and influenza viruses to the host cells. PMID:26035023

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering.

PubMed

Haddad, Yazan; Dostalova, Simona; Kudr, Jiri; Zitka, Ondrej; Heger, Zbynek; Adam, Vojtech

2017-11-09

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

Synthesis and cytotoxicity of azo nano-materials as new biosensors for L-Arginine determination.

PubMed

Shang, Xuefang; Luo, Leiming; Ren, Kui; Wei, Xiaofang; Feng, Yaqian; Li, Xin; Xu, Xiufang

2015-06-01

Inspired from biological counterparts, chemical modification of azo derivatives with function groups may provide a highly efficient method to detect amino acid. Herein, we have designed and prepared a series of azo nano-materials involving -NO2, -COOH, -SO3H and naphthyl group, which showed high response for Arginine (Arg) among normal twenty kinds of (Alanine, Valine, Leucine, Isoleucine, Methionine, Aspartic acid, Glutamic acid, Arginine, Glycine, Serine, Threonine, Asparagine, Phenylalanine, Histidine, Tryptophan, Proline, Lysine, Glutamine, Tyrosine and Cysteine). Furthermore, theoretical investigation further illustrated the possible binding mode in the host-guest interaction and the roles of molecular frontier orbitals in molecular interplay. In addition, nano-material 3 exhibited high binding ability for Arg and low cytotoxicity to KYSE450 cells over a concentration range of 5-50μmol·L(-1) which may be used a biosensor for the Arg detection in vivo. Copyright © 2015 Elsevier B.V. All rights reserved.

Porous photonic crystal external cavity laser biosensor

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

Huang, Qinglan; Peh, Jessie; Hergenrother, Paul J.

2016-08-15

We report the design, fabrication, and testing of a photonic crystal (PC) biosensor structure that incorporates a porous high refractive index TiO{sub 2} dielectric film that enables immobilization of capture proteins within an enhanced surface-area volume that spatially overlaps with the regions of resonant electromagnetic fields where biomolecular binding can produce the greatest shifts in photonic crystal resonant wavelength. Despite the nanoscale porosity of the sensor structure, the PC slab exhibits narrowband and high efficiency resonant reflection, enabling the structure to serve as a wavelength-tunable element of an external cavity laser. In the context of sensing small molecule interactions withmore » much larger immobilized proteins, we demonstrate that the porous structure provides 3.7× larger biosensor signals than an equivalent nonporous structure, while the external cavity laser (ECL) detection method provides capability for sensing picometer-scale shifts in the PC resonant wavelength caused by small molecule binding. The porous ECL achieves a record high figure of merit for label-free optical biosensors.« less

Preparation of highly stable zeolite-alginate foam composite for strontium(90Sr) removal from seawater and evaluation of Sr adsorption performance.

PubMed

Hong, Hye-Jin; Kim, Byoung-Gyu; Ryu, Jungho; Park, In-Su; Chung, Kang-Sup; Lee, Sang Moon; Lee, Jin-Bae; Jeong, Hyeon Su; Kim, Hyunchul; Ryu, Taegong

2018-01-01

Alginate bead is a promising strontium (Sr) adsorbent in seawater, but highly concentrated Na ions caused over-swelling and damaged the hydrogel bead. To improve the mechanical stability of alginate bead, flexible foam-type zeolite-alginate composite was synthesized and Sr adsorption performance was evaluated in seawater; 1-10% zeolite immobilized alginate foams were prepared by freeze-dry technique. Immobilization of zeolite into alginate foam converted macro-pores to meso-pores which lead to more compact structure. It resulted in less swollen composite in seawater medium and exhibited highly improved mechanical stability compared with alginate bead. Besides, Sr adsorption efficiency and selectivity were enhanced by immobilization of zeolite in alginate foam due to the increase of Sr binding sites (zeolite). In particular, Sr selectivity against Na was highly improved. The 10% zeolite-alginate foam exhibited a higher log K d of 3.3, while the pure alginate foam exhibited 2.7 in the presence of 0.1 M Na. Finally, in the real seawater, the 10% zeolite-alginate foam exhibited 1.5 times higher Sr adsorption efficiency than the pure alginate foam. This result reveals that zeolite-alginate foam composite is appropriate material for Sr removal in seawater due to its swelling resistance as well as improved Sr adsorption performance in complex media. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification of the bioactive and consensus peptide motif from Momordica charantia insulin receptor-binding protein.

PubMed

Lo, Hsin-Yi; Li, Chia-Cheng; Ho, Tin-Yun; Hsiang, Chien-Yun

2016-08-01

Many food bioactive peptides with diverse functions have been discovered by studying plant proteins. We have previously identified a 68-residue insulin receptor (IR)-binding protein (mcIRBP) from Momordica charantia that exhibits hypoglycemic effects in mice via interaction with IR. By in vitro digestion, we found that mcIRBP-19, spanning residues 50-68 of mcIRBP, enhanced the binding of insulin to IR, stimulated the phosphorylation of PDK1 and Akt, induced the expression of glucose transporter 4, and stimulated both the uptake of glucose in cells and the clearance of glucose in diabetic mice. Furthermore, mcIRBP-19 homologs were present in various plants and shared similar β-hairpin structures and IR kinase-activating abilities to mcIRBP-19. In conclusion, our findings suggested that mcIRBP-19 is a blood glucose-lowering bioactive peptide that exhibits IR-binding potentials. Moreover, we newly identified novel IR-binding bioactive peptides in various plants which belonged to different taxonomic families. Copyright © 2016 Elsevier Ltd. All rights reserved.

Force fields for describing the solution-phase synthesis of shape-selective metal nanoparticles

NASA Astrophysics Data System (ADS)

Zhou, Ya; Al-Saidi, Wissam; Fichthorn, Kristen

2013-03-01

Polyvinylpyrrolidone (PVP) and polyethylene oxide (PEO) are structure-directing agents that exhibit different performance in the polyol synthesis of Ag nanostructures. The success of these structure-directing agents in selective nanostructure synthesis is often attributed to their selective binding to Ag(100) facets. We use first-principles, density-functional theory (DFT) calculations in a vacuum environment to show that PVP has a stronger preference to bind to Ag(100) than to Ag(111), whereas PEO exhibits much weaker selectivity. To understand the role of solvent in the surface-sensitive binding, we develop classical force fields to describe the interactions of the structure-directing (PVP and PEO) and solvent (ethylene glycol) molecules with various Ag substrates. We parameterize the force fields through force-and-energy matching to DFT results using simulated annealing. We validate the force fields by comparisons to DFT and experimental binding energies. Our force fields reproduce the surface-sensitive binding predicted by DFT calculations. Molecular dynamics simulations based on these force fields can be used to reveal the role of solvent, polymer chain length, and polymer concentration in the selective synthesis of Ag nanostructures.

Molecular coevolution of mammalian ribosomal gene terminator sequences and the transcription termination factor TTF-I.

PubMed Central

Evers, R; Grummt, I

1995-01-01

Both the DNA elements and the nuclear factors that direct termination of ribosomal gene transcription exhibit species-specific differences. Even between mammals--e.g., human and mouse--the termination signals are not identical and the respective transcription termination factors (TTFs) which bind to the terminator sequence are not fully interchangeable. To elucidate the molecular basis for this species-specificity, we have cloned TTF-I from human and mouse cells and compared their structural and functional properties. Recombinant TTF-I exhibits species-specific DNA binding and terminates transcription both in cell-free transcription assays and in transfection experiments. Chimeric constructs of mouse TTF-I and human TTF-I reveal that the major determinant for species-specific DNA binding resides within the C terminus of TTF-I. Replacing 31 C-terminal amino acids of mouse TTF-I with the homologous human sequences relaxes the DNA-binding specificity and, as a consequence, allows the chimeric factor to bind the human terminator sequence and to specifically stop rDNA transcription. Images Fig. 2 Fig. 3 Fig. 4 PMID:7597036

Structure-guided development of a high-affinity human Programmed Cell Death-1: Implications for tumor immunotherapy

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

Lázár-Molnár, Eszter; Scandiuzzi, Lisa; Basu, Indranil

Programmed Cell Death-1 (PD-1) is an inhibitory immune receptor, which plays critical roles in T cell co-inhibition and exhaustion upon binding to its ligands PD-L1 and PD-L2. We report the crystal structure of the human PD-1 ectodomain and the mapping of the PD-1 binding interface. Mutagenesis studies confirmed the crystallographic interface, and resulted in mutant PD-1 receptors with altered affinity and ligand-specificity. In particular, a high-affinity mutant PD-1 (HA PD-1) exhibited 45 and 30-fold increase in binding to PD-L1 and PD-L2, respectively, due to slower dissociation rates. This mutant (A132L) was used to engineer a soluble chimeric Ig fusion proteinmore » for cell-based and in vivo studies. HA PD-1 Ig showed enhanced binding to human dendritic cells, and increased T cell proliferation and cytokine production in a mixed lymphocyte reaction (MLR) assay. Moreover, in an experimental model of murine Lewis lung carcinoma, HA PD-1 Ig treatment synergized with radiation therapy to decrease local and metastatic tumor burden, as well as in the establishment of immunological memory responses. Our studies highlight the value of structural considerations in guiding the design of a high-affinity chimeric PD-1 Ig fusion protein with robust immune modulatory properties, and underscore the power of combination therapies to selectively manipulate the PD-1 pathway for tumor immunotherapy.« less

Development of an enzyme-linked immunosorbent assay and a beta-1 adrenergic receptor-based assay for monitoring the drug atenolol.

PubMed

Sapir, A; Shalev, A Hariton; Skalka, N; Bronshtein, A; Altstein, M

2013-03-01

Two approaches for monitoring atenolol (ATL) were applied: an immunochemical assay and a competitive-binding assay, based on the interaction between ATL and its target receptor, β1 adrenergic receptor (β1AR). Polyclonal antibodies (Abs) for ATL were generated, and a highly specific microplate immunochemical assay, that is, an enzyme-linked immunosorbent assay (ELISA), for its detection was developed. The ATL ELISA exhibited I50 and limit of detection (I20) values of 0.15 ± 0.048 and 0.032 ± 0.016 ng/ml, respectively, and the Abs did not cross-react with any of the tested beta-blocker drugs. Furthermore, a human β1AR (h-β1AR) was stably expressed in Spodoptera frugiperda cells (Sf9). The receptor was employed to develop a competitive-binding assay that monitored binding of ATL in the presence of isoproteranol by quantification of secondary messenger, cyclic adenosine monophosphate (cAMP), levels in the transfected cells. The assay showed that the recombinant h-β1AR was functional, could bind the agonistic ligand isoproterenol as well as the antagonist ATL, as indicated by a dose-dependent elevation of cAMP in the presence of isoproteranol, and decrease after ATL addition. The highly efficient and sensitive ELISA and the receptor assay represent two methods suitable for efficient and cost-effective large-scale, high-throughput monitoring of ATL in environmental, agricultural, and biological samples. Copyright © 2012 SETAC.

Modeling of Arylamide Helix Mimetics in the p53 Peptide Binding Site of hDM2 Suggests Parallel and Anti-Parallel Conformations Are Both Stable

PubMed Central

Fuller, Jonathan C.; Jackson, Richard M.; Edwards, Thomas A.; Wilson, Andrew J.; Shirts, Michael R.

2012-01-01

The design of novel α-helix mimetic inhibitors of protein-protein interactions is of interest to pharmaceuticals and chemical genetics researchers as these inhibitors provide a chemical scaffold presenting side chains in the same geometry as an α-helix. This conformational arrangement allows the design of high affinity inhibitors mimicking known peptide sequences binding specific protein substrates. We show that GAFF and AutoDock potentials do not properly capture the conformational preferences of α-helix mimetics based on arylamide oligomers and identify alternate parameters matching solution NMR data and suitable for molecular dynamics simulation of arylamide compounds. Results from both docking and molecular dynamics simulations are consistent with the arylamides binding in the p53 peptide binding pocket. Simulations of arylamides in the p53 binding pocket of hDM2 are consistent with binding, exhibiting similar structural dynamics in the pocket as simulations of known hDM2 binders Nutlin-2 and a benzodiazepinedione compound. Arylamide conformations converge towards the same region of the binding pocket on the 20 ns time scale, and most, though not all dihedrals in the binding pocket are well sampled on this timescale. We show that there are two putative classes of binding modes for arylamide compounds supported equally by the modeling evidence. In the first, the arylamide compound lies parallel to the observed p53 helix. In the second class, not previously identified or proposed, the arylamide compound lies anti-parallel to the p53 helix. PMID:22916232

The correlation between ouabain binding and potassium pump inhibition in human and sheep erythrocytes.

PubMed Central

Joiner, C H; Lauf, P K

1978-01-01

1. [3H]Ouabain binding to human and sheep red blood cells was shown to be specific for receptors associated with Na/K transport. Virtually all tritium binding was abolished by dilution with unlabelled drug. Saturation levels of binding were independent of glycoside concentration and were identical to those associated with 100% inhibition of K pumping. 2. [3H]Ouabain binding and 42K influx were measured simultaneously in order to correlate the degree of K pump inhibition with the amount of glycoside bound. Results by this method agreed exactly with those obtained by pre-exposing cells to drug, followed by washing and then measuring K influx. 3. Plots of [3H]oubain binding vs. K pump inhibition were rectilinear for human and low K (LK) sheep red cells, indicating one glycoside receptor per K pump site and functional homogeneity of pump sites. High K (HK) sheep red cells exhibited curved plots of binding versus inhibition, which were best explained in terms of one receptor per pump, but a heterogeneous population of pump sites. 4. External K reduced the rate of glycoside binding, but did not alter the relationship between binding and inhibition. 5. The number of K pump sites was estimated as 450--500 per human cell and 30--50 per LK sheep cell. HK sheep cells had 90--130 sites per cell, of which eighty to ninety were functionally dominant. The number of K pump sites on LK sheep cells was not changed by anti-L, although the maximum velocity of pump turnover was increased. PMID:722573

The Myelin and Lymphocyte Protein MAL Is Required for Binding and Activity of Clostridium perfringens ε-Toxin

PubMed Central

Oo, Myat Lin; Anrather, Josef; Schaeren-Wiemers, Nicole; Alonso, Miguel A.; Fischetti, Vincent A.; McClain, Mark S.; Vartanian, Timothy

2015-01-01

Clostridium perfringens ε-toxin (ETX) is a potent pore-forming toxin responsible for a central nervous system (CNS) disease in ruminant animals with characteristics of blood-brain barrier (BBB) dysfunction and white matter injury. ETX has been proposed as a potential causative agent for Multiple Sclerosis (MS), a human disease that begins with BBB breakdown and injury to myelin forming cells of the CNS. The receptor for ETX is unknown. Here we show that both binding of ETX to mammalian cells and cytotoxicity requires the tetraspan proteolipid Myelin and Lymphocyte protein (MAL). While native Chinese Hamster Ovary (CHO) cells are resistant to ETX, exogenous expression of MAL in CHO cells confers both ETX binding and susceptibility to ETX-mediated cell death. Cells expressing rat MAL are ~100 times more sensitive to ETX than cells expressing similar levels of human MAL. Insertion of the FLAG sequence into the second extracellular loop of MAL abolishes ETX binding and cytotoxicity. ETX is known to bind specifically and with high affinity to intestinal epithelium, renal tubules, brain endothelial cells and myelin. We identify specific binding of ETX to these structures and additionally show binding to retinal microvasculature and the squamous epithelial cells of the sclera in wild-type mice. In contrast, there is a complete absence of ETX binding to tissues from MAL knockout (MAL-/-) mice. Furthermore, MAL-/- mice exhibit complete resistance to ETX at doses in excess of 1000 times the symptomatic dose for wild-type mice. We conclude that MAL is required for both ETX binding and cytotoxicity. PMID:25993478

The Myelin and Lymphocyte Protein MAL Is Required for Binding and Activity of Clostridium perfringens ε-Toxin.

PubMed

Rumah, Kareem Rashid; Ma, Yinghua; Linden, Jennifer R; Oo, Myat Lin; Anrather, Josef; Schaeren-Wiemers, Nicole; Alonso, Miguel A; Fischetti, Vincent A; McClain, Mark S; Vartanian, Timothy

2015-05-01

Clostridium perfringens ε-toxin (ETX) is a potent pore-forming toxin responsible for a central nervous system (CNS) disease in ruminant animals with characteristics of blood-brain barrier (BBB) dysfunction and white matter injury. ETX has been proposed as a potential causative agent for Multiple Sclerosis (MS), a human disease that begins with BBB breakdown and injury to myelin forming cells of the CNS. The receptor for ETX is unknown. Here we show that both binding of ETX to mammalian cells and cytotoxicity requires the tetraspan proteolipid Myelin and Lymphocyte protein (MAL). While native Chinese Hamster Ovary (CHO) cells are resistant to ETX, exogenous expression of MAL in CHO cells confers both ETX binding and susceptibility to ETX-mediated cell death. Cells expressing rat MAL are ~100 times more sensitive to ETX than cells expressing similar levels of human MAL. Insertion of the FLAG sequence into the second extracellular loop of MAL abolishes ETX binding and cytotoxicity. ETX is known to bind specifically and with high affinity to intestinal epithelium, renal tubules, brain endothelial cells and myelin. We identify specific binding of ETX to these structures and additionally show binding to retinal microvasculature and the squamous epithelial cells of the sclera in wild-type mice. In contrast, there is a complete absence of ETX binding to tissues from MAL knockout (MAL-/-) mice. Furthermore, MAL-/- mice exhibit complete resistance to ETX at doses in excess of 1000 times the symptomatic dose for wild-type mice. We conclude that MAL is required for both ETX binding and cytotoxicity.

Weakly Hydrated Surfaces and the Binding Interactions of Small Biological Solutes

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

Brady, J. W.; Tavagnacco, L.; Ehrlich, L.

2012-04-01

Extended planar hydrophobic surfaces, such as are found in the side chains of the amino acids histidine, phenylalanine, tyrosine, and tryptophan, exhibit an affinity for the weakly hydrated faces of glucopyranose. In addition, molecular species such as these, including indole, caffeine, and imidazole, exhibit a weak tendency to pair together by hydrophobic stacking in aqueous solution. These interactions can be partially understood in terms of recent models for the hydration of extended hydrophobic faces and should provide insight into the architecture of sugar-binding sites in proteins.

Weakly hydrated surfaces and the binding interactions of small biological solutes.

PubMed

Brady, John W; Tavagnacco, Letizia; Ehrlich, Laurent; Chen, Mo; Schnupf, Udo; Himmel, Michael E; Saboungi, Marie-Louise; Cesàro, Attilio

2012-04-01

Extended planar hydrophobic surfaces, such as are found in the side chains of the amino acids histidine, phenylalanine, tyrosine, and tryptophan, exhibit an affinity for the weakly hydrated faces of glucopyranose. In addition, molecular species such as these, including indole, caffeine, and imidazole, exhibit a weak tendency to pair together by hydrophobic stacking in aqueous solution. These interactions can be partially understood in terms of recent models for the hydration of extended hydrophobic faces and should provide insight into the architecture of sugar-binding sites in proteins.

Diurnal rhythm of melatonin binding in the rat suprachiasmatic nucleus

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

Laitinen, J.T.; Castren, E.; Vakkuri, O.

1989-03-01

We used quantitative in vitro autoradiography to localize and characterize 2-/sup 125/I-melatonin binding sites in the rat suprachiasmatic nuclei in relation to pineal melatonin production. In a light:dark cycle of 12:12 h, binding density exhibited significant diurnal variation with a peak at the dark-light transition and a trough 12 hours later. Saturation studies suggested that the decreased binding at light-dark transition might be due to a shift of the putative melatonin receptor to a low affinity state.

Broadening the cofactor specificity of a thermostable alcohol dehydrogenase using rational protein design introduces novel kinetic transient behavior.

PubMed

Campbell, Elliot; Wheeldon, Ian R; Banta, Scott

2010-12-01

Cofactor specificity in the aldo-keto reductase (AKR) superfamily has been well studied, and several groups have reported the rational alteration of cofactor specificity in these enzymes. Although most efforts have focused on mesostable AKRs, several putative AKRs have recently been identified from hyperthermophiles. The few that have been characterized exhibit a strong preference for NAD(H) as a cofactor, in contrast to the NADP(H) preference of the mesophilic AKRs. Using the design rules elucidated from mesostable AKRs, we introduced two site-directed mutations in the cofactor binding pocket to investigate cofactor specificity in a thermostable AKR, AdhD, which is an alcohol dehydrogenase from Pyrococcus furiosus. The resulting double mutant exhibited significantly improved activity and broadened cofactor specificity as compared to the wild-type. Results of previous pre-steady-state kinetic experiments suggest that the high affinity of the mesostable AKRs for NADP(H) stems from a conformational change upon cofactor binding which is mediated by interactions between a canonical arginine and the 2'-phosphate of the cofactor. Pre-steady-state kinetics with AdhD and the new mutants show a rich conformational behavior that is independent of the canonical arginine or the 2'-phosphate. Additionally, experiments with the highly active double mutant using NADPH as a cofactor demonstrate an unprecedented transient behavior where the binding mechanism appears to be dependent on cofactor concentration. These results suggest that the structural features involved in cofactor specificity in the AKRs are conserved within the superfamily, but the dynamic interactions of the enzyme with cofactors are unexpectedly complex. © 2010 Wiley Periodicals, Inc.

Structural characterization of framework-gas interactions in the metal-organic framework Co2(dobdc) by in situ single-crystal X-ray diffraction.

PubMed

Gonzalez, Miguel I; Mason, Jarad A; Bloch, Eric D; Teat, Simon J; Gagnon, Kevin J; Morrison, Gregory Y; Queen, Wendy L; Long, Jeffrey R

2017-06-01

The crystallographic characterization of framework-guest interactions in metal-organic frameworks allows the location of guest binding sites and provides meaningful information on the nature of these interactions, enabling the correlation of structure with adsorption behavior. Here, techniques developed for in situ single-crystal X-ray diffraction experiments on porous crystals have enabled the direct observation of CO, CH 4 , N 2 , O 2 , Ar, and P 4 adsorption in Co 2 (dobdc) (dobdc 4- = 2,5-dioxido-1,4-benzenedicarboxylate), a metal-organic framework bearing coordinatively unsaturated cobalt(ii) sites. All these molecules exhibit such weak interactions with the high-spin cobalt(ii) sites in the framework that no analogous molecular structures exist, demonstrating the utility of metal-organic frameworks as crystalline matrices for the isolation and structural determination of unstable species. Notably, the Co-CH 4 and Co-Ar interactions observed in Co 2 (dobdc) represent, to the best of our knowledge, the first single-crystal structure determination of a metal-CH 4 interaction and the first crystallographically characterized metal-Ar interaction. Analysis of low-pressure gas adsorption isotherms confirms that these gases exhibit mainly physisorptive interactions with the cobalt(ii) sites in Co 2 (dobdc), with differential enthalpies of adsorption as weak as -17(1) kJ mol -1 (for Ar). Moreover, the structures of Co 2 (dobdc)·3.8N 2 , Co 2 (dobdc)·5.9O 2 , and Co 2 (dobdc)·2.0Ar reveal the location of secondary (N 2 , O 2 , and Ar) and tertiary (O 2 ) binding sites in Co 2 (dobdc), while high-pressure CO 2 , CO, CH 4 , N 2 , and Ar adsorption isotherms show that these binding sites become more relevant at elevated pressures.

Highly selective isolation and purification of heme proteins in biological samples using multifunctional magnetic nanospheres.

PubMed

Liu, Yating; Li, Yan; Wei, Yun

2014-12-01

Magnetic particles with suitable surface modification are capable of binding proteins selectively, and magnetic separations have advantages of rapidity, convenience, and high selectivity. In this paper, new magnetic nanoparticles modified with imidazolium ionic liquid (Fe3O4 @SiO2 @ILs) were successfully fabricated. N-Methylimidazolium was immobilized onto silica-coated magnetic nanoparticles via γ-chloropropyl modification as a magnetic nanoadsorbent for heme protein separation. The particle size was about 90 nm without significant aggregation during the preparation process. Hemoglobin as one of heme proteins used in this experiment was compared with other nonheme proteins. It has been found that the magnetic nanoparticles can be used for more rapid, efficient, and specific adsorption of hemoglobin with a binding capacity as high as 5.78 mg/mg. In comparison with other adsorption materials of proteins in the previous reports, Fe3 O4 @SiO2 @ILs magnetic nanoparticles exhibit the excellent performance in isolation of heme proteins with higher binding capacity and selectivity. In addition, a short separation time makes the functionalized nanoparticles suitable for purifying unstable proteins, as well as having other potential applications in a variety of biomedical fields. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

High Fractional Occupancy of a Tandem Maf Recognition Element and Its Role in Long-Range β-Globin Gene Regulation

PubMed Central

Stees, Jared R.; Hossain, Mir A.; Sunose, Tomoki; Kudo, Yasushi; Pardo, Carolina E.; Nabilsi, Nancy H.; Darst, Russell P.; Poudyal, Rosha; Igarashi, Kazuhiko; Kladde, Michael P.

2015-01-01

Enhancers and promoters assemble protein complexes that ultimately regulate the recruitment and activity of RNA polymerases. Previous work has shown that at least some enhancers form stable protein complexes, leading to the formation of enhanceosomes. We analyzed protein-DNA interactions in the murine β-globin gene locus using the methyltransferase accessibility protocol for individual templates (MAPit). The data show that a tandem Maf recognition element (MARE) in locus control region (LCR) hypersensitive site 2 (HS2) reveals a remarkably high degree of occupancy during differentiation of mouse erythroleukemia cells. Most of the other transcription factor binding sites in LCR HS2 or in the adult β-globin gene promoter regions exhibit low fractional occupancy, suggesting highly dynamic protein-DNA interactions. Targeting of an artificial zinc finger DNA-binding domain (ZF-DBD) to the HS2 tandem MARE caused a reduction in the association of MARE-binding proteins and transcription complexes at LCR HS2 and the adult βmajor-globin gene promoter but did not affect expression of the βminor-globin gene. The data demonstrate that a stable MARE-associated footprint in LCR HS2 is important for the recruitment of transcription complexes to the adult βmajor-globin gene promoter during erythroid cell differentiation. PMID:26503787

A Water‐Soluble Tetraazaperopyrene Dye as Strong G‐Quadruplex DNA Binder

PubMed Central

Hahn, Lena

2016-01-01

Abstract The interactions of the water‐soluble tetraazaperopyrene dye 1 with ct‐DNA, duplex‐[(dAdT)12 ⋅(dAdT)12], duplex‐[(dGdC)12 ⋅(dGdC)12] as well as with two G‐quadruplex‐forming sequences, namely the human telomeric 22AG and the promotor sequence c‐myc, were investigated by means of UV/visible and fluorescence spectroscopy, isothermal titration calorimetry (ITC) and molecular docking studies. Dye 1 exhibits a high affinity for G‐quadruplex structures over duplex DNA structures. Furthermore, the ligand shows promising G‐quadruplex discrimination, with an affinity towards c‐myc of 2×107  m −1 (i.e., K d=50 nm), which is higher than for 22AG (4×106  m −1). The ITC data reveal that compound 1 interacts with c‐myc in a stoichiometric ratio of 1:1 but also indicate the presence of two identical lower affinity secondary binding sites per quadruplex. In 22AG, there are two high affinity binding sites per quadruplex, that is, one on each side, with a further four weaker binding sites. For both quadruplex structures, the high affinity interactions between compound 1 and the quadruplex‐forming nucleic acid structures are weakly endothermic. Molecular docking studies suggest an end‐stacking binding mode for compound 1 interacting with quadruplex structures, and a higher affinity for the parallel conformation of c‐myc than for the mixed‐hybrid conformation of 22AG. In addition, docking studies also suggest that the reduced affinity for duplex DNA structures is due to the non‐viability of an intercalative binding mode. PMID:26997208

Site-selective conjugation of an anticoagulant aptamer to recombinant albumins and maintenance of neonatal Fc receptor binding

NASA Astrophysics Data System (ADS)

Schmøkel, Julie; Voldum, Anders; Tsakiridou, Georgia; Kuhlmann, Matthias; Cameron, Jason; Sørensen, Esben S.; Wengel, Jesper; Howard, Kenneth A.

2017-05-01

Aptamers are an attractive molecular medicine that offers high target specificity. Nucleic acid-based aptamers, however, are prone to nuclease degradation and rapid renal excretion that require blood circulatory half-life extension enabling technologies. The long circulatory half-life, predominately facilitated by engagement with the cellular recycling neonatal Fc receptor (FcRn), and ligand transport properties of albumin promote it as an attractive candidate to improve the pharmacokinetic profile of aptamers. This study investigates the effect of Cys34 site-selective covalent attachment of a factor IXa anticoagulant aptamer on aptamer functionality and human FcRn (hFcRn) engagement using recombinant human albumin (rHA) of either a wild type (WT) or an engineered human FcRn high binding variant (HB). Albumin-aptamer conjugates, connected covalently through a heterobifunctional succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate linker, were successfully prepared and purified by high performance liquid chromatography as confirmed by gel electrophoresis band-shift analysis and matrix-assisted laser desorption/ionization time of flight. Minimal reduction (∼25%) in activity of WT-linked aptamer to that of aptamer alone was found using an anticoagulant activity assay measuring temporal levels of activated partial thrombin. Covalent albumin-aptamer conjugation, however, substantially compromized binding to hFcRn, to 10% affinity of that of non-conjugated WT, determined by biolayer interferometry. Binding could be rescued by aptamer conjugation to recombinant albumin engineered for higher FcRn affinity (HB) that exhibited an 8-fold affinity compared to WT alone. This work describes a novel albumin-based aptamer delivery system whose hFcRn binding can be increased using a HB engineered albumin.

Site-selective conjugation of an anticoagulant aptamer to recombinant albumins and maintenance of neonatal Fc receptor binding.

PubMed

Schmøkel, Julie; Voldum, Anders; Tsakiridou, Georgia; Kuhlmann, Matthias; Cameron, Jason; Sørensen, Esben S; Wengel, Jesper; Howard, Kenneth A

2017-05-19

Aptamers are an attractive molecular medicine that offers high target specificity. Nucleic acid-based aptamers, however, are prone to nuclease degradation and rapid renal excretion that require blood circulatory half-life extension enabling technologies. The long circulatory half-life, predominately facilitated by engagement with the cellular recycling neonatal Fc receptor (FcRn), and ligand transport properties of albumin promote it as an attractive candidate to improve the pharmacokinetic profile of aptamers. This study investigates the effect of Cys34 site-selective covalent attachment of a factor IXa anticoagulant aptamer on aptamer functionality and human FcRn (hFcRn) engagement using recombinant human albumin (rHA) of either a wild type (WT) or an engineered human FcRn high binding variant (HB). Albumin-aptamer conjugates, connected covalently through a heterobifunctional succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate linker, were successfully prepared and purified by high performance liquid chromatography as confirmed by gel electrophoresis band-shift analysis and matrix-assisted laser desorption/ionization time of flight. Minimal reduction (∼25%) in activity of WT-linked aptamer to that of aptamer alone was found using an anticoagulant activity assay measuring temporal levels of activated partial thrombin. Covalent albumin-aptamer conjugation, however, substantially compromized binding to hFcRn, to 10% affinity of that of non-conjugated WT, determined by biolayer interferometry. Binding could be rescued by aptamer conjugation to recombinant albumin engineered for higher FcRn affinity (HB) that exhibited an 8-fold affinity compared to WT alone. This work describes a novel albumin-based aptamer delivery system whose hFcRn binding can be increased using a HB engineered albumin.

Low-basicity 5-HT7 Receptor Agonists Synthesized Using the van Leusen Multicomponent Protocol.

PubMed

Hogendorf, Adam S; Hogendorf, Agata; Kurczab, Rafał; Satała, Grzegorz; Lenda, Tomasz; Walczak, Maria; Latacz, Gniewomir; Handzlik, Jadwiga; Kieć-Kononowicz, Katarzyna; Wierońska, Joanna M; Woźniak, Monika; Cieślik, Paulina; Bugno, Ryszard; Staroń, Jakub; Bojarski, Andrzej J

2017-05-04

A series of 5-aryl-1-alkylimidazole derivatives was synthesized using the van Leusen multicomponent reaction. The chemotype is the first example of low-basicity scaffolds exhibiting high affinity for 5-HT 7 receptor together with agonist function. The chosen lead compounds 3-(1-ethyl-1H-imidazol-5-yl)-5-iodo-1H-indole (AGH-107, 1o, K i 5-HT7  = 6 nM, EC 50  = 19 nM, 176-fold selectivity over 5-HT 1A R) and 1e (5-methoxy analogue, K i 5-HT7  = 30 nM, EC 50  = 60 nM) exhibited high selectivity over related CNS targets, high metabolic stability and low toxicity in HEK-293 and HepG2 cell cultures. A rapid absorption to the blood, high blood-brain barrier permeation and a very high peak concentration in the brain (C max  = 2723 ng/g) were found for 1o after i.p. (5 mg/kg) administration in mice. The compound was found active in novel object recognition test in mice, at 0.5, 1 and 5 mg/kg. Docking to 5-HT 7 R homology models indicated a plausible binding mode which explain the unusually high selectivity over the related CNS targets. Halogen bond formation between the most potent derivatives and the receptor is consistent with both the docking results and SAR. 5-Chlorine, bromine and iodine substitution resulted in a 13, 27 and 89-fold increase in binding affinities, respectively, and in enhanced 5-HT 1A R selectivity.

Water-soluble Manganese and Iron Mesotetrakis(carboxyl)porphyrin: DNA Binding, Oxidative Cleavage, and Cytotoxic Activities.

PubMed

Shi, Lei; Jiang, Yi-Yu; Jiang, Tao; Yin, Wei; Yang, Jian-Ping; Cao, Man-Li; Fang, Yu-Qi; Liu, Hai-Yang

2017-06-29

Two new water-soluble metal carboxyl porphyrins, manganese (III) meso -tetrakis (carboxyl) porphyrin and iron (III) meso -tetrakis (carboxyl) porphyrin, were synthesized and characterized. Their interactions with ct-DNA were investigated by UV-Vis titration, fluorescence spectra, viscosity measurement and CD spectra. The results showed they can strongly bind to ct-DNA via outside binding mode. Electrophoresis experiments revealed that both complexes can cleave pBR322 DNA efficiently in the presence of hydrogen peroxide, albeit 2-Mn exhibited a little higher efficiency. The inhibitor tests suggest the oxidative DNA cleavage by these two complexes may involve hydroxyl radical active intermediates. Notably, 2-Mn exhibited considerable photocytotoxicity against Hep G2 cell via triggering a significant generation of ROS and causing disruption of MMP after irradiation.

Influence of sulfhydryl sites on metal binding by bacteria

NASA Astrophysics Data System (ADS)

Nell, Ryan M.; Fein, Jeremy B.

2017-02-01

The role of sulfhydryl sites within bacterial cell envelopes is still unknown, but the sites may control the fate and bioavailability of metals. Organic sulfhydryl compounds are important complexing ligands in aqueous systems and they can influence metal speciation in natural waters. Though representing only approximately 5-10% of the total available binding sites on bacterial surfaces, sulfhydryl sites exhibit high binding affinities for some metals. Due to the potential importance of bacterial sulfhydryl sites in natural systems, metal-bacterial sulfhydryl site binding constants must be determined in order to construct accurate models of the fate and distribution of metals in these systems. To date, only Cd-sulfhydryl binding has been quantified. In this study, the thermodynamic stabilities of Mn-, Co-, Ni-, Zn-, Sr- and Pb-sulfhydryl bacterial cell envelope complexes were determined for the bacterial species Shewanella oneidensis MR-1. Metal adsorption experiments were conducted as a function of both pH, ranging from 5.0 to 7.0, and metal loading, from 0.5 to 40.0 μmol/g (wet weight) bacteria, in batch experiments in order to determine if metal-sulfhydryl binding occurs. Initially, the data were used to calculate the value of the stability constants for the important metal-sulfhydryl bacterial complexes for each metal-loading condition studied, assuming a single binding reaction for the dominant metal-binding site type under the pH conditions of the experiments. For most of the metals that we studied, these calculated stability constant values increased significantly with decreasing metal loading, strongly suggesting that our initial assumption was not valid and that more than one type of binding occurs at the assumed binding site. We then modeled each dataset with two distinct site types with identical acidity constants: one site with a high metal-site stability constant value, which we take to represent metal-sulfhydryl binding and which dominates under low metal loading conditions, and another more abundant site that we term non-sulfhydryl sites that becomes important at high metal loadings. The resulting calculated stability constants do not vary significantly as a function of metal loading and yield reasonable fits to the observed adsorption behaviors as a function of both pH and metal loading. We use the results to calculate the speciation of metals bound by the bacterial envelope in realistic bacteria-bearing, heavy metal contaminated systems in order to demonstrate the potential importance of metal-sulfhydryl binding in the budget of bacterially-adsorbed metals under low metal-loading conditions.

Structural basis for the ligand-binding specificity of fatty acid-binding proteins (pFABP4 and pFABP5) in gentoo penguin.

PubMed

Lee, Chang Woo; Kim, Jung Eun; Do, Hackwon; Kim, Ryeo-Ok; Lee, Sung Gu; Park, Hyun Ho; Chang, Jeong Ho; Yim, Joung Han; Park, Hyun; Kim, Il-Chan; Lee, Jun Hyuck

2015-09-11

Fatty acid-binding proteins (FABPs) are involved in transporting hydrophobic fatty acids between various aqueous compartments of the cell by directly binding ligands inside their β-barrel cavities. Here, we report the crystal structures of ligand-unbound pFABP4, linoleate-bound pFABP4, and palmitate-bound pFABP5, obtained from gentoo penguin (Pygoscelis papua), at a resolution of 2.1 Å, 2.2 Å, and 2.3 Å, respectively. The pFABP4 and pFABP5 proteins have a canonical β-barrel structure with two short α-helices that form a cap region and fatty acid ligand binding sites in the hydrophobic cavity within the β-barrel structure. Linoleate-bound pFABP4 and palmitate-bound pFABP5 possess different ligand-binding modes and a unique ligand-binding pocket due to several sequence dissimilarities (A76/L78, T30/M32, underlining indicates pFABP4 residues) between the two proteins. Structural comparison revealed significantly different conformational changes in the β3-β4 loop region (residues 57-62) as well as the flipped Phe60 residue of pFABP5 than that in pFABP4 (the corresponding residue is Phe58). A ligand-binding study using fluorophore displacement assays shows that pFABP4 has a relatively strong affinity for linoleate as compared to pFABP5. In contrast, pFABP5 exhibits higher affinity for palmitate than that for pFABP4. In conclusion, our high-resolution structures and ligand-binding studies provide useful insights into the ligand-binding preferences of pFABPs based on key protein-ligand interactions. Copyright © 2015 Elsevier Inc. All rights reserved.

Improving specific activity and thermostability of Escherichia coli phytase by structure-based rational design.

PubMed

Wu, Tzu-Hui; Chen, Chun-Chi; Cheng, Ya-Shan; Ko, Tzu-Ping; Lin, Cheng-Yen; Lai, Hui-Lin; Huang, Ting-Yung; Liu, Je-Ruei; Guo, Rey-Ting

2014-04-10

Escherichia coli phytase (EcAppA) which hydrolyzes phytate has been widely applied in the feed industry, but the need to improve the enzyme activity and thermostability remains. Here, we conduct rational design with two strategies to enhance the EcAppA performance. First, residues near the substrate binding pocket of EcAppA were modified according to the consensus sequence of two highly active Citrobacter phytases. One out of the eleven mutants, V89T, exhibited 17.5% increase in catalytic activity, which might be a result of stabilized protein folding. Second, the EcAppA glycosylation pattern was modified in accordance with the Citrobacter phytases. An N-glycosylation motif near the substrate binding site was disrupted to remove spatial hindrance for phytate entry and product departure. The de-glycosylated mutants showed 9.6% increase in specific activity. On the other hand, the EcAppA mutants that adopt N-glycosylation motifs from CbAppA showed improved thermostability that three mutants carrying single N-glycosylation motif exhibited 5.6-9.5% residual activity after treatment at 80°C (1.8% for wild type). Furthermore, the mutant carrying all three glycosylation motifs exhibited 27% residual activity. In conclusion, a successful rational design was performed to obtain several useful EcAppA mutants with better properties for further applications. Copyright © 2014 Elsevier B.V. All rights reserved.

Adsorption of biometals to monosodium titanate in biological environments

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

HOBBS, D.T.; MESSER, R. L. W.; LEWIS, J. B.

2005-06-06

Monosodium titanate (MST) is an inorganic sorbent/ion exchanger developed for the removal of radionuclides from nuclear wastes. We investigated the ability of MST to bind Cd(II), Hg(II), or Au(III) to establish the utility of MST for applications in environmental decontamination or medical therapy (drug delivery). Adsorption isotherms for MST were determined at pH 7-7.5 in water or phosphate-buffered saline. The extent of metal binding was determined spectroscopically by measuring the concentrations of the metals in solution before and after contact with the MST. Cytotoxic responses to MST were assessed using THP1 monocytes and succinate dehydrogenase activity. Monocytic activation by MSTmore » was assessed by TNF{alpha} secretion (ELISA) with or without lipopolysaccharide (LPS) activation. MST sorbed Cd(II), Hg(II), and Au(III) under conditions similar to that in physiological systems. MST exhibited the highest affinity for Cd(II) followed by Hg(II) and Au (III). MST (up to 100 mg/L) exhibited only minor (< 25% suppression of succinate dehydrogenase) cytotoxicity and did not trigger TNF{alpha} secretion nor modulate LPS-induced TNF{alpha} secretion from monocytes. MST exhibits high affinity for biometals with no significant biological liabilities in these introductory studies. MST deserves further scrutiny as a substance with the capacity to decontaminate biological environments or deliver metals in a controlled fashion.« less

Changes in conformational dynamics of basic side chains upon protein-DNA association.

PubMed

Esadze, Alexandre; Chen, Chuanying; Zandarashvili, Levani; Roy, Sourav; Pettitt, B Montgometry; Iwahara, Junji

2016-08-19

Basic side chains play major roles in recognition of nucleic acids by proteins. However, dynamic properties of these positively charged side chains are not well understood. In this work, we studied changes in conformational dynamics of basic side chains upon protein-DNA association for the zinc-finger protein Egr-1. By nuclear magnetic resonance (NMR) spectroscopy, we characterized the dynamics of all side-chain cationic groups in the free protein and in the complex with target DNA. Our NMR order parameters indicate that the arginine guanidino groups interacting with DNA bases are strongly immobilized, forming rigid interfaces. Despite the strong short-range electrostatic interactions, the majority of the basic side chains interacting with the DNA phosphates exhibited high mobility, forming dynamic interfaces. In particular, the lysine side-chain amino groups exhibited only small changes in the order parameters upon DNA-binding. We found a similar trend in the molecular dynamics (MD) simulations for the free Egr-1 and the Egr-1-DNA complex. Using the MD trajectories, we also analyzed side-chain conformational entropy. The interfacial arginine side chains exhibited substantial entropic loss upon binding to DNA, whereas the interfacial lysine side chains showed relatively small changes in conformational entropy. These data illustrate different dynamic characteristics of the interfacial arginine and lysine side chains. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Actions of alpha2 adrenoceptor ligands at alpha2A and 5-HT1A receptors: the antagonist, atipamezole, and the agonist, dexmedetomidine, are highly selective for alpha2A adrenoceptors.

PubMed

Newman-Tancredi, A; Nicolas, J P; Audinot, V; Gavaudan, S; Verrièle, L; Touzard, M; Chaput, C; Richard, N; Millan, M J

1998-08-01

This study examined the activity of chemically diverse alpha2 adrenoceptor ligands at recombinant human (h) and native rat (r) alpha2A adrenoceptors compared with 5-HT1A receptors. First, in competition binding experiments at h alpha2A and h5-HT1A receptors expressed in CHO cells, several compounds, including the antagonists 1-(2-pyrimidinyl)piperazine (1-PP), (+/-)-idazoxan, benalfocin (SKF 86466), yohimbine and RX 821,002, displayed preference for h alpha2A versus h5-HT1A receptors of only 1.4-, 3.6-, 4-, 10- and 11-fold, respectively (based on differences in pKi values). Clonidine, brimonidine (UK 14304), the benzopyrrolidine fluparoxan and the guanidines guanfacine and guanabenz exhibited intermediate selectivity (22- to 31-fold) for h alpha2A receptors. Only the antagonist atipamezole and the agonist dexmedetomidine (DMT) displayed high preference for alpha2 adrenoceptors (1290- and 91-fold, respectively). Second, the compounds were tested for their ability to induce h5-HT1A receptor-mediated G-protein activation, as indicated by the stimulation of [35S]GTPgammaS binding. All except atipamezole and RX 821,002 exhibited agonist activity, with potencies which correlated with their affinity for h5-HT1A receptors. Relative efficacies (Emax values) were 25-35% for guanabenz, guanfacine, WB 4101 and benalfocin, 50-65% for 1-PP, (+/-)-idazoxan and clonidine, and over 70% for fluparoxan, oxymetazoline and yohimbine (relative to 5-HT = 100%). Yohimbine-induced [35S]GTPgammaS binding was inhibited by the selective 5-HT1A receptor antagonist WAY 100,635. In contrast, RX 821,002 was the only ligand which exhibited antagonist activity at h5-HT1A receptors, inhibiting 5-HT-stimulated [35S]GTPgammaS binding. Atipamezole, which exhibited negligeable affinity for 5-HT1A receptors, was inactive. Third, the affinities for r alpha2A differed considerably from the affinities for h alpha2A receptors whereas the affinities for r5-HT1A differed much less from the affinities for h5-HT1A receptors. This affected markedly the affinity ratios of certain compounds. For example, (+/-)-idazoxan was only 3.6-fold selective for h alpha2A versus h5-HT1A but 51-fold selective for r alpha2A versus r5-HT1A receptors. Conversely, yohimbine was tenfold selective for h alpha2A versus h5-HT1A adrenoceptors but 4.2-fold selective for r alpha2A versus r5-HT1A receptors. Nevertheless, both atipamezole and DMT were highly selective for both rat and human alpha2A versus rat or human 5-HT1A receptors. In conclusion, these data indicate that: (1) the agonist DMT and the antagonist atipamezole are the ligands of choice to distinguish alpha2-mediated from 5-HT1A-mediated actions, whilst several of the other compounds show only low or modest selectivity for alpha2A over 5-HT1A receptors; (2) caution should be exercised in experimental and clinical interpretation of the actions of traditionally employed alpha2 ligands, such as clonidine, yohimbine and (+/-)-idazoxan, which exhibit marked agonist activity at 5-HT1A receptors.

Chiral mutagenesis of insulin. Contribution of the B20-B23 beta-turn to activity and stability.

PubMed

Nakagawa, Satoe H; Hua, Qing-xin; Hu, Shi-Quan; Jia, Wenhua; Wang, Shuhua; Katsoyannis, Panayotis G; Weiss, Michael A

2006-08-04

Insulin contains a beta-turn (residues B20-B23) interposed between two receptor-binding elements, the central alpha-helix of the B chain (B9-B19) and its C-terminal beta-strand (B24-B28). The turn contains conserved glycines at B20 and B23. Although insulin exhibits marked conformational variability among crystal forms, these glycines consistently maintain positive phi dihedral angles within a classic type-I beta-turn. Because the Ramachandran conformations of GlyB20 and GlyB23 are ordinarily forbidden to L-amino acids, turn architecture may contribute to structure or function. Here, we employ "chiral mutagenesis," comparison of corresponding D- and L-Ala substitutions, to investigate this turn. Control substitutions are introduced at GluB21, a neighboring residue exhibiting a conventional (negative) phi angle. The D- and L-Ala substitutions at B23 are associated with a marked stereospecific difference in activity. Whereas the D-AlaB23 analog retains native activity, the L analog exhibits a 20-fold decrease in receptor binding. By contrast, D- and L-AlaB20 analogs each exhibit high activity. Stereospecific differences between the thermodynamic stabilities of the analogs are nonetheless more pronounced at B20 (delta deltaG(u) 2.0 kcal/mole) than at B23 (delta deltaG(u) 0.7 kcal/mole). Control substitutions at B21 are well tolerated without significant stereospecificity. Chiral mutagenesis thus defines the complementary contributions of these conserved glycines to protein stability (GlyB20) or receptor recognition (GlyB23).

Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form

PubMed Central

Zheng, Heping; Mandal, Arabinda; Shumilin, Igor A.; Chordia, Mahendra D.; Panneerdoss, Subbarayalu; Herr, John C.; Minor, Wladek

2016-01-01

Sperm Lysozyme-Like Protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75Å in diameter with a 25Å central pore comprised of six monomers per helix turn repeating every 33Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally-observed SLLP1/SAS1B interaction involved in fertilization. PMID:26198801

Switch-on fluorescent/FRET probes to study human histidine triad nucleotide binding protein 1 (hHint1), a novel target for opioid tolerance and neuropathic pain.

PubMed

Shah, Rachit; Zhou, Andrew; Wagner, Carston R

2017-12-13

Histidine Triad Nucleotide Binding Protein 1 (Hint1) has emerged to be an important post-synaptic protein associated with a variety of central nervous system disorders such as pain, addiction, and schizophrenia. Recently, inhibition of histidine nucleotide binding protein 1 (Hint1) with a small nucleoside inhibitor has shown promise as a new therapeutic strategy for the treatment of neuropathic pain. Herein, we describe the first rationally designed small molecule switch-on probes with dual fluorescence and FRET properties to study Hint1. Two non-natural fluorescent nucleosides with a fluorescent lifetime of 20 and 25 ns were each coupled through a linker to the indole ring, i.e. probes 7 and 8. Both probes were found to be water soluble and quenched intramolecularly via photoinduced electron transfer (PET) resulting in minimal background fluorescence. Upon incubating with Hint1, compound 7 and 8 exhibited a 40- and 16-fold increase in the fluorescence intensity compared to the control. Compounds 7 and 8 bind Hint1 with a dissociation constant of 0.121 ± 0.02 and 2.2 ± 0.36 μM, respectively. We demonstrate that probe 8 exhibits a switch-on FRET property with an active site tryptophan residue (W123). We show the utility of probes in performing quantitative ligand displacement studies, as well as in selective detection of Hint1 in the cell lysates. These probes should be useful for studying the dynamics of the active site, as well as for the development of fluorescence lifetime based high throughput screening assay to identify novel inhibitors for Hint1 in future.

Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form.

PubMed

Zheng, H; Mandal, A; Shumilin, I A; Chordia, M D; Panneerdoss, S; Herr, J C; Minor, W

2015-07-01

Sperm lysozyme-like protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15 Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75 Å in diameter with a 25 Å central pore comprised of six monomers per helix turn repeating every 33 Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan-binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan-binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally observed SLLP1/SAS1B interaction involved in fertilization. © 2015 American Society of Andrology and European Academy of Andrology.

A BPTTF-based self-assembled electron-donating triangle capable of C60 binding.

PubMed

Goeb, Sébastien; Bivaud, Sébastien; Dron, Paul Ionut; Balandier, Jean-Yves; Chas, Marcos; Sallé, Marc

2012-03-25

A kinetically stable self-assembled redox-active triangle is isolated. The resulting electron-donating cavity, which incorporates three BPTTF units, exhibits a remarkable binding ability for electron-deficient C(60), supported by a favorable combination of structural and electronic features.

17 CFR 230.402 - Number of copies; binding; signatures.

Code of Federal Regulations, 2010 CFR

2010-04-01

...; signatures. 230.402 Section 230.402 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION...; binding; signatures. (a) Three copies of the complete registration statement, including exhibits and all... bound and may contain facsimile versions of manual signatures in accordance with paragraph (e) of this...

The cytotoxic effect of spiroflavanone derivatives, their binding ability to human serum albumin (HSA) and a DFT study on the mechanism of their synthesis

NASA Astrophysics Data System (ADS)

Budzisz, Elzbieta; Paneth, Piotr; Geromino, Inacrist; Muzioł, Tadeusz; Rozalski, Marek; Krajewska, Urszula; Pipiak, Paulina; Ponczek, Michał B.; Małecka, Magdalena; Kupcewicz, Bogumiła

2017-06-01

This paper examines the cytotoxic effect of nine compounds with spiropyrazoline structures, and determines the reaction mechanism between diazomethane and selected benzylideneflavanones, their lipophilicity, and their binding ability to human serum albumin. The cytotoxic effect was determined on two human leukaemia cell lines (HL-60 and NALM-6) and melanoma WM-115 cells, as well as on normal human umbilical vein endothelial cells (HUVEC). The highest cytotoxicity was exhibited by compound B7: it was found to have an IC50 of less than 10 μM for all three cancer cell lines, with five to 12-fold lower sensitivity against normal cells (HUVEC). All the compounds exhibit comparable affinity energy in human serum albumin binding (from -8.1 to -8.6 kcal mol-1) but vary in their binding sites depending on the substituent. X-ray crystallography of two derivatives confirmed their synthetic pathway, and their structures were carefully examined.

Cooperative binding of Annexin A5 to phosphatidylserine on apoptotic cell membranes

NASA Astrophysics Data System (ADS)

Janko, Christina; Jeremic, Ivica; Biermann, Mona; Chaurio, Ricardo; Schorn, Christine; Muñoz, Luis E.; Herrmann, Martin

2013-12-01

Healthy cells exhibit an asymmetric plasma membrane with phosphatidylserine (PS) located on the cytoplasmic leaflet of the plasma membrane bilayer. Annexin A5-FITC, a PS binding protein, is commonly used to evaluate apoptosis in flow cytometry. PS exposed by apoptotic cells serves as a major ‘eat-me’ signal for phagocytes. Although exposition of PS has been observed after alternative stimuli, no clearance of viable, PS exposing cells has been detected. Thus, besides PS exposure, membranes of viable and apoptotic cells might exhibit specific characteristics. Here, we show that Annexin A5 binds in a cooperative manner to different types of dead cells. Shrunken apoptotic cells thereby showed the highest Hill coefficient values. Contrarily, parafomaldehyde fixation of apoptotic cells completely abrogates the cooperativity effect seen with dead and dying cells. We tend to speculate that the cooperative binding of Annexin A5 to the membranes of apoptotic cells reflects higher fluidity of the exposed membranes facilitating PS clustering.

Evidence for co-operativity in coenzyme binding to tetrameric Sulfolobus solfataricus alcohol dehydrogenase and its structural basis: fluorescence, kinetic and structural studies of the wild-type enzyme and non-co-operative N249Y mutant.

PubMed

Giordano, Antonietta; Febbraio, Ferdinando; Russo, Consiglia; Rossi, Mosè; Raia, Carlo A

2005-06-01

The interaction of coenzyme with thermostable homotetrameric NAD(H)-dependent alcohol dehydrogenase from the thermoacidophilic sulphur-dependent crenarchaeon Sulfolobus solfataricus (SsADH) and its N249Y (Asn-249-->Tyr) mutant was studied using the high fluorescence sensitivity of its tryptophan residues Trp-95 and Trp-117 to the binding of coenzyme moieties. Fluorescence quenching studies performed at 25 degrees C show that SsADH exhibits linearity in the NAD(H) binding [the Hill coefficient (h) approximately 1) at pH 9.8 and at moderate ionic strength, in addition to positive co-operativity (h=2.0-2.4) at pH 7.8 and 6.8, and at pH 9.8 in the presence of salt. Furthermore, NADH binding is positively co-operative below 20 degrees C (h approximately 3) and negatively co-operative at 40-50 degrees C (h approximately 0.7), as determined at moderate ionic strength and pH 9.8. Steady-state kinetic measurements show that SsADH displays standard Michaelis-Menten kinetics between 35 and 45 degrees C, but exhibits positive and negative co-operativity for NADH oxidation below (h=3.3 at 20 degrees C) and above (h=0.7 at 70-80 degrees C) this range of temperatures respectively. However, N249Y SsADH displays non-co-operative behaviour in coenzyme binding under the same experimental conditions used for the wild-type enzyme. In loop 270-275 of the coenzyme domain and segments at the interface of dimer A-B, analyses of the wild-type and mutant SsADH structures identified the structural elements involved in the intersubunit communication and suggested a possible structural basis for co-operativity. This is the first report of co-operativity in a tetrameric ADH and of temperature-induced co-operativity in a thermophilic enzyme.

Predicted structure of MIF/CD74 and RTL1000/CD74 complexes.

PubMed

Meza-Romero, Roberto; Benedek, Gil; Leng, Lin; Bucala, Richard; Vandenbark, Arthur A

2016-04-01

Macrophage migration inhibitory factor (MIF) is a key cytokine in autoimmune and inflammatory diseases that attracts and then retains activated immune cells from the periphery to the tissues. MIF exists as a homotrimer and its effects are mediated through its primary receptor, CD74 (the class II invariant chain that exhibits a highly structured trimerization domain), present on class II expressing cells. Although a number of binding residues have been identified between MIF and CD74 trimers, their spatial orientation has not been established. Using a docking program in silico, we have modeled binding interactions between CD74 and MIF as well as CD74 and a competitive MIF inhibitor, RTL1000, a partial MHC class II construct that is currently in clinical trials for multiple sclerosis. These analyses revealed 3 binding sites on the MIF trimer that each were predicted to bind one CD74 trimer through interactions with two distinct 5 amino acid determinants. Surprisingly, predicted binding of one CD74 trimer to a single RTL1000 antagonist utilized the same two 5 residue determinants, providing strong suggestive evidence in support of the MIF binding regions on CD74. Taken together, our structural modeling predicts a new MIF(CD74)3 dodecamer that may provide the basis for increased MIF potency and the requirement for ~3-fold excess RTL1000 to achieve full antagonism.

Stopped-flow kinetic studies of poly(amidoamine) dendrimer-calf thymus DNA to form dendriplexes.

PubMed

Dey, Debabrata; Kumar, Santosh; Maiti, Souvik; Dhara, Dibakar

2013-11-07

Poly(amidoamine) (PAMAM) dendrimers are known to be highly efficient nonviral carriers in gene delivery. Dendrimer-mediated transfection is known to be a function of the dendrimer to DNA charge ratio as well as the size of the dendrimer. In the present study, the binding kinetics of four PAMAM dendrimers (G1, G2, G3, and G4) with calf thymus DNA (CT-DNA) has been studied using stopped-flow fluorescence spectroscopy. The effect of dendrimer-to-DNA charge ratio and dendrimer generation on the binding kinetics was investigated. In most cases, the results of dendrimer-CT-DNA binding can be explained by a two-step reaction mechanism: a rapid electrostatic binding between the dendrimer and DNA, followed by a conformational change of the dendrimer-DNA complex that ultimately leads to DNA condensation. It was observed that the charge ratio on the dendrimer and the DNA phosphate groups, as well as the dendrimer generation (size), has a marked effect on the kinetics of binding between the DNA and the dendrimers. The rate constant (k'1) of the first step was much higher compared to that of the second step (k'2), and both were found to increase with an increase in dendrimer concentration. Among the four generations of dendrimers, G4 exhibited significantly faster binding kinetics compared to the three smaller generation dendrimers.

Essential slow degrees of freedom in protein-surface simulations: A metadynamics investigation.

PubMed

Prakash, Arushi; Sprenger, K G; Pfaendtner, Jim

2018-03-29

Many proteins exhibit strong binding affinities to surfaces, with binding energies much greater than thermal fluctuations. When modelling these protein-surface systems with classical molecular dynamics (MD) simulations, the large forces that exist at the protein/surface interface generally confine the system to a single free energy minimum. Exploring the full conformational space of the protein, especially finding other stable structures, becomes prohibitively expensive. Coupling MD simulations with metadynamics (enhanced sampling) has fast become a common method for sampling the adsorption of such proteins. In this paper, we compare three different flavors of metadynamics, specifically well-tempered, parallel-bias, and parallel-tempering in the well-tempered ensemble, to exhaustively sample the conformational surface-binding landscape of model peptide GGKGG. We investigate the effect of mobile ions and ion charge, as well as the choice of collective variable (CV), on the binding free energy of the peptide. We make the case for explicitly biasing ions to sample the true binding free energy of biomolecules when the ion concentration is high and the binding free energies of the solute and ions are similar. We also make the case for choosing CVs that apply bias to all atoms of the solute to speed up calculations and obtain the maximum possible amount of information about the system. Copyright © 2017 Elsevier Inc. All rights reserved.

A human transcription factor in search mode.

PubMed

Hauser, Kevin; Essuman, Bernard; He, Yiqing; Coutsias, Evangelos; Garcia-Diaz, Miguel; Simmerling, Carlos

2016-01-08

Transcription factors (TF) can change shape to bind and recognize DNA, shifting the energy landscape from a weak binding, rapid search mode to a higher affinity recognition mode. However, the mechanism(s) driving this conformational change remains unresolved and in most cases high-resolution structures of the non-specific complexes are unavailable. Here, we investigate the conformational switch of the human mitochondrial transcription termination factor MTERF1, which has a modular, superhelical topology complementary to DNA. Our goal was to characterize the details of the non-specific search mode to complement the crystal structure of the specific binding complex, providing a basis for understanding the recognition mechanism. In the specific complex, MTERF1 binds a significantly distorted and unwound DNA structure, exhibiting a protein conformation incompatible with binding to B-form DNA. In contrast, our simulations of apo MTERF1 revealed significant flexibility, sampling structures with superhelical pitch and radius complementary to the major groove of B-DNA. Docking these structures to B-DNA followed by unrestrained MD simulations led to a stable complex in which MTERF1 was observed to undergo spontaneous diffusion on the DNA. Overall, the data support an MTERF1-DNA binding and recognition mechanism driven by intrinsic dynamics of the MTERF1 superhelical topology. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Characterization of dFOXO binding sites upstream of the Insulin Receptor P2 promoter across the Drosophila phylogeny

PubMed Central

Orengo, Dorcas J.; Aguadé, Montserrat

2017-01-01

The insulin/TOR signal transduction pathway plays a critical role in determining such important traits as body and organ size, metabolic homeostasis and life span. Although this pathway is highly conserved across the animal kingdom, the affected traits can exhibit important differences even between closely related species. Evolutionary studies of regulatory regions require the reliable identification of transcription factor binding sites. Here we have focused on the Insulin Receptor (InR) expression from its P2 promoter in the Drosophila genus, which in D. melanogaster is up-regulated by hypophosphorylated Drosophila FOXO (dFOXO). We have finely characterized this transcription factor binding sites in vitro along the 1.3 kb region upstream of the InR P2 promoter in five Drosophila species. Moreover, we have tested the effect of mutations in the characterized dFOXO sites of D. melanogaster in transgenic flies. The number of experimentally established binding sites varies across the 1.3 kb region of any particular species, and their distribution also differs among species. In D. melanogaster, InR expression from P2 is differentially affected by dFOXO binding sites at the proximal and distal halves of the species 1.3 kb fragment. The observed uneven distribution of binding sites across this fragment might underlie their differential contribution to regulate InR transcription. PMID:29200426

Regulation of uterine progesterone receptors by the nonsteroidal anti-androgen hydroxyflutamide

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

Chandrasekhar, Y.; Armstrong, D.T.

1991-07-01

The authors have recently reported that the anti-androgen hydroxyflutamide causes delayed implantation and exhibits antideciduogenic activity in the rat. The present experiments were conducted to examine whether hydroxyflutamide binds to the uterine progesterone receptors and/or alters the progesterone binding sites in the uterus. Cytosol and nuclear fractions from decidualized rat uterus were incubated with (3H)-R5020 without or with increasing concentrations of radioinert R5020, RU486, dihydrotestosterone, or hydroxyflutamide. From the log-dose inhibition curves, the relative binding affinity of both hydroxyflutamide and dihydrotestosterone was less than 0.1% and 2%, compared with R5020 (100%) for displacing (3H)-R5020 bound to uterine cytosol and nuclearmore » fractions, respectively. Injection of estradiol-17 beta (1 microgram/rat) to ovariectomized prepubertal rats induced a 1.85-fold increase in uterine weight by 24 h. Hydroxyflutamide at 2.5 or 5.0 mg did not significantly alter the estrogen-induced increase in uterine weight. Compared to vehicle alone, estrogen induced an approximately 5-fold increase in uterine cytosolic progesterone binding sites. Hydroxyflutamide at both 2.5- and 5.0-mg doses significantly attenuated the estrogen-induced elevation in uterine progesterone binding sites. These studies demonstrate that hydroxyflutamide does not bind with high affinity to progesterone receptors, but suppresses the estrogen-induced elevation in progesterone receptor levels in the uterus.« less

Analyzing Intracellular Binding and Diffusion with Continuous Fluorescence Photobleaching

PubMed Central

Wachsmuth, Malte; Weidemann, Thomas; Müller, Gabriele; Hoffmann-Rohrer, Urs W.; Knoch, Tobias A.; Waldeck, Waldemar; Langowski, Jörg

2003-01-01

Transport and binding of molecules to specific sites are necessary for the assembly and function of ordered supramolecular structures in cells. For analyzing these processes in vivo, we have developed a confocal fluorescence fluctuation microscope that allows both imaging of the spatial distribution of fluorescent molecules with confocal laser scanning microscopy and probing their mobility at specific positions in the cell with fluorescence correlation spectroscopy and continuous fluorescence photobleaching (CP). Because fluorescence correlation spectroscopy is restricted to rapidly diffusing particles and CP to slower processes, these two methods complement each other. For the analysis of binding-related contributions to mobility we have derived analytical expressions for the temporal behavior of CP curves from which the bound fraction and/or the dissociation rate or residence time at binding sites, respectively, can be obtained. In experiments, we investigated HeLa cells expressing different fluorescent proteins: Although enhanced green fluorescent protein (EGFP) shows high mobility, fusions of histone H2B with the yellow fluorescent protein are incorporated into chromatin, and these nuclei exhibit the presence of a stably bound and a freely diffusing species. Nonpermanent binding was found for mTTF-I, a transcription termination factor for RNA polymerase I, fused with EGFP. The cells show fluorescent nucleoli, and binding is transient. CP yields residence times for mTTF-I-EGFP of ∼13 s. PMID:12719264

Analyzing intracellular binding and diffusion with continuous fluorescence photobleaching.

PubMed

Wachsmuth, Malte; Weidemann, Thomas; Müller, Gabriele; Hoffmann-Rohrer, Urs W; Knoch, Tobias A; Waldeck, Waldemar; Langowski, Jörg

2003-05-01

Transport and binding of molecules to specific sites are necessary for the assembly and function of ordered supramolecular structures in cells. For analyzing these processes in vivo, we have developed a confocal fluorescence fluctuation microscope that allows both imaging of the spatial distribution of fluorescent molecules with confocal laser scanning microscopy and probing their mobility at specific positions in the cell with fluorescence correlation spectroscopy and continuous fluorescence photobleaching (CP). Because fluorescence correlation spectroscopy is restricted to rapidly diffusing particles and CP to slower processes, these two methods complement each other. For the analysis of binding-related contributions to mobility we have derived analytical expressions for the temporal behavior of CP curves from which the bound fraction and/or the dissociation rate or residence time at binding sites, respectively, can be obtained. In experiments, we investigated HeLa cells expressing different fluorescent proteins: Although enhanced green fluorescent protein (EGFP) shows high mobility, fusions of histone H2B with the yellow fluorescent protein are incorporated into chromatin, and these nuclei exhibit the presence of a stably bound and a freely diffusing species. Nonpermanent binding was found for mTTF-I, a transcription termination factor for RNA polymerase I, fused with EGFP. The cells show fluorescent nucleoli, and binding is transient. CP yields residence times for mTTF-I-EGFP of approximately 13 s.

Structural properties of the intrinsically disordered, multiple calcium ion-binding otolith matrix macromolecule-64 (OMM-64).

PubMed

Poznar, Monika; Hołubowicz, Rafał; Wojtas, Magdalena; Gapiński, Jacek; Banachowicz, Ewa; Patkowski, Adam; Ożyhar, Andrzej; Dobryszycki, Piotr

2017-11-01

Fish otoliths are calcium carbonate biominerals that are involved in hearing and balance sensing. An organic matrix plays a crucial role in their formation. Otolith matrix macromolecule-64 (OMM-64) is a highly acidic, calcium-binding protein (CBP) found in rainbow trout otoliths. It is a component of high-molecular-weight aggregates, which influence the size, shape and polymorph of calcium carbonate in vitro. In this study, a protocol for the efficient expression and purification of OMM-64 was developed. For the first time, the complete structural characteristics of OMM-64 were described. Various biophysical methods were combined to show that OMM-64 occurs as an intrinsically disordered monomer. Under denaturing conditions (pH, temperature) OMM-64 exhibits folding propensity. It was determined that OMM-64 binds approximately 61 calcium ions with millimolar affinity. The folding-unfolding experiments showed that calcium ions induced the collapse of OMM-64. The effect of other counter ions present in trout endolymph on OMM-64 conformational changes was studied. The significance of disordered properties of OMM-64 and the possible function of this protein is discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo

PubMed Central

Plamont, Marie-Aude; Billon-Denis, Emmanuelle; Maurin, Sylvie; Gauron, Carole; Pimenta, Frederico M.; Specht, Christian G.; Shi, Jian; Quérard, Jérôme; Pan, Buyan; Rossignol, Julien; Moncoq, Karine; Morellet, Nelly; Volovitch, Michel; Lescop, Ewen; Chen, Yong; Triller, Antoine; Vriz, Sophie; Le Saux, Thomas; Jullien, Ludovic; Gautier, Arnaud

2016-01-01

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling. PMID:26711992

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo.

PubMed

Plamont, Marie-Aude; Billon-Denis, Emmanuelle; Maurin, Sylvie; Gauron, Carole; Pimenta, Frederico M; Specht, Christian G; Shi, Jian; Quérard, Jérôme; Pan, Buyan; Rossignol, Julien; Moncoq, Karine; Morellet, Nelly; Volovitch, Michel; Lescop, Ewen; Chen, Yong; Triller, Antoine; Vriz, Sophie; Le Saux, Thomas; Jullien, Ludovic; Gautier, Arnaud

2016-01-19

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

Synthesis and Monkey-PET Study of (R)- and (S)-18F-Labeled 2-Arylbenzoheterocyclic Derivatives as Amyloid Probes with Distinctive in Vivo Kinetics.

PubMed

Yang, Yanping; Wang, Xuedan; Yang, Hui; Fu, Hualong; Zhang, Jinming; Zhang, Xiaojun; Dai, Jiapei; Zhang, Zhiyong; Lin, Chunping; Guo, Yuzhi; Cui, Mengchao

2016-11-07

This study describes an effective strategy to improve pharmacokinetics of Aβ imaging agents, offering a novel class of (R)- and (S)- 18 F-labeled 2-arylbenzoheterocyclic derivatives which bear an additional chiral hydroxyl group on the side chain. These ligands displayed binding abilities toward Aβ aggregates with K i values ranging from 3.2 to 195.6 nM. Chirality-related discrepancy was observed in biodistribution, and (S)-2-phenylbenzoxazole enantiomers exhibited vastly improved brain clearance with washout ratios higher than 20. Notably, (S)-[ 18 F]28 possessed high binding potency (K i = 7.6 nM) and exceptional brain kinetics (9.46% ID/g at 2 min, brain 2min /brain 60min = 27.8) that is superior to well-established [ 18 F]AV45. The excellent pharmacokinetics and low nonspecific binding of (S)-[ 18 F]28 were testified by dynamic PET/CT scans in monkey brains. In addition, (S)-[ 18 F]28 clearly labeled Aβ plaques both in vitro and ex vivo. These results might qualify (S)-[ 18 F]28 to detect Aβ plaques with high signal-to-noise ratio.

Linking CO 2 Sorption Performance to Polymer Morphology in Aminopolymer/Silica Composites through Neutron Scattering

DOE PAGES

Holewinski, Adam; Sakwa-Novak, Miles A.; Jones, Christopher W.

2015-08-26

Composites of poly(ethylenimine) (PEI) and mesoporous silica are effective, reversible adsorbents for CO 2, both from flue gas and in direct air-capture applications. The morphology of the PEI within the silica can strongly impact the overall carbon capture efficiency and rate of saturation. Here, we directly probe the spatial distribution of the supported polymer through small-angle neutron scattering (SANS). Combined with textural characterization from physisorption analysis, the data indicate that PEI first forms a thin conformal coating on the pore walls, but all additional polymer aggregates into plug(s) that grow along the pore axis. This model is consistent with observedmore » trends in amine-efficiency (CO 2/N binding ratio) and pore size distributions, and points to a trade-off between achieving high chemical accessibility of the amine binding sites, which are inaccessible when they strongly interact with the silica, and high accessibility for mass transport, which can be hampered by diffusion through PEI plugs. In conclusion, we illustrate this design principle by demonstrating higher CO 2 capacity and uptake rate for PEI supported in a hydrophobically modified silica, which exhibits repulsive interactions with the PEI, freeing up binding sites.« less

Design of HIV-1 Protease Inhibitors with Amino-bis-tetrahydrofuran Derivatives as P2-Ligands to Enhance Backbone-Binding Interactions. Synthesis, Biological Evaluation, and Protein-Ligand X-ray Studies

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

Ghosh, Arun K.; Martyr, Cuthbert D.; Osswald, Heather L.

Structure-based design, synthesis, and biological evaluation of a series of very potent HIV-1 protease inhibitors are described. In an effort to improve backbone ligand–binding site interactions, we have incorporated basic-amines at the C4 position of the bis-tetrahydrofuran (bis-THF) ring. We speculated that these substituents would make hydrogen bonding interactions in the flap region of HIV-1 protease. Synthesis of these inhibitors was performed diastereoselectively. A number of inhibitors displayed very potent enzyme inhibitory and antiviral activity. Inhibitors 25f, 25i, and 25j were evaluated against a number of highly-PI-resistant HIV-1 strains, and they exhibited improved antiviral activity over darunavir. Two high resolutionmore » X-ray structures of 25f- and 25g-bound HIV-1 protease revealed unique hydrogen bonding interactions with the backbone carbonyl group of Gly48 as well as with the backbone NH of Gly48 in the flap region of the enzyme active site. These ligand–binding site interactions are possibly responsible for their potent activity.« less

Binding of ReO[subscript 4];#8722; with an engineered MoO[subscript 4 superscript 2];#8722;-binding protein: towards a new approach in radiopharmaceutical applications

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

Aryal, Baikuntha P.; Brugarolas, Pedro; He, Chuan

2012-05-25

Radiolabeled biomolecules are routinely used for clinical diagnostics. {sup 99m}Tc is the most commonly used radioactive tracer in radiopharmaceuticals. {sup 188}Re and {sup 186}Re are also commonly used as radioactive tracers in medicine. However, currently available methods for radiolabeling are lengthy and involve several steps in bioconjugation processes. In this work we present a strategy to engineer proteins that may selectively recognize the perrhenate (ReO{sub 4}{sup -}) ion as a new way to label proteins. We found that a molybdate (MoO{sub 4}{sup 2-})-binding protein (ModA) from Escherichia coli can bind perrhenate with high affinity. Using fluorescence and isothermal titration calorimetrymore » measurements, we determined the dissociation constant of ModA for ReO{sub 4}{sup -} to be 541 nM and we solved a crystal structure of ModA with a bound ReO{sub 4}{sup -}. On the basis of the structure we created a mutant protein containing a disulfide linkage, which exhibited increased affinity for perrhenate (K{sub d} = 104 nM). High-resolution crystal structures of ModA (1.7 {angstrom}) and A11C/R153C mutant (2.0 {angstrom}) were solved with bound perrhenate. Both structures show that a perrhenate ion occupies the molybdate binding site using the same amino acid residues that are involved in molybdate binding. The overall structure of the perrhenate-bound ModA is unchanged compared with that of the molybdate-bound form. In the mutant protein, the bound perrhenate is further stabilized by the engineered disulfide bond.« less

Characterization of Clostridium botulinum Type B Neurotoxin Associated with Infant Botulism in Japan

PubMed Central

Kozaki, Shunji; Kamata, Yoichi; Nishiki, Tei-ichi; Kakinuma, Hiroaki; Maruyama, Hiromi; Takahashi, Hiroaki; Karasawa, Tadahiro; Yamakawa, Kiyotaka; Nakamura, Shinichi

1998-01-01

The neurotoxin of strain 111 (111/NT) associated with type B infant botulism showed antigenic and biological properties different from that (Okra/NT) produced by a food-borne botulism-related strain, Okra. The specific toxicity of 111/NT was found to be about 10 times lower than that of Okra/NT. The monoclonal antibodies recognizing the light chain cross-reacted with both neurotoxins, whereas most of the antibodies recognizing the carboxyl-terminal half of the heavy chain of Okra/NT did not react to 111/NT. Binding experiments with rat brain synaptosomes revealed that 125I-labeled 111/NT bound to a single binding site with a dissociation constant (Kd) of 2.5 nM; the value was rather lower than that (0.42 nM) of 125I-Okra/NT for the high-affinity binding site. In the lipid vesicles reconstituted with ganglioside GT1b, 125I-Okra/NT interacted with the amino-terminal domain of synaptotagmin 1 (Stg1N) or synaptotagmin 2 (Stg2N), fused with the maltose-binding protein, in the same manner as the respective full-length synaptotagmins, and the Kd values accorded with those of the low- and high-affinity binding sites in synaptosomes. However, 125I-111/NT only exhibited a low capacity for binding to the lipid vesicles containing Stg2N, but not Stg1N, in the presence of ganglioside GT1b. Moreover, synaptobrevin-2, an intracellular target protein, was digested to the same extent by the light chains of both neurotoxins in a concentration-dependent manner. These findings indicate that the 111/NT molecule possesses the receptor-recognition site structurally different from Okra/NT, probably causing a decreased specific toxicity. PMID:9746583

Unraveling the Molecular Mechanism of Benzothiophene and Benzofuran scaffold merged compounds binding to anti-apoptotic Myeloid cell leukemia 1.

PubMed

Marimuthu, Parthiban; Singaravelu, Kalaimathy

2018-05-10

Myeloid cell leukemia 1 (Mcl1), is an anti-apoptotic member of the Bcl-2 family proteins, has gained considerable importance due to its overexpression activity prevents the oncogenic cells to undergo apoptosis. This overexpression activity of Mcl1 eventually develops strong resistance to a wide variety of anticancer agents. Therefore, designing novel inhibitors with potentials to elicit higher binding affinity and specificity to inhibit Mcl1 activity is of greater importance. Thus, Mcl1 acts as an attractive cancer target. Despite recent experimental advancement in the identification and characterization of Benzothiophene and Benzofuran scaffold merged compounds the molecular mechanisms of their binding to Mcl1 are yet to be explored. The current study demonstrates an integrated approach -pharmacophore-based 3D-QSAR, docking, Molecular Dynamics (MD) simulation and free-energy estimation- to access the precise and comprehensive effects of current inhibitors targeting Mcl1 together with its known activity values. The pharmacophore -ANRRR.240- based 3D-QSAR model from the current study provided high confidence (R 2 =0.9154, Q 2 =0.8736, and RMSE=0.3533) values. Furthermore, the docking correctly predicted the binding mode of highly active compound 42. Additionally, the MD simulation for docked complex under explicit-solvent conditions together with free-energy estimation exhibited stable interaction and binding strength over the time period. Also, the decomposition analysis revealed potential energy contributing residues -M231, M250, V253, R265, L267, and F270- to the complex stability. Overall, the current investigation might serve as a valuable insight, either to (i) improve the binding affinity of the current compounds or (ii) discover new generation anti-cancer agents that can effectively downregulate Mcl1 activity.

Subunit Dissociation and Metal Binding by Escherichia coli apo-Manganese Superoxide Dismutase

PubMed Central

Whittaker, Mei M.; Lerch, Thomas F.; Kirillova, Olga; Chapman, Michael S.; Whittaker, James W.

2010-01-01

Metal binding by apo-manganese superoxide dismutase (apo-MnSOD) is essential for functional maturation of the enzyme. Previous studies have demonstrated that metal binding by apo-MnSOD is conformationally gated, requiring protein reorganization for the metal to bind. We have now solved the X-ray crystal structure of apo-MnSOD at 1.9 Å resolution. The organization of active site residues is independent of the presence of the metal cofactor, demonstrating that protein itself templates the unusual metal coordination geometry. Electrophoretic analysis of mixtures of apo- and (Mn2)-MnSOD, dye-conjugated protein, or C-terminal Strep-tag II fusion protein reveals a dynamic subunit exchange process associated with cooperative metal binding by the two subunits of the dimeric protein. In contrast, (S126C) (SS) apo-MnSOD, which contains an inter-subunit covalent disulfide crosslink, exhibits anticooperative metal binding. The protein concentration dependence of metal uptake kinetics implies that protein dissociation is involved in metal binding by the wild type apo-protein, although other processes may also contribute to gating metal uptake. Protein concentration dependent small-zone size exclusion chromatography is consistent with apo-MnSOD dimer dissociation at low protein concentration (KD = 1×10−6 M). Studies on metal uptake by apo-MnSOD in Escherichia coli cells show that the protein exhibits similar behavior in vivo and in vitro. PMID:21044611

Stimulation of iodine organification in porcine thyroid cells by thyroid stimulators.

PubMed

Ginsberg, J; Shewring, G; Howells, R; Smith, B R; Hall, R

Several Graves' sera were simultaneously assessed in a bioassay based on the ability of porcine thyroid cells to organify 125I and in a radioreceptor assay for TSH receptor binding activity. Both assay systems were sensitive to 1 mcU/ml (final concentration) of unlabelled bovine TSH. Six Graves' sera were studied in detail over a wide (0-1.0 mcl sera) dose response range in repeat determinations. Two sera exhibited parallel binding and stimulating. However, two sera revealed significant inhibition of 125I-TSH binding prior to the demonstration of stimulation and the other two sera showed stimulatory capabilities before significant binding was evident. IgG was prepared from one serum by ammonium sulphate precipitation and chromatography on Sepharose 6B and then subjected to preparative isoelectric focusing. The isoelectric distribution of the two activities were found to be identical with major peaks of activity at pl=9.5 and pl=8.5. In summary: 1) each Graves' sera exhibits different dose-response curves with respect to binding and stimulation, 2) at certain concentrations of sera, only binding or stimulation were evident, 3) neither assay was consistently more sensitive for the presence of Graves' immunoglobulins, 4) for one Graves' sera, binding and stimulation could not be separated by isoelectric focusing. These studies would suggest each Graves' immunoglobulin has inherently different characteristics in its interaction with the TSH receptor.

Computer-aided rational design of novel EBF analogues with an aromatic ring.

PubMed

Wang, Shanshan; Sun, Yufeng; Du, Shaoqing; Qin, Yaoguo; Duan, Hongxia; Yang, Xinling

2016-06-01

Odorant binding proteins (OBPs) are important in insect olfactory recognition. These proteins bind specifically to insect semiochemicals and induce their seeking, mating, and alarm behaviors. Molecular docking and molecular dynamics simulations were performed to provide computational insight into the interaction mode between AgamOBP7 and novel (E)-β-farnesene (EBF) analogues with an aromatic ring. The ligand-binding cavity in OBP7 was found to be mostly hydrophobic due to the presence of several nonpolar residues. The interactions between the EBF analogues and the hydrophobic residues in the binding cavity increased in strength as the distance between them decreased. The EBF analogues with an N-methyl formamide or ester linkage had higher docking scores than those with an amide linkage. Moreover, delocalized π-π and electrostatic interactions were found to contribute significantly to the binding between the ligand benzene ring and nearby protein residues. To design new compounds with higher activity, four EBF analogues D1-D4 with a benzene ring were synthesized and evaluated based on their docking scores and binding affinities. D2, which had an N-methyl formamide group linkage, exhibited stronger binding than D1, which had an amide linkage. D4 exhibited particularly strong binding due to multiple hydrophobic interactions with the protein. This study provides crucial foundations for designing novel EBF analogues based on the OBP structure. Graphical abstract The design strategy of new EBF analogues based on the OBP7 structure.

Characterization of the APLF FHA–XRCC1 phosphopeptide interaction and its structural and functional implications

PubMed Central

Kim, Kyungmin; Pedersen, Lars C.; Kirby, Thomas W.; DeRose, Eugene F.

2017-01-01

Abstract Aprataxin and PNKP-like factor (APLF) is a DNA repair factor containing a forkhead-associated (FHA) domain that supports binding to the phosphorylated FHA domain binding motifs (FBMs) in XRCC1 and XRCC4. We have characterized the interaction of the APLF FHA domain with phosphorylated XRCC1 peptides using crystallographic, NMR, and fluorescence polarization studies. The FHA–FBM interactions exhibit significant pH dependence in the physiological range as a consequence of the atypically high pK values of the phosphoserine and phosphothreonine residues and the preference for a dianionic charge state of FHA-bound pThr. These high pK values are characteristic of the polyanionic peptides typically produced by CK2 phosphorylation. Binding affinity is greatly enhanced by residues flanking the crystallographically-defined recognition motif, apparently as a consequence of non-specific electrostatic interactions, supporting the role of XRCC1 in nuclear cotransport of APLF. The FHA domain-dependent interaction of XRCC1 with APLF joins repair scaffolds that support single-strand break repair and non-homologous end joining (NHEJ). It is suggested that for double-strand DNA breaks that have initially formed a complex with PARP1 and its binding partner XRCC1, this interaction acts as a backup attempt to intercept the more error-prone alternative NHEJ repair pathway by recruiting Ku and associated NHEJ factors. PMID:29059378

Methanobactin and the Link between Copper and Bacterial Methane Oxidation

PubMed Central

Semrau, Jeremy D.; Murrell, J. Colin; Gallagher, Warren H.; Dennison, Christopher; Vuilleumier, Stéphane

2016-01-01

SUMMARY Methanobactins (mbs) are low-molecular-mass (<1,200 Da) copper-binding peptides, or chalkophores, produced by many methane-oxidizing bacteria (methanotrophs). These molecules exhibit similarities to certain iron-binding siderophores but are expressed and secreted in response to copper limitation. Structurally, mbs are characterized by a pair of heterocyclic rings with associated thioamide groups that form the copper coordination site. One of the rings is always an oxazolone and the second ring an oxazolone, an imidazolone, or a pyrazinedione moiety. The mb molecule originates from a peptide precursor that undergoes a series of posttranslational modifications, including (i) ring formation, (ii) cleavage of a leader peptide sequence, and (iii) in some cases, addition of a sulfate group. Functionally, mbs represent the extracellular component of a copper acquisition system. Consistent with this role in copper acquisition, mbs have a high affinity for copper ions. Following binding, mbs rapidly reduce Cu2+ to Cu1+. In addition to binding copper, mbs will bind most transition metals and near-transition metals and protect the host methanotroph as well as other bacteria from toxic metals. Several other physiological functions have been assigned to mbs, based primarily on their redox and metal-binding properties. In this review, we examine the current state of knowledge of this novel type of metal-binding peptide. We also explore its potential applications, how mbs may alter the bioavailability of multiple metals, and the many roles mbs may play in the physiology of methanotrophs. PMID:26984926

Modulating the DNA affinity of Elk-1 with computationally selected mutations.

PubMed

Park, Sheldon; Boder, Eric T; Saven, Jeffery G

2005-04-22

In order to regulate gene expression, transcription factors must first bind their target DNA sequences. The affinity of this binding is determined by both the network of interactions at the interface and the entropy change associated with the complex formation. To study the role of structural fluctuation in fine-tuning DNA affinity, we performed molecular dynamics simulations of two highly homologous proteins, Elk-1 and SAP-1, that exhibit different sequence specificity. Simulation studies show that several residues in Elk have significantly higher main-chain root-mean-square deviations than their counterparts in SAP. In particular, a single residue, D69, may contribute to Elk's lower DNA affinity for P(c-fos) by structurally destabilizing the carboxy terminus of the recognition helix. While D69 does not contact DNA directly, the increased mobility in the region may contribute to its weaker binding. We measured the ability of single point mutants of Elk to bind P(c-fos) in a reporter assay, in which D69 of wild-type Elk has been mutated to other residues with higher helix propensity in order to stabilize the local conformation. The gains in transcriptional activity and the free energy of binding suggested from these measurements correlate well with stability gains computed from helix propensity and charge-macrodipole interactions. The study suggests that residues that are distal to the binding interface may indirectly modulate the binding affinity by stabilizing the protein scaffold required for efficient DNA interaction.

Specific Interactions of Antitumor Metallocenes with Deoxydinucleoside Monophosphates

NASA Astrophysics Data System (ADS)

Eberle, Rahel P.; Hari, Yvonne; Schürch, Stefan

2017-09-01

Bent metallocenes Cp2MCl2 (M = Ti, V, Nb, Mo) are known to exhibit cytotoxic activity against a variety of cancer types. Though the mechanism of action is not fully understood yet, the accumulation of the metal ions in the nucleus points towards DNA as one of the primary targets. A set of eight deoxydinucleoside monophosphates was used to study the adduct yields with metallocenes and cisplatin. The binding affinities are reflected by the relative intensities of the adducts and were found to follow the order of Pt > V > Ti > Mo (no adducts were detected with Nb). High-resolution tandem mass spectrometry was applied to locate the binding patterns in the deoxydinucleoside monophosphates. Whereas cisplatin binds to the soft nitrogen atoms in the purine nucleobases, the metallocenes additionally interact with the hard phosphate oxygen, which is in good agreement with the hard and soft (Lewis) acids and bases (HSAB) concept. However, the binding specificities were found to be unique for each metallocene. The hard Lewis acids titanium and vanadium predominantly bind to the deprotonated phosphate oxygen, whereas molybdenum, an intermediate Lewis acid, preferentially interacts with the nucleobases. Nucleobases comprise alternative binding sites for titanium and vanadium, presumably oxygen atoms for the first and nitrogen atoms for the latter. In summary, the intrinsic binding behavior of the different metallodrugs is reflected by the gas-phase dissociation of the adducts. Consequently, MS/MS can provide insights into therapeutically relevant interactions between metallodrugs and their cellular targets. [Figure not available: see fulltext.

Comprehensive understanding of acetohydroxyacid synthase inhibition by different herbicide families.

PubMed

Garcia, Mario D; Nouwens, Amanda; Lonhienne, Thierry G; Guddat, Luke W

2017-02-14

Five commercial herbicide families inhibit acetohydroxyacid synthase (AHAS, E.C. 2.2.1.6), which is the first enzyme in the branched-chain amino acid biosynthesis pathway. The popularity of these herbicides is due to their low application rates, high crop vs. weed selectivity, and low toxicity in animals. Here, we have determined the crystal structures of Arabidopsis thaliana AHAS in complex with two members of the pyrimidinyl-benzoate (PYB) and two members of the sulfonylamino-carbonyl-triazolinone (SCT) herbicide families, revealing the structural basis for their inhibitory activity. Bispyribac, a member of the PYBs, possesses three aromatic rings and these adopt a twisted "S"-shaped conformation when bound to A. thaliana AHAS ( At AHAS) with the pyrimidinyl group inserted deepest into the herbicide binding site. The SCTs bind such that the triazolinone ring is inserted deepest into the herbicide binding site. Both compound classes fill the channel that leads to the active site, thus preventing substrate binding. The crystal structures and mass spectrometry also show that when these herbicides bind, thiamine diphosphate (ThDP) is modified. When the PYBs bind, the thiazolium ring is cleaved, but when the SCTs bind, ThDP is modified to thiamine 2-thiazolone diphosphate. Kinetic studies show that these compounds not only trigger reversible accumulative inhibition of AHAS, but also can induce inhibition linked with ThDP degradation. Here, we describe the features that contribute to the extraordinarily powerful herbicidal activity exhibited by four classes of AHAS inhibitors.

Comprehensive understanding of acetohydroxyacid synthase inhibition by different herbicide families

PubMed Central

Nouwens, Amanda; Lonhienne, Thierry G.; Guddat, Luke W.

2017-01-01

Five commercial herbicide families inhibit acetohydroxyacid synthase (AHAS, E.C. 2.2.1.6), which is the first enzyme in the branched-chain amino acid biosynthesis pathway. The popularity of these herbicides is due to their low application rates, high crop vs. weed selectivity, and low toxicity in animals. Here, we have determined the crystal structures of Arabidopsis thaliana AHAS in complex with two members of the pyrimidinyl-benzoate (PYB) and two members of the sulfonylamino-carbonyl-triazolinone (SCT) herbicide families, revealing the structural basis for their inhibitory activity. Bispyribac, a member of the PYBs, possesses three aromatic rings and these adopt a twisted “S”-shaped conformation when bound to A. thaliana AHAS (AtAHAS) with the pyrimidinyl group inserted deepest into the herbicide binding site. The SCTs bind such that the triazolinone ring is inserted deepest into the herbicide binding site. Both compound classes fill the channel that leads to the active site, thus preventing substrate binding. The crystal structures and mass spectrometry also show that when these herbicides bind, thiamine diphosphate (ThDP) is modified. When the PYBs bind, the thiazolium ring is cleaved, but when the SCTs bind, ThDP is modified to thiamine 2-thiazolone diphosphate. Kinetic studies show that these compounds not only trigger reversible accumulative inhibition of AHAS, but also can induce inhibition linked with ThDP degradation. Here, we describe the features that contribute to the extraordinarily powerful herbicidal activity exhibited by four classes of AHAS inhibitors. PMID:28137884

Mass Spectrometric Determination of ILPR G-quadruplex Binding Sites in Insulin and IGF-2

PubMed Central

Xiao, JunFeng

2009-01-01

The insulin-linked polymorphic region (ILPR) of the human insulin gene promoter region forms G-quadruplex structures in vitro. Previous studies show that insulin and insulin-like growth factor-2 (IGF-2) exhibit high affinity binding in vitro to 2-repeat sequences of ILPR variants a and h, but negligible binding to variant i. Two-repeat sequences of variants a and h form intramolecular G-quadruplex structures that are not evidenced for variant i. Here we report on the use of protein digestion combined with affinity capture and MALDI-MS detection to pinpoint ILPR binding sites in insulin and IGF-2. Peptides captured by ILPR variants a and h were sequenced by MALDI-MS/MS, LC-MS and in silico digestion. On-bead digestion of insulin-ILPR variant a complexes supported the conclusions. The results indicate that the sequence VCG(N)RGF is generally present in the captured peptides and is likely involved in the affinity binding interactions of the proteins with the ILPR G-quadruplexes. The significance of arginine in the interactions was studied by comparing the affinities of synthesized peptides VCGERGF and VCGEAGF with ILPR variant a. Peptides from other regions of the proteins that are connected through disulfide linkages were also detected in some capture experiments. Identification of binding sites could facilitate design of DNA binding ligands for capture and detection of insulin and IGF-2. The interactions may have biological significance as well. PMID:19747845

Genome-wide identification of novel expression signatures reveal distinct patterns and prevalence of binding motifs for p53, nuclear factor-κB and other signal transcription factors in head and neck squamous cell carcinoma

PubMed Central

Yan, Bin; Yang, Xinping; Lee, Tin-Lap; Friedman, Jay; Tang, Jun; Van Waes, Carter; Chen, Zhong

2007-01-01

Background Differentially expressed gene profiles have previously been observed among pathologically defined cancers by microarray technologies, including head and neck squamous cell carcinomas (HNSCCs). However, the molecular expression signatures and transcriptional regulatory controls that underlie the heterogeneity in HNSCCs are not well defined. Results Genome-wide cDNA microarray profiling of ten HNSCC cell lines revealed novel gene expression signatures that distinguished cancer cell subsets associated with p53 status. Three major clusters of over-expressed genes (A to C) were defined through hierarchical clustering, Gene Ontology, and statistical modeling. The promoters of genes in these clusters exhibited different patterns and prevalence of transcription factor binding sites for p53, nuclear factor-κB (NF-κB), activator protein (AP)-1, signal transducer and activator of transcription (STAT)3 and early growth response (EGR)1, as compared with the frequency in vertebrate promoters. Cluster A genes involved in chromatin structure and function exhibited enrichment for p53 and decreased AP-1 binding sites, whereas clusters B and C, containing cytokine and antiapoptotic genes, exhibited a significant increase in prevalence of NF-κB binding sites. An increase in STAT3 and EGR1 binding sites was distributed among the over-expressed clusters. Novel regulatory modules containing p53 or NF-κB concomitant with other transcription factor binding motifs were identified, and experimental data supported the predicted transcriptional regulation and binding activity. Conclusion The transcription factors p53, NF-κB, and AP-1 may be important determinants of the heterogeneous pattern of gene expression, whereas STAT3 and EGR1 may broadly enhance gene expression in HNSCCs. Defining these novel gene signatures and regulatory mechanisms will be important for establishing new molecular classifications and subtyping, which in turn will promote development of targeted therapeutics for HNSCC. PMID:17498291

Dynamic CRM occupancy reflects a temporal map of developmental progression.

PubMed

Wilczyński, Bartek; Furlong, Eileen E M

2010-06-22

Development is driven by tightly coordinated spatio-temporal patterns of gene expression, which are initiated through the action of transcription factors (TFs) binding to cis-regulatory modules (CRMs). Although many studies have investigated how spatial patterns arise, precise temporal control of gene expression is less well understood. Here, we show that dynamic changes in the timing of CRM occupancy is a prevalent feature common to all TFs examined in a developmental ChIP time course to date. CRMs exhibit complex binding patterns that cannot be explained by the sequence motifs or expression of the TFs themselves. The temporal changes in TF binding are highly correlated with dynamic patterns of target gene expression, which in turn reflect transitions in cellular function during different stages of development. Thus, it is not only the timing of a TF's expression, but also its temporal occupancy in refined time windows, which determines temporal gene expression. Systematic measurement of dynamic CRM occupancy may therefore serve as a powerful method to decode dynamic changes in gene expression driving developmental progression.

Novel N-allyl/propargyl tetrahydroquinolines: Synthesis via Three-component Cationic Imino Diels-Alder Reaction, Binding Prediction, and Evaluation as Cholinesterase Inhibitors.

PubMed

Rodríguez, Yeray A; Gutiérrez, Margarita; Ramírez, David; Alzate-Morales, Jans; Bernal, Cristian C; Güiza, Fausto M; Romero Bohórquez, Arnold R

2016-10-01

New N-allyl/propargyl 4-substituted 1,2,3,4-tetrahydroquinolines derivatives were efficiently synthesized using acid-catalyzed three components cationic imino Diels-Alder reaction (70-95%). All compounds were tested in vitro as dual acetylcholinesterase and butyryl-cholinesterase inhibitors and their potential binding modes, and affinity, were predicted by molecular docking and binding free energy calculations (∆G) respectively. The compound 4af (IC50 = 72 μm) presented the most effective inhibition against acetylcholinesterase despite its poor selectivity (SI = 2), while the best inhibitory activity on butyryl-cholinesterase was exhibited by compound 4ae (IC50 = 25.58 μm) with considerable selectivity (SI = 0.15). Molecular docking studies indicated that the most active compounds fit in the reported acetylcholinesterase and butyryl-cholinesterase active sites. Moreover, our computational data indicated a high correlation between the calculated ∆G and the experimental activity values in both targets. © 2016 The Authors Chemical Biology & Drug Design Published by John Wiley & Sons Ltd.

Development of binding assays for the SH2 domain of Grb7 and Grb2 using fluorescence polarization.

PubMed

Luzy, Jean-Philippe; Chen, Huixiong; Gril, Brunilde; Liu, Wang-Qing; Vidal, Michel; Perdereau, Dominique; Burnol, Anne-Françoise; Garbay, Christiane

2008-02-01

Adaptor proteins Grb7 and Grb2 have been implicated as being 2 potential therapeutic targets in several human cancers, especially those that overexpress ErbB2. These 2 proteins contain both a SH2 domain (Src homology 2) that binds to phosphorylated tyrosine residues contained within ErbB2 and other specific protein targets. Two assays based on enzyme-linked immunosorbent assay and fluorescence polarization methods have been developed and validated to find and rank inhibitors for both proteins binding to the pY(1139). Fluorescence polarization assays allowed the authors to determine quickly and reproducibly affinities of peptides from low nanomolar to high micromolar range and to compare them directly for Grb7 and Grb2. As a result, the assays have identified a known peptidomimetic Grb2 SH2 inhibitor (mAZ-pTyr-(alphaMe)pTyr-Asn-NH(2)) that exhibits the most potent affinity for the Grb7 SH2 domain described to date.

Structural insights into Aspergillus fumigatus lectin specificity: AFL binding sites are functionally non-equivalent.

PubMed

Houser, Josef; Komarek, Jan; Cioci, Gianluca; Varrot, Annabelle; Imberty, Anne; Wimmerova, Michaela

2015-03-01

The Aspergillus fumigatus lectin AFL was recently described as a new member of the AAL lectin family. As a lectin from an opportunistic pathogen, it might play an important role in the interaction of the pathogen with the human host. A detailed study of structures of AFL complexed with several monosaccharides and oligosaccharides, including blood-group epitopes, was combined with affinity data from SPR and discussed in the context of previous findings. Its six binding sites are non-equivalent, and owing to minor differences in amino-acid composition they exhibit a marked difference in specific ligand recognition. AFL displays a high affinity in the micromolar range towards oligosaccharides which were detected in plants and also those bound on the human epithelia. All of these results indicate AFL to be a complex member of the lectin family and a challenging target for future medical research and, owing to its binding properties, a potentially useful tool in specific biotechnological applications.

Conformational Plasticity in Broadly Neutralizing HIV-1 Antibodies Triggers Polyreactivity.

PubMed

Prigent, Julie; Jarossay, Annaëlle; Planchais, Cyril; Eden, Caroline; Dufloo, Jérémy; Kök, Ayrin; Lorin, Valérie; Vratskikh, Oxana; Couderc, Thérèse; Bruel, Timothée; Schwartz, Olivier; Seaman, Michael S; Ohlenschläger, Oliver; Dimitrov, Jordan D; Mouquet, Hugo

2018-05-29

Human high-affinity antibodies to pathogens often recognize unrelated ligands. The molecular origin and the role of this polyreactivity are largely unknown. Here, we report that HIV-1 broadly neutralizing antibodies (bNAbs) are frequently polyreactive, cross-reacting with non-HIV-1 molecules, including self-antigens. Mutating bNAb genes to increase HIV-1 binding and neutralization also results in de novo polyreactivity. Unliganded paratopes of polyreactive bNAbs with improved HIV-1 neutralization exhibit a conformational flexibility, which contributes to enhanced affinity of bNAbs to various HIV-1 envelope glycoproteins and non-HIV antigens. Binding adaptation of polyreactive bNAbs to the divergent ligands mainly involves hydrophophic interactions. Plasticity of bNAbs' paratopes may, therefore, facilitate accommodating divergent viral variants, but it simultaneously triggers promiscuous binding to non-HIV-1 antigens. Thus, a certain level of polyreactivity can be a mark of adaptable antibodies displaying optimal pathogens' recognition. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Synergistic activation of human pregnane X receptor by binary cocktails of pharmaceutical and environmental compounds

PubMed Central

Delfosse, Vanessa; Dendele, Béatrice; Huet, Tiphaine; Grimaldi, Marina; Boulahtouf, Abdelhay; Gerbal-Chaloin, Sabine; Beucher, Bertrand; Roecklin, Dominique; Muller, Christina; Rahmani, Roger; Cavaillès, Vincent; Daujat-Chavanieu, Martine; Vivat, Valérie; Pascussi, Jean-Marc; Balaguer, Patrick; Bourguet, William

2015-01-01

Humans are chronically exposed to multiple exogenous substances, including environmental pollutants, drugs and dietary components. Many of these compounds are suspected to impact human health, and their combination in complex mixtures could exacerbate their harmful effects. Here we demonstrate that a pharmaceutical oestrogen and a persistent organochlorine pesticide, both exhibiting low efficacy when studied separately, cooperatively bind to the pregnane X receptor, leading to synergistic activation. Biophysical analysis shows that each ligand enhances the binding affinity of the other, so the binary mixture induces a substantial biological response at doses at which each chemical individually is inactive. High-resolution crystal structures reveal the structural basis for the observed cooperativity. Our results suggest that the formation of ‘supramolecular ligands' within the ligand-binding pocket of nuclear receptors contributes to the synergistic toxic effect of chemical mixtures, which may have broad implications for the fields of endocrine disruption, toxicology and chemical risk assessment. PMID:26333997

Colorimetric detection of copper in water using a Schiff base derivative

NASA Astrophysics Data System (ADS)

Peralta Domínguez, D.; Ramos-Ortiz, G.; Maldonado, J. L.; Rodriguez, M.; Meneses-Nava, M. A.; Barbosa-Garcia, O.; Santillan, R.; Farfán, N.

2013-09-01

Organic molecular sensors have the advantage of being used through an easy, fast, economical and reliable optical method for detecting toxic metal ions in our environment. In this work, we present a simple but highly specific organic ligand compound 5-Chloro-2-((E)-((E)-3-(4-(dimethylamino)phenyl)allylidene)amino)phenol (L1) that acts as a colorimetric sensor for ions in a mixture of acetonitrile/water (ratio 10:1, v:v). Binding interaction between L1 and various metal-ions has been established by ultraviolet-visible spectroscopic measurements that indicate favorable coordination of the ligand with selective metal ions, particularly, with copper. These results showed that the electronic transition band shape of L1 change after binding with copper in aqueous solution. L1 exhibited binding-induced color changes from yellow to pink one detected by the naked eye. This new sensor presented 2.5 × 10-6 M as limit detection, even under the presence of other metal ions.

The synthetic diazonamide DZ-2384 has distinct effects on microtubule curvature and dynamics without neurotoxicity

PubMed Central

Wieczorek, Michal; Tcherkezian, Joseph; Bernier, Cynthia; Prota, Andrea E.; Chaaban, Sami; Rolland, Yannève; Godbout, Claude; Hancock, Mark A.; Arezzo, Joseph C.; Ocal, Ozhan; Rocha, Cecilia; Olieric, Natacha; Hall, Anita; Ding, Hui; Bramoullé, Alexandre; Annis, Matthew G.; Zogopoulos, George; Harran, Patrick G.; Wilkie, Thomas M.; Brekken, Rolf A.; Siegel, Peter M.; Steinmetz, Michel O.; Shore, Gordon C.; Brouhard, Gary J.; Roulston, Anne

2017-01-01

Microtubule-targeting agents (MTAs) are widely used anticancer agents, but toxicities such as neuropathy limit their clinical use. MTAs bind to and alter the stability of microtubules, causing cell death in mitosis. We describe DZ-2384, a preclinical compound that exhibits potent antitumor activity in models of multiple cancer types. It has an unusually high safety margin and lacks neurotoxicity in rats at effective plasma concentrations. DZ-2384 binds the vinca domain of tubulin in a distinct way, imparting structurally and functionally different effects on microtubule dynamics compared to other vinca-binding compounds. X-ray crystallography and electron microscopy studies demonstrate that DZ-2384 causes straightening of curved protofilaments, an effect proposed to favor polymerization of tubulin. Both DZ-2384 and the vinca alkaloid vinorelbine inhibit microtubule growth rate; however, DZ-2384 increases the rescue frequency and preserves the microtubule network in nonmitotic cells and in primary neurons. This differential modulation of tubulin results in a potent MTA therapeutic with enhanced safety. PMID:27856798

Atrial natriuretic peptide receptor heterogeneity and effects on cyclic GMP accumulation

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

Leitman, D.C.

1988-01-01

The effects of atrial natriuretic peptide (ANP), oxytocin (OT) and vasopressin (AVP) on guanylate cyclase activity and cyclic GMP accumulation were examined, since these hormones appear to be intimately associated with blood pressure and intravascular volume homeostasis. ANP was found to increase cyclic GMP accumulation in ten cell culture systems, which were derived from blood vessels, adrenal cortex, kidney, lung, testes and mammary gland. ANP receptors were characterized in intact cultured cells using {sup 125}I-ANP{sub 8-33}. Specific {sup 125}I-ANP binding was saturable and of high affinity. Scratchard analysis of the binding data for all cell types exhibited a straight line,more » indicating that these cells possessed a single class of binding sites. Despite the presence of linear Scatchard plots, these studies demonstrated that cultured cells possess two functionally and physically distinct ANP-binding sites. Most of the ANP-binding sites in cultured cells have a molecular size of 66,000 daltons under reducing conditions. The identification of cultured cell types in which hormones (ANP and oxytocin) regulate guanylate cyclase activity and increase cyclic GMP synthesis will provide valuable systems to determine the mechanisms of hormone-receptor coupling to guanylate cyclase and the cellular processes regulated by cyclic GMP.« less

MalE of Group A Streptococcus Participates in the Rapid Transport of Maltotriose and Longer Maltodextrins▿ †

PubMed Central

Shelburne, Samuel A.; Fang, Han; Okorafor, Nnaja; Sumby, Paul; Sitkiewicz, Izabela; Keith, David; Patel, Payal; Austin, Celest; Graviss, Edward A.; Musser, James M.; Chow, Dar-Chone

2007-01-01

Study of the maltose/maltodextrin binding protein MalE in Escherichia coli has resulted in fundamental insights into the molecular mechanisms of microbial transport. Whether gram-positive bacteria employ a similar pathway for maltodextrin transport is unclear. The maltodextrin binding protein MalE has previously been shown to be key to the ability of group A Streptococcus (GAS) to colonize the oropharynx, the major site of GAS infection in humans. Here we used a multifaceted approach to elucidate the function and binding characteristics of GAS MalE. We found that GAS MalE is a central part of a highly efficient maltodextrin transport system capable of transporting linear maltodextrins that are up to at least seven glucose molecules long. Of the carbohydrates tested, GAS MalE had the highest affinity for maltotriose, a major breakdown product of starch in the human oropharynx. The thermodynamics and fluorescence changes induced by GAS MalE-maltodextrin binding were essentially opposite those reported for E. coli MalE. Moreover, unlike E. coli MalE, GAS MalE exhibited no specific binding of maltose or cyclic maltodextrins. Our data show that GAS developed a transport system optimized for linear maltodextrins longer than two glucose molecules that has several key differences from its well-studied E. coli counterpart. PMID:17259319

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

Totoritis, Rachel; Duraiswami, Chaya; Taylor, Amy N.

The continual bacterial adaptation to antibiotics creates an ongoing medical need for the development of novel therapeutics. Polypeptide deformylase (PDF) is a highly conserved bacterial enzyme, which is essential for viability. It has previously been shown that PDF inhibitors represent a promising new area for the development of antimicrobial agents, and that many of the best PDF inhibitors demonstrate slow, time-dependent binding. To improve our understanding of the mechanistic origin of this time-dependent inhibition, we examined in detail the kinetics of PDF catalysis and inhibition by several different PDF inhibitors. Varying pH and solvent isotope led to clear changes inmore » time-dependent inhibition parameters, as did inclusion of NaCl, which binds to the active site metal of PDF. Quantitative analysis of these results demonstrated that the observed time dependence arises from slow binding of the inhibitors to the active site metal. However, we also found several metal binding inhibitors that exhibited rapid, non-time-dependent onset of inhibition. By a combination of structural and chemical modification studies, we show that metal binding is only slow when the rest of the inhibitor makes optimal hydrogen bonds within the subsites of PDF. Both of these interactions between the inhibitor and enzyme were found to be necessary to observe time-dependent inhibition, as elimination of either leads to its loss.« less

Substitutional impact on biological activity of new water soluble Ni(II) complexes: Preparation, spectral characterization, X-ray crystallography, DNA/protein binding, antibacterial activity and in vitro cytotoxicity.

PubMed

Umadevi, C; Kalaivani, P; Puschmann, H; Murugan, S; Mohan, P S; Prabhakaran, R

2017-02-01

A series of new water soluble nickel(II) complexes containing triphenylphosphine and 4-methoxysalicylaldehyde-4(N)-substituted thiosemicarbazones were synthesized and characterized. Crystallographic investigations confirmed the structure of the complexes (1-4) having the general structure [Ni(4-Msal-Rtsc)(PPh 3 )] (Where R=H (1); CH 3 (2); C 2 H 5 (3); C 6 H 5 (4)) which showed that thiosemicarbazone ligands coordinated to nickel(II) ion as ONS tridentate bibasic donor. DNA/BSA protein binding ability of the ligands and their new complexes were studied by taking calf-thymus DNA (CT-DNA) and Bovine serum albumin (BSA) through absorption and emission titrations. Ethidium bromide (EB) displacement study showed the intercalative binding trend of the complexes to DNA. From the albumin binding studies, the mechanism of quenching was found as static and the alterations in the secondary structure of BSA by the compounds were confirmed with synchronous spectral studies. The binding affinity of the complexes to CT-DNA and BSA has the order of [Ni(4-Msal-etsc)(PPh 3 )] (3) >[Ni(4-Msal-mtsc)(PPh 3 )] (2) >[Ni(4-Msal-tsc)(PPh 3 )] (1) >[Ni(4-Msal-ptsc)(PPh 3 )] (4). In vitro cytotoxicity of the complexes was tested on human lung cancer cells (A549), human cervical cancer cells (HeLa), human liver carcinoma cells (Hep G2). All the complexes exhibited significant activity against three cancer cells. Among them, complex 4 exhibited almost 2.5 fold activity than cisplatin in A549 and HepG2 cell lines. In HeLa cell line, the complexes exhibited significant activity which is less than cisplatin. While comparing the activity of the complexes in A549 and HepG2 cell lines it falls in the order 4>1>2>3>cisplatin. The results obtained from DNA, protein binding and cytotoxicity studies, it is concluded that the cytotoxicity of the complexes as determined by MTT assay were not unduly influenced by the complexes having different binding efficiency with DNA and protein. The complexes exhibited good spectrum of antibacterial activity against four pathogenic bacteria such as E. faecalis (gram +ve), S. aureus (gram +ve), E. coli (gram -ve) and P. aeruginosa (gram -ve). Copyright © 2016 Elsevier B.V. All rights reserved.

A web server for analysis, comparison and prediction of protein ligand binding sites.

PubMed

Singh, Harinder; Srivastava, Hemant Kumar; Raghava, Gajendra P S

2016-03-25

One of the major challenges in the field of system biology is to understand the interaction between a wide range of proteins and ligands. In the past, methods have been developed for predicting binding sites in a protein for a limited number of ligands. In order to address this problem, we developed a web server named 'LPIcom' to facilitate users in understanding protein-ligand interaction. Analysis, comparison and prediction modules are available in the "LPIcom' server to predict protein-ligand interacting residues for 824 ligands. Each ligand must have at least 30 protein binding sites in PDB. Analysis module of the server can identify residues preferred in interaction and binding motif for a given ligand; for example residues glycine, lysine and arginine are preferred in ATP binding sites. Comparison module of the server allows comparing protein-binding sites of multiple ligands to understand the similarity between ligands based on their binding site. This module indicates that ATP, ADP and GTP ligands are in the same cluster and thus their binding sites or interacting residues exhibit a high level of similarity. Propensity-based prediction module has been developed for predicting ligand-interacting residues in a protein for more than 800 ligands. In addition, a number of web-based tools have been integrated to facilitate users in creating web logo and two-sample between ligand interacting and non-interacting residues. In summary, this manuscript presents a web-server for analysis of ligand interacting residue. This server is available for public use from URL http://crdd.osdd.net/raghava/lpicom .

Analysis of SOX10 mutations identified in Waardenburg-Hirschsprung patients: Differential effects on target gene regulation.

PubMed

Chan, Kwok Keung; Wong, Corinne Kung Yen; Lui, Vincent Chi Hang; Tam, Paul Kwong Hang; Sham, Mai Har

2003-10-15

SOX10 is a member of the SOX gene family related by homology to the high-mobility group (HMG) box region of the testis-determining gene SRY. Mutations of the transcription factor gene SOX10 lead to Waardenburg-Hirschsprung syndrome (Waardenburg-Shah syndrome, WS4) in humans. A number of SOX10 mutations have been identified in WS4 patients who suffer from different extents of intestinal aganglionosis, pigmentation, and hearing abnormalities. Some patients also exhibit signs of myelination deficiency in the central and peripheral nervous systems. Although the molecular bases for the wide range of symptoms displayed by the patients are still not clearly understood, a few target genes for SOX10 have been identified. We have analyzed the impact of six different SOX10 mutations on the activation of SOX10 target genes by yeast one-hybrid and mammalian cell transfection assays. To investigate the transactivation activities of the mutant proteins, three different SOX target binding sites were introduced into luciferase reporter gene constructs and examined in our series of transfection assays: consensus HMG domain protein binding sites; SOX10 binding sites identified in the RET promoter; and Sox10 binding sites identified in the P0 promoter. We found that the same mutation could have different transactivation activities when tested with different target binding sites and in different cell lines. The differential transactivation activities of the SOX10 mutants appeared to correlate with the intestinal and/or neurological symptoms presented in the patients. Among the six mutant SOX10 proteins tested, much reduced transactivation activities were observed when tested on the SOX10 binding sites from the RET promoter. Of the two similar mutations X467K and 1400del12, only the 1400del12 mutant protein exhibited an increase of transactivation through the P0 promoter. While the lack of normal SOX10 mediated activation of RET transcription may lead to intestinal aganglionosis, overexpression of genes coding for structural myelin proteins such as P0 due to mutant SOX10 may explain the dysmyelination phenotype observed in the patients with an additional neurological disorder. Copyright 2003 Wiley-Liss, Inc.

Exploring the Molecular Design of Protein Interaction Sites with Molecular Dynamics Simulations and Free Energy Calculations†

PubMed Central

Liang, Shide; Li, Liwei; Hsu, Wei-Lun; Pilcher, Meaghan N.; Uversky, Vladimir; Zhou, Yaoqi; Dunker, A. Keith; Meroueh, Samy O.

2009-01-01

The significant work that has been invested toward understanding protein–protein interaction has not translated into significant advances in structure-based predictions. In particular redesigning protein surfaces to bind to unrelated receptors remains a challenge, partly due to receptor flexibility, which is often neglected in these efforts. In this work, we computationally graft the binding epitope of various small proteins obtained from the RCSB database to bind to barnase, lysozyme, and trypsin using a previously derived and validated algorithm. In an effort to probe the protein complexes in a realistic environment, all native and designer complexes were subjected to a total of nearly 400 ns of explicit-solvent molecular dynamics (MD) simulation. The MD data led to an unexpected observation: some of the designer complexes were highly unstable and decomposed during the trajectories. In contrast, the native and a number of designer complexes remained consistently stable. The unstable conformers provided us with a unique opportunity to define the structural and energetic factors that lead to unproductive protein–protein complexes. To that end we used free energy calculations following the MM-PBSA approach to determine the role of nonpolar effects, electrostatics and entropy in binding. Remarkably, we found that a majority of unstable complexes exhibited more favorable electrostatics than native or stable designer complexes, suggesting that favorable electrostatic interactions are not prerequisite for complex formation between proteins. However, nonpolar effects remained consistently more favorable in native and stable designer complexes reinforcing the importance of hydrophobic effects in protein–protein binding. While entropy systematically opposed binding in all cases, there was no observed trend in the entropy difference between native and designer complexes. A series of alanine scanning mutations of hot-spot residues at the interface of native and designer complexes showed less than optimal contacts of hot-spot residues with their surroundings in the unstable conformers, resulting in more favorable entropy for these complexes. Finally, disorder predictions revealed that secondary structures at the interface of unstable complexes exhibited greater disorder than the stable complexes. PMID:19113835

Mechanistic Characterization and Molecular Modeling of Hepatitis B Virus Polymerase Resistance to Entecavir

PubMed Central

Walsh, Ann W.; Langley, David R.; Colonno, Richard J.; Tenney, Daniel J.

2010-01-01

Background Entecavir (ETV) is a deoxyguanosine analog competitive inhibitor of hepatitis B virus (HBV) polymerase that exhibits delayed chain termination of HBV DNA. A high barrier to entecavir-resistance (ETVr) is observed clinically, likely due to its potency and a requirement for multiple resistance changes to overcome suppression. Changes in the HBV polymerase reverse-transcriptase (RT) domain involve lamivudine-resistance (LVDr) substitutions in the conserved YMDD motif (M204V/I ± L180M), plus an additional ETV-specific change at residues T184, S202 or M250. These substitutions surround the putative dNTP binding site or primer grip regions of the HBV RT. Methods/Principal Findings To determine the mechanistic basis for ETVr, wildtype, lamivudine-resistant (M204V, L180M) and ETVr HBVs were studied using in vitro RT enzyme and cell culture assays, as well as molecular modeling. Resistance substitutions significantly reduced ETV incorporation and chain termination in HBV DNA and increased the ETV-TP inhibition constant (Ki) for HBV RT. Resistant HBVs exhibited impaired replication in culture and reduced enzyme activity (kcat) in vitro. Molecular modeling of the HBV RT suggested that ETVr residue T184 was adjacent to and stabilized S202 within the LVDr YMDD loop. ETVr arose through steric changes at T184 or S202 or by disruption of hydrogen-bonding between the two, both of which repositioned the loop and reduced the ETV-triphosphate (ETV-TP) binding pocket. In contrast to T184 and S202 changes, ETVr at primer grip residue M250 was observed during RNA-directed DNA synthesis only. Experimentally, M250 changes also impacted the dNTP-binding site. Modeling suggested a novel mechanism for M250 resistance, whereby repositioning of the primer-template component of the dNTP-binding site shifted the ETV-TP binding pocket. No structural data are available to confirm the HBV RT modeling, however, results were consistent with phenotypic analysis of comprehensive substitutions of each ETVr position. Conclusions Altogether, ETVr occurred through exclusion of ETV-TP from the dNTP-binding site, through different, novel mechanisms that involved lamivudine-resistance, ETV-specific substitutions, and the primer-template. PMID:20169198

Mechanistic characterization and molecular modeling of hepatitis B virus polymerase resistance to entecavir.

PubMed

Walsh, Ann W; Langley, David R; Colonno, Richard J; Tenney, Daniel J

2010-02-12

Entecavir (ETV) is a deoxyguanosine analog competitive inhibitor of hepatitis B virus (HBV) polymerase that exhibits delayed chain termination of HBV DNA. A high barrier to entecavir-resistance (ETVr) is observed clinically, likely due to its potency and a requirement for multiple resistance changes to overcome suppression. Changes in the HBV polymerase reverse-transcriptase (RT) domain involve lamivudine-resistance (LVDr) substitutions in the conserved YMDD motif (M204V/I +/- L180M), plus an additional ETV-specific change at residues T184, S202 or M250. These substitutions surround the putative dNTP binding site or primer grip regions of the HBV RT. To determine the mechanistic basis for ETVr, wildtype, lamivudine-resistant (M204V, L180M) and ETVr HBVs were studied using in vitro RT enzyme and cell culture assays, as well as molecular modeling. Resistance substitutions significantly reduced ETV incorporation and chain termination in HBV DNA and increased the ETV-TP inhibition constant (K(i)) for HBV RT. Resistant HBVs exhibited impaired replication in culture and reduced enzyme activity (k(cat)) in vitro. Molecular modeling of the HBV RT suggested that ETVr residue T184 was adjacent to and stabilized S202 within the LVDr YMDD loop. ETVr arose through steric changes at T184 or S202 or by disruption of hydrogen-bonding between the two, both of which repositioned the loop and reduced the ETV-triphosphate (ETV-TP) binding pocket. In contrast to T184 and S202 changes, ETVr at primer grip residue M250 was observed during RNA-directed DNA synthesis only. Experimentally, M250 changes also impacted the dNTP-binding site. Modeling suggested a novel mechanism for M250 resistance, whereby repositioning of the primer-template component of the dNTP-binding site shifted the ETV-TP binding pocket. No structural data are available to confirm the HBV RT modeling, however, results were consistent with phenotypic analysis of comprehensive substitutions of each ETVr position. Altogether, ETVr occurred through exclusion of ETV-TP from the dNTP-binding site, through different, novel mechanisms that involved lamivudine-resistance, ETV-specific substitutions, and the primer-template.

Sex Differences in Serotonin 1 Receptor Binding in Rat Brain

NASA Astrophysics Data System (ADS)

Fischette, Christine T.; Biegon, Anat; McEwen, Bruce S.

1983-10-01

Male and female rats exhibit sex differences in binding by serotonin 1 receptors in discrete areas of the brain, some of which have been implicated in the control of ovulation and of gonadotropin release. The sex-specific changes in binding, which occur in response to the same hormonal (estrogenic) stimulus, are due to changes in the number of binding sites. Castration alone also affects the number of binding sites in certain areas. The results lead to the conclusion that peripheral hormones modulate binding by serotonin 1 receptors. The status of the serotonin receptor system may affect the reproductive capacity of an organism and may be related to sex-linked emotional disturbances in humans.

Mimetics of beta-galactosylceramide with simple ceramide substitutes: Synthesis and binding togp 120 of HIV-1, and, Enactment of chemistry knowledge by a high school student at a summer program

NASA Astrophysics Data System (ADS)

Augustin, Line A.

This thesis is the account of two research works. The first part reports the synthesis of O- and C- and aza-C-glycosides of beta-Galactosylceramide (GalCer) that contain simple ceramide substitutes, and the initial results of their binding with gp120 of HIV-1. The O-glycosides were prepared via an established procedure. The C- and aza-C-glycosides originated from a central C1-substituted galactal precursor, and their synthesis is illustrative of a potentially general method for pairs of C- and aza-C-beta-galactosides. They aza-C-glycoside with a simple C-17 hydrocarbon chain exhibited significant higher affinity than GalCer, whereas the corresponding C-glycoside was as active as GalCer. The second part describes the ethnographic study of the enactment of the chemistry knowledge of a high school student at a summer program and the influence of a cultural practice, othermothering, on her ability to perform well on her chemistry Regents Exams. Kelly, an 11th grade student exhibited very good understanding of the chemistry curriculum in the classroom, the laboratory period and the tutoring sessions where she plays a caring role for her peers. The same level of understanding was not reflected on the paper pencil exams taken during the summer program.

Intrinsic Nucleic Acid Dynamics Modulates HIV-1 Nucleocapsid Protein Binding to Its Targets

PubMed Central

Bazzi, Ali; Zargarian, Loussiné; Chaminade, Françoise; De Rocquigny, Hugues; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

2012-01-01

HIV-1 nucleocapsid protein (NC) is involved in the rearrangement of nucleic acids occurring in key steps of reverse transcription. The protein, through its two zinc fingers, interacts preferentially with unpaired guanines in single-stranded sequences. In mini-cTAR stem-loop, which corresponds to the top half of the cDNA copy of the transactivation response element of the HIV-1 genome, NC was found to exhibit a clear preference for the TGG sequence at the bottom of mini-cTAR stem. To further understand how this site was selected among several potential binding sites containing unpaired guanines, we probed the intrinsic dynamics of mini-cTAR using 13C relaxation measurements. Results of spin relaxation time measurements have been analyzed using the model-free formalism and completed by dispersion relaxation measurements. Our data indicate that the preferentially recognized guanine in the lower part of the stem is exempt of conformational exchange and highly mobile. In contrast, the unrecognized unpaired guanines of mini-cTAR are involved in conformational exchange, probably related to transient base-pairs. These findings support the notion that NC preferentially recognizes unpaired guanines exhibiting a high degree of mobility. The ability of NC to discriminate between close sequences through their dynamic properties contributes to understanding how NC recognizes specific sites within the HIV genome. PMID:22745685

Whole-Genome and Epigenomic Landscapes of Etiologically Distinct Subtypes of Cholangiocarcinoma

PubMed Central

Jusakul, Apinya; Cutcutache, Ioana; Yong, Chern Han; Lim, Jing Quan; Huang, Mi Ni; Padmanabhan, Nisha; Nellore, Vishwa; Kongpetch, Sarinya; Ng, Alvin Wei Tian; Ng, Ley Moy; Choo, Su Pin; Myint, Swe Swe; Thanan, Raynoo; Nagarajan, Sanjanaa; Lim, Weng Khong; Ng, Cedric Chuan Young; Boot, Arnoud; Liu, Mo; Ong, Choon Kiat; Rajasegaran, Vikneswari; Lie, Stefanus; Lim, Alvin Soon Tiong; Lim, Tse Hui; Tan, Jing; Loh, Jia Liang; McPherson, John R.; Khuntikeo, Narong; Bhudhisawasdi, Vajaraphongsa; Yongvanit, Puangrat; Wongkham, Sopit; Totoki, Yasushi; Nakamura, Hiromi; Arai, Yasuhito; Yamasaki, Satoshi; Chow, Pierce Kah-Hoe; Chung, Alexander Yaw Fui; Ooi, London Lucien Peng Jin; Lim, Kiat Hon; Dima, Simona; Duda, Dan G.; Popescu, Irinel; Broet, Philippe; Hsieh, Sen-Yung; Yu, Ming-Chin; Scarpa, Aldo; Lai, Jiaming; Luo, Di-Xian; Carvalho, André Lopes; Vettore, André Luiz; Rhee, Hyungjin; Park, Young Nyun; Alexandrov, Ludmil B.; Gordân, Raluca; Rozen, Steven G.; Shibata, Tatsuhiro; Pairojkul, Chawalit; Teh, Bin Tean; Tan, Patrick

2017-01-01

Cholangiocarcinoma (CCA) is a hepatobiliary malignancy exhibiting high incidence in countries with endemic liver-fluke infection. We analysed 489 CCAs from 10 countries, combining whole-genome (71 cases), targeted/exome, copy-number, gene expression, and DNA methylation information. Integrative clustering defined four CCA clusters – Fluke-Positive CCAs (Clusters 1/2) are enriched in ERBB2 amplifications and TP53 mutations, conversely Fluke-Negative CCAs (Clusters 3/4) exhibit high copy-number alterations and PD-1/PD-L2 expression, or epigenetic mutations (IDH1/2, BAP1) and FGFR/PRKA-related gene rearrangements. Whole-genome analysis highlighted FGFR2 3′UTR deletion as a mechanism of FGFR2 upregulation. Integration of non-coding promoter mutations with protein-DNA binding profiles demonstrates pervasive modulation of H3K27me3-associated sites in CCA. Clusters 1 and 4 exhibit distinct DNA hypermethylation patterns targeting either CpG islands or shores – mutation signature and subclonality analysis suggests that these reflect different mutational pathways. Our results exemplify how genetics, epigenetics and environmental carcinogens can interplay across different geographies to generate distinct molecular subtypes of cancer. PMID:28667006

Enhanced aging properties of HKUST-1 in hydrophobic mixed-matrix membranes for ammonia adsorption.

PubMed

DeCoste, Jared B; Denny, Michael S; Peterson, Gregory W; Mahle, John J; Cohen, Seth M

2016-04-21

Metal-organic frameworks (MOFs) in their free powder form have exhibited superior capacities for many gases when compared to other materials, due to their tailorable functionality and high surface areas. Specifically, the MOF HKUST-1 binds small Lewis bases, such as ammonia, with its coordinatively unsaturated copper sites. We describe here the use of HKUST-1 in mixed-matrix membranes (MMMs) prepared from polyvinylidene difluoride (PVDF) for the removal of ammonia gas. These MMMs exhibit ammonia capacities similar to their hypothetical capacities based on the weight percent of HKUST-1 in each MMM. HKUST-1 in its powder form is unstable toward humid conditions; however, upon exposure to humid environments for prolonged periods of time, the HKUST-1 MMMs exhibit outstanding structural stability, and maintain their ammonia capacity. Overall, this study has achieved all of the critical and combined elements for real-world applications of MOFs: high MOF loadings, fully accessible MOF surfaces, enhanced MOF stabilization, recyclability, mechanical stability, and processability. This study is a critical step in advancing MOFs to a stable, usable, and enabling technology.

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

Jusakul, Apinya; Cutcutache, Ioana; Yong, Chern Han

Cholangiocarcinoma (CCA) is a hepatobiliary malignancy exhibiting high incidence in countries with endemic liver-fluke infection. We analysed 489 CCAs from 10 countries, combining whole-genome (71 cases), targeted/exome, copy-number, gene expression, and DNA methylation information. Integrative clustering defined four CCA clusters - Fluke- Positive CCAs (Clusters 1/2) are enriched in ERBB2 amplifications and TP53 mutations, conversely Fluke-Negative CCAs (Clusters 3/4) exhibit high copy-number alterations and PD-1/PD-L2 expression, or epigenetic mutations (IDH1/2, BAP1) and FGFR/PRKA-related gene rearrangements. Whole-genome analysis highlighted FGFR2 3’UTR deletion as a mechanism of FGFR2 upregulation. Integration of non-coding promoter mutations with protein-DNA binding profiles demonstrates pervasive modulation ofmore » H3K27me3-associated sites in CCA. Clusters 1 and 4 exhibit distinct DNA hypermethylation patterns targeting either CpG islands or shores - mutation signature and subclonality analysis suggests that these reflect different mutational pathways. Lastly, our results exemplify how genetics, epigenetics and environmental carcinogens can interplay across different geographies to generate distinct molecular subtypes of cancer.« less

Synthesis and preliminary evaluation of [3H]PSB-0413, a selective antagonist radioligand for platelet P2Y12 receptors.

PubMed

El-Tayeb, Ali; Griessmeier, Kerstin J; Müller, Christa E

2005-12-15

The selective antagonist radioligand [(3)H]2-propylthioadenosine-5'-adenylic acid (1,1-dichloro-1-phosphonomethyl-1-phosphonyl) anhydride ([(3)H]PSB-0413) was prepared by catalytic hydrogenation of its propargyl precursor with a high specific radioactivity of 74Ci/mmol. In preliminary saturation binding studies, [(3)H]PSB-0413 showed high affinity for platelet P2Y(12) receptors with a K(D) value of 4.57nM. Human platelets had a high density of P2Y(12) receptors exhibiting a B(max) value of 7.66pmol/mg of protein.

New tris(dopamine) derivative as an iron chelator. Synthesis, solution thermodynamic stability, and antioxidant research.

PubMed

Zhang, Qingchun; Jin, Bo; Shi, Zhaotao; Wang, Xiaofang; Lei, Shan; Tang, Xingyan; Liang, Hua; Liu, Qiangqiang; Gong, Mei; Peng, Rufang

2017-06-01

A new tris(dopamine) derivative, containing three dopamine chelate moieties which were attached to a trimesic acid molecular scaffold, has been prepared and fully characterized by NMR, FTIR and HRMS. The solution thermodynamic stability of the chelator with Fe(III), Mg(II), Zn(II) and Fe(II) ions was investigated. Results demonstrated that the chelator exhibited effective binding ability and improved selectivity to Fe(III) ion. The chelator possessed affinity similar to that of diethylenetriaminepentaacetic acid chelator for Fe(III) ion. The high affinity could be attributed to the favorable geometric arrangement between the chelator and Fe(III) ion coordination preference. The chelator also exhibited high antioxidant activity and nontoxicity to neuron-like rat pheochromocytoma cells. Hence, the chelator could be used as chelating agent for iron overload situations without depleting essential metal ions, such as Mg(II) and Zn(II) ions. Copyright © 2017. Published by Elsevier Inc.

Prostate-specific membrane antigen targeted protein contrast agents for molecular imaging of prostate cancer by MRI

NASA Astrophysics Data System (ADS)

Pu, Fan; Salarian, Mani; Xue, Shenghui; Qiao, Jingjuan; Feng, Jie; Tan, Shanshan; Patel, Anvi; Li, Xin; Mamouni, Kenza; Hekmatyar, Khan; Zou, Juan; Wu, Daqing; Yang, Jenny J.

2016-06-01

Prostate-specific membrane antigen (PSMA) is one of the most specific cell surface markers for prostate cancer diagnosis and targeted treatment. However, achieving molecular imaging using non-invasive MRI with high resolution has yet to be achieved due to the lack of contrast agents with significantly improved relaxivity for sensitivity, targeting capabilities and metal selectivity. We have previously reported our creation of a novel class of protein Gd3+ contrast agents, ProCA32, which displayed significantly improved relaxivity while exhibiting strong Gd3+ binding selectivity over physiological metal ions. In this study, we report our effort in further developing biomarker-targeted protein MRI contrast agents for molecular imaging of PSMA. Among three PSMA targeted contrast agents engineered with addition of different molecular recognition sequences, ProCA32.PSMA exhibits a binding affinity of 1.1 +/- 0.1 μM for PSMA while the metal binding affinity is maintained at 0.9 +/- 0.1 × 10-22 M. In addition, ProCA32.PSMA exhibits r1 of 27.6 mM-1 s-1 and r2 of 37.9 mM-1 s-1 per Gd (55.2 and 75.8 mM-1 s-1 per molecule r1 and r2, respectively) at 1.4 T. At 7 T, ProCA32.PSMA also has r2 of 94.0 mM-1 s-1 per Gd (188.0 mM-1 s-1 per molecule) and r1 of 18.6 mM-1 s-1 per Gd (37.2 mM-1 s-1 per molecule). This contrast capability enables the first MRI enhancement dependent on PSMA expression levels in tumor bearing mice using both T1 and T2-weighted MRI at 7 T. Further development of these PSMA-targeted contrast agents are expected to be used for the precision imaging of prostate cancer at an early stage and to monitor disease progression and staging, as well as determine the effect of therapeutic treatment by non-invasive evaluation of the PSMA level using MRI.Prostate-specific membrane antigen (PSMA) is one of the most specific cell surface markers for prostate cancer diagnosis and targeted treatment. However, achieving molecular imaging using non-invasive MRI with high resolution has yet to be achieved due to the lack of contrast agents with significantly improved relaxivity for sensitivity, targeting capabilities and metal selectivity. We have previously reported our creation of a novel class of protein Gd3+ contrast agents, ProCA32, which displayed significantly improved relaxivity while exhibiting strong Gd3+ binding selectivity over physiological metal ions. In this study, we report our effort in further developing biomarker-targeted protein MRI contrast agents for molecular imaging of PSMA. Among three PSMA targeted contrast agents engineered with addition of different molecular recognition sequences, ProCA32.PSMA exhibits a binding affinity of 1.1 +/- 0.1 μM for PSMA while the metal binding affinity is maintained at 0.9 +/- 0.1 × 10-22 M. In addition, ProCA32.PSMA exhibits r1 of 27.6 mM-1 s-1 and r2 of 37.9 mM-1 s-1 per Gd (55.2 and 75.8 mM-1 s-1 per molecule r1 and r2, respectively) at 1.4 T. At 7 T, ProCA32.PSMA also has r2 of 94.0 mM-1 s-1 per Gd (188.0 mM-1 s-1 per molecule) and r1 of 18.6 mM-1 s-1 per Gd (37.2 mM-1 s-1 per molecule). This contrast capability enables the first MRI enhancement dependent on PSMA expression levels in tumor bearing mice using both T1 and T2-weighted MRI at 7 T. Further development of these PSMA-targeted contrast agents are expected to be used for the precision imaging of prostate cancer at an early stage and to monitor disease progression and staging, as well as determine the effect of therapeutic treatment by non-invasive evaluation of the PSMA level using MRI. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr09071g

Highly stable biocompatible inorganic nanoparticles by self-assembly of triblock-copolymer ligands.

PubMed

Pöselt, Elmar; Fischer, Steffen; Foerster, Stephan; Weller, Horst

2009-12-15

A novel type of ligand for biofunctionalization of nanoparticles is presented that comprises tailor-made triblock-copolymers consisting of a polyethylene imine binding block, a hydrophobic polycaprolactone and a terminal functionalized polyethelene oxide block. Phase transfer to water occurs simply by ligand and water addition and removal of the organic solvents. It is shown that the intermediate polycaprolacton block favors the attachment to the particle surface and shields the binding groups effectively from the solution. As a consequence, the particles exhibit an outstanding stability in various aqueous media for biological studies and give easy access to specific coupling reactions at the terminal end groups of the polyethylene oxide block. Controlling the ligand exchange parameters leads to self-assembly to either individual encapsulated nanoparticles or to multifunctional nanobeads.

Comparison of antigenic and allergenic components of Holoptelea integrifolia pollen collected from different source materials.

PubMed

Malik, P; Singh, A B; Gangal, S V; Babu, C R

1991-05-01

Antigenic extracts prepared from pollen samples collected at weekly intervals during the same season did not exhibit significant variation in protein concentration. Stored pollen samples from different years, however, showed highly significant variations in protein concentration. The protein content of samples from different ecozones of India also varied (CV = +/- 32%). The IEF and SDS-PAGE patterns were almost identical in samples from the same season, but were variable in the samples stored from different years and different parts of India. IgE binding proteins from different samples also varied depending on the overall protein profiles. Almost all the patients, however, showed IgE binding to four proteins at 50, 60, 66 and 70 kD, indicating the important allergenic components of Holoptelea integrifolia.

Different mechanisms are involved in the antibody mediated inhibition of ligand binding to the urokinase receptor: a study based on biosensor technology.

PubMed

List, K; Høyer-Hansen, G; Rønne, E; Danø, K; Behrendt, N

1999-01-01

Certain monoclonal antibodies are capable of inhibiting the biological binding reactions of their target proteins. At the molecular level, this type of effect may be brought about by completely different mechanisms, such as competition for common binding determinants, steric hindrance or interference with conformational properties of the receptor critical for ligand binding. This distinction is central when employing the antibodies as tools in the elucidation of the structure-function relationship of the protein in question. We have studied the effect of monoclonal antibodies against the urokinase plasminogen activator receptor (uPAR), a protein located on the surface of various types of malignant and normal cells which is involved in the direction of proteolytic degradation reactions in the extracellular matrix. We show that surface plasmon resonance/biomolecular interaction analysis (BIA) can be employed as a highly useful tool to characterize the inhibitory mechanism of specific antagonist antibodies. Two inhibitory antibodies against uPAR, mAb R3 and mAb R5, were shown to exhibit competitive and non-competitive inhibition, respectively, of ligand binding to the receptor. The former antibody efficiently blocked the receptor against subsequent ligand binding but was unable to promote the dissociation of a preformed receptor-ligand complex. The latter antibody was capable of binding the preformed complex, forming a transient trimolecular assembly, and promoting the dissociation of the uPA/uPAR complex. The continuous recording of binding and dissociation, obtained in BIA, is central in characterizing these phenomena. The identification of a non-competitive inhibitory mechanism against this receptor reveals the presence of a determinant which influences the binding properties of a remote site in the molecular structure and which could be an important target for a putative synthetic antagonist.

Strains of Actinomyces naeslundii and Actinomyces viscosus Exhibit Structurally Variant Fimbrial Subunit Proteins and Bind to Different Peptide Motifs in Salivary Proteins

PubMed Central

Li, Tong; Johansson, Ingegerd; Hay, Donald I.; Strömberg, Nicklas

1999-01-01

Oral strains of Actinomyces spp. express type 1 fimbriae, which are composed of major FimP subunits, and bind preferentially to salivary acidic proline-rich proteins (APRPs) or to statherin. We have mapped genetic differences in the fimP subunit genes and the peptide recognition motifs within the host proteins associated with these differential binding specificities. The fimP genes were amplified by PCR from Actinomyces viscosus ATCC 19246, with preferential binding to statherin, and from Actinomyces naeslundii LY7, P-1-K, and B-1-K, with preferential binding to APRPs. The fimP gene from the statherin-binding strain 19246 is novel and has about 80% nucleotide and amino acid sequence identity to the highly conserved fimP genes of the APRP-binding strains (about 98 to 99% sequence identity). The novel FimP protein contains an amino-terminal signal peptide, randomly distributed single-amino-acid substitutions, and structurally different segments and ends with a cell wall-anchoring and a membrane-spanning region. When agarose beads with CNBr-linked host determinant-specific decapeptides were used, A. viscosus 19246 bound to the Thr42Phe43 terminus of statherin and A. naeslundii LY7 bound to the Pro149Gln150 termini of APRPs. Furthermore, while the APRP-binding A. naeslundii strains originate from the human mouth, A. viscosus strains isolated from the oral cavity of rat and hamster hosts showed preferential binding to statherin and contained the novel fimP gene. Thus, A. viscosus and A. naeslundii display structurally variant fimP genes whose protein products are likely to interact with different peptide motifs and to determine animal host tropism. PMID:10225854

Functional Regulation of Sugar Assimilation by N-Glycan-specific Interaction of Pancreatic α-Amylase with Glycoproteins of Duodenal Brush Border Membrane*

PubMed Central

Asanuma-Date, Kimie; Hirano, Yuki; Le, Na; Sano, Kotone; Kawasaki, Nana; Hashii, Noritaka; Hiruta, Yoko; Nakayama, Ken-ichi; Umemura, Mariko; Ishikawa, Kazuhiko; Sakagami, Hiromi; Ogawa, Haruko

2012-01-01

Porcine pancreatic α-amylase (PPA) binds to N-linked glycans of glycoproteins (Matsushita, H., Takenaka, M., and Ogawa, H. (2002) J. Biol Chem., 277, 4680–4686). Immunostaining revealed that PPA is located at the brush-border membrane (BBM) of enterocytes in the duodenum and that the binding is inhibited by mannan but not galactan, indicating that PPA binds carbohydrate-specifically to BBM. The ligands for PPA in BBM were identified as glycoprotein N-glycans that are significantly involved in the assimilation of glucose, including sucrase-isomaltase (SI) and Na+/Glc cotransporter 1 (SGLT1). Binding of SI and SGLT1 in BBM to PPA was dose-dependent and inhibited by mannan. Using BBM vesicles, we found functional changes in PPA and its ligands in BBM due to the N-glycan-specific interaction. The starch-degrading activity of PPA and maltose-degrading activity of SI were enhanced to 240 and 175%, respectively, while Glc uptake by SGLT1 was markedly inhibited by PPA at high but physiologically possible concentrations, and the binding was attenuated by the addition of mannose-specific lectins, especially from Galanthus nivalis. Additionally, recombinant human pancreatic α-amylases expressed in yeast and purified by single-step affinity chromatography exhibited the same carbohydrate binding specificity as PPA in binding assays with sugar-biotinyl polymer probes. The results indicate that mammalian pancreatic α-amylases share a common carbohydrate binding activity and specifically bind to the intestinal BBM. Interaction with N-glycans in the BBM activated PPA and SI to produce much Glc on the one hand and to inhibit Glc absorption by enterocytes via SGLT1 in order to prevent a rapid increase in blood sugar on the other. PMID:22584580

Molecular Dynamics Simulations of Family 7 Cellobiohydrolase Mutants Aimed at Reducing Product Inhibition.

PubMed

Silveira, Rodrigo L; Skaf, Munir S

2015-07-23

Enzymatic conversion of lignocellulosic biomass into biofuels and chemicals constitutes a potential route for sustainable development. Cellobiohydrolases are key enzymes used in industrial cocktails for depolymerization of crystalline cellulose, and their mechanism of action has been intensely studied in the past several years. Provided with a tunnel-like substrate-binding cavity, cellobiohydrolases possess the ability to processively hydrolyze glycosidic bonds of crystalline cellulose, yielding one molecule of cellobiose per catalytic cycle. As such, cellobiose expulsion from the product binding site is a necessary step in order to allow for the processive hydrolysis mechanism. However, the high-affinity binding of cellobiose to the enzyme impairs the process and causes activity inhibition due to reaction products. Here, we use molecular dynamics simulations to study the binding of cellobiose to the Trichoderma reesei Cel7A (TrCel7A) cellobiohydrolase and the effects of mutations that reduce cellobiose binding, without affecting the structural and dynamical integrities of the enzyme. We observe that the product binding site exhibits an intrinsic flexibility that can sterically hinder cellobiose release. Several point mutations in the product binding site reduce cellobiose-enzyme interactions, but not all modifications are able to maintain the structural integrity of the enzyme. In particular, mutation of charged residues in the TrCel7A product binding site causes perturbations that affect the structure of the loops that form the substrate-binding tunnel of the enzyme and, hence, may affect TrCel7A function in other steps of the hydrolysis mechanism. Our results suggest there is a trade-off between product inhibition and catalytic efficiency, and they provide directions for cellulases engineering.

Analysis of nucleoside-binding proteins by ligand-specific elution from dye resin: application to Mycobacterium tuberculosis aldehyde dehydrogenases.

PubMed

Kim, Chang-Yub; Webster, Cecelia; Roberts, Justin K M; Moon, Jin Ho; Alipio Lyon, Emily Z; Kim, Heungbok; Yu, Minmin; Hung, Li-Wei; Terwilliger, Thomas C

2009-12-01

We show that Cibacron Blue F3GA dye resin chromatography can be used to identify ligands that specifically interact with proteins from Mycobacterium tuberculosis, and that the identification of these ligands can facilitate structure determination by enhancing the quality of crystals. Four native Mtb proteins of the aldehyde dehydrogenase (ALDH) family were previously shown to be specifically eluted from a Cibacron Blue F3GA dye resin with nucleosides. In this study we characterized the nucleoside-binding specificity of one of these ALDH isozymes (recombinant Mtb Rv0223c) and compared these biochemical results with co-crystallization experiments with different Rv0223c-nucleoside pairings. We found that the strongly interacting ligands (NAD and NADH) aided formation of high-quality crystals, permitting solution of the first Mtb ALDH (Rv0223c) structure. Other nucleoside ligands (AMP, FAD, adenosine, GTP and NADP) exhibited weaker binding to Rv0223c, and produced co-crystals diffracting to lower resolution. Difference electron density maps based on crystals of Rv0223c with various nucleoside ligands show most share the binding site where the natural ligand NAD binds. From the high degree of similarity of sequence and structure compared to human mitochondrial ALDH-2 (BLAST Z-score = 53.5 and RMSD = 1.5 A), Rv0223c appears to belong to the ALDH-2 class. An altered oligomerization domain in the Rv0223c structure seems to keep this protein as monomer whereas native human ALDH-2 is a multimer.

Cell-specific targeting by heterobivalent ligands.

PubMed

Josan, Jatinder S; Handl, Heather L; Sankaranarayanan, Rajesh; Xu, Liping; Lynch, Ronald M; Vagner, Josef; Mash, Eugene A; Hruby, Victor J; Gillies, Robert J

2011-07-20

Current cancer therapies exploit either differential metabolism or targeting to specific individual gene products that are overexpressed in aberrant cells. The work described herein proposes an alternative approach--to specifically target combinations of cell-surface receptors using heteromultivalent ligands ("receptor combination approach"). As a proof-of-concept that functionally unrelated receptors can be noncovalently cross-linked with high avidity and specificity, a series of heterobivalent ligands (htBVLs) were constructed from analogues of the melanocortin peptide ligand ([Nle(4), dPhe(7)]-α-MSH) and the cholecystokinin peptide ligand (CCK-8). Binding of these ligands to cells expressing the human Melanocortin-4 receptor and the Cholecystokinin-2 receptor was analyzed. The MSH(7) and CCK(6) were tethered with linkers of varying rigidity and length, constructed from natural and/or synthetic building blocks. Modeling data suggest that a linker length of 20-50 Å is needed to simultaneously bind these two different G-protein coupled receptors (GPCRs). These ligands exhibited up to 24-fold enhancement in binding affinity to cells that expressed both (bivalent binding), compared to cells with only one (monovalent binding) of the cognate receptors. The htBVLs had up to 50-fold higher affinity than that of a monomeric CCK ligand, i.e., Ac-CCK(6)-NH(2). Cell-surface targeting of these two cell types with labeled heteromultivalent ligand demonstrated high avidity and specificity, thereby validating the receptor combination approach. This ability to noncovalently cross-link heterologous receptors and target individual cells using a receptor combination approach opens up new possibilities for specific cell targeting in vivo for therapy or imaging.

Cell-Specific Targeting by Heterobivalent Ligands

PubMed Central

Josan, Jatinder S.; Handl, Heather L.; Sankaranarayanan, Rajesh; Xu, Liping; Lynch, Ronald M.; Vagner, Josef; Mash, Eugene A.; Hruby, Victor J.; Gillies, Robert J.

2012-01-01

Current cancer therapies exploit either differential metabolism or targeting to specific individual gene products that are overexpressed in aberrant cells. The work described herein proposes an alternative approach—to specifically target combinations of cell-surface receptors using heteromultivalent ligands (“receptor combination approach”). As a proof-of-concept that functionally unrelated receptors can be noncovalently cross-linked with high avidity and specificity, a series of heterobivalent ligands (htBVLs) were constructed from analogues of the melanocortin peptide ligand ([Nle4, DPhe7]-α-MSH) and the cholecystokinin peptide ligand (CCK-8). Binding of these ligands to cells expressing the human Melanocortin-4 receptor and the Cholecystokinin-2 receptor was analyzed. The MSH(7) and CCK(6) were tethered with linkers of varying rigidity and length, constructed from natural and/or synthetic building blocks. Modeling data suggest that a linker length of 20–50 Å is needed to simultaneously bind these two different G-protein coupled receptors (GPCRs). These ligands exhibited up to 24-fold enhancement in binding affinity to cells that expressed both (bivalent binding), compared to cells with only one (monovalent binding) of the cognate receptors. The htBVLs had up to 50-fold higher affinity than that of a monomeric CCK ligand, i.e., Ac-CCK(6)-NH2. Cell-surface targeting of these two cell types with labeled heteromultivalent ligand demonstrated high avidity and specificity, thereby validating the receptor combination approach. This ability to noncovalently cross-link heterologous receptors and target individual cells using a receptor combination approach opens up new possibilities for specific cell targeting in vivo for therapy or imaging. PMID:21639139

Autoradiographic labelling of P2 purinoceptors in the guinea-pig cochlea.

PubMed

Mockett, B G; Bo, X; Housley, G D; Thorne, P R; Burnstock, G

1995-04-01

Two different radioligands were used to identify extracellular ATP binding sites specific to P2 purinoceptors in guinea-pig cochlear tissue. Deoxyadenosine 5'-(alpha-[35S]thio)triphosphate ([35S]dATP alpha S; 10 nM) provided a high activity probe for the P2y purinoceptor subtype on the basis of selective block by 2-methylthio-ATP (2MeSATP; 100 microM). [3H]alpha, beta-methylene-ATP (10 nM), a high affinity probe for a P2x purinoceptor subtype was selectively blocked by inclusion of the related compound beta, gamma-methylene-ATP (100 microM). Both probes labelled the organ of Corti, stria vascularis and spiral prominence regions. The P2x purinoceptor probe also bound to lateral wall tissue below the spiral prominence and insertion point of the basilar membrane within the scala tympani compartment, a region which failed to show significant binding using [35S]dATP alpha S. Frozen sections of whole cochlea permitted analysis of radioligand binding to the cell body region (spiral ganglion in Rosenthal's canal) of the primary auditory afferents and the auditory nerve itself, which lies within the central region of the modiolus of the cochlea. Both these regions exhibited 2MeSATP blockable [35S]dATP alpha S binding whereas specific [3H]alpha, beta-methylene-ATP binding was absent from spiral ganglion and minimal in the auditory nerve region. These results demonstrate a mixed P2 purinoceptor distribution in cochlear tissues and suggest that complex purine-mediated neurohumoral mechanisms may influence cochlear function at a number of sites.

Plasmin substrate binding site cooperativity guides the design of potent peptide aldehyde inhibitors.

PubMed

Swedberg, Joakim E; Harris, Jonathan M

2011-10-04

Perioperative bleeding is a cause of major blood loss and is associated with increased rates of postoperative morbidity and mortality. To combat this, antifibrinolytic inhibitors of the serine protease plasmin are commonly used to reduce bleeding during surgery. The most effective and previously widely used of these is the broad range serine protease inhibitor aprotinin. However, adverse clinical outcomes have led to use of alternative serine lysine analogues to inhibit plasmin. These compounds suffer from low selectivity and binding affinity. Consequently, a concerted effort to discover potent and selective plasmin inhibitors has developed. This study used a noncombinatorial peptide library to define plasmin's extended substrate specificity and guide the design of potent transition state analogue inhibitors. The various substrate binding sites of plasmin were found to exhibit a higher degree of cooperativity than had previously been appreciated. Peptide sequences capitalizing on these features produced high-affinity inhibitors of plasmin. The most potent of these, Lys-Met(sulfone)-Tyr-Arg-H [KM(O(2))YR-H], inhibited plasmin with a K(i) of 3.1 nM while maintaining 25-fold selectivity over plasma kallikrein. Furthermore, 125 nM (0.16 μg/mL) KM(O(2))YR-H attenuated fibrinolysis in vitro with an efficacy similar to that of 15 nM (0.20 μg/mL) aprotinin. To date, this is the most potent peptide inhibitor of plasmin that exhibits selectivity against plasma kallikrein, making this compound an attractive candidate for further therapeutic development.

Identification and characterization of Escherichia coli RS218-derived islands in the pathogenesis of E. coli meningitis.

PubMed

Xie, Yi; Kolisnychenko, Vitaliy; Paul-Satyaseela, Maneesh; Elliott, Simon; Parthasarathy, Geetha; Yao, Yufeng; Plunkett, Guy; Blattner, Frederick R; Kim, Kwang Sik

2006-08-01

Escherichia coli K1 is the most common gram-negative bacterium causing neonatal meningitis, but the mechanisms by which E. coli K1 causes meningitis are not clear. We identified 22 E. coli RS218-derived genomic islands (RDIs), using a comparative genome analysis of meningitis-causing E. coli K1 strain RS218 (O18:K1:H7) and laboratory K-12 strain MG1655. Series of RDI deletion mutants were constructed and examined for phenotypes relevant to E. coli K1 meningitis. We identified 9 RDI deletion mutants (RDI 1, 4, 7, 12, 13, 16, 20, 21, and 22) that exhibited defects in meningitis development. RDI 16 and 21 mutants had profound defects in the induction of a high level of bacteremia in neonatal rats, and RDI 4 mutants exhibited a moderate defect in the induction of bacteremia. RDI 1 and 22 mutants showed defects in the ability to invade human brain microvascular endothelial cells (HBMECs), and RDI 12 mutants were defective in the ability to bind to HBMECs. RDI 13 and 20 mutants were defective in the ability to both bind to and invade HBMECs. RDI 7 mutants were defective in the induction of bacteremia and in the ability to both bind to and invade HBMECs. These results provide a framework for the future discovery and analysis of bacteremia and meningitis caused by E. coli K1 strain RS218.

CTCF regulates the human p53 gene through direct interaction with its natural antisense transcript, Wrap53

PubMed Central

Saldaña-Meyer, Ricardo; González-Buendía, Edgar; Guerrero, Georgina; Narendra, Varun; Bonasio, Roberto; Recillas-Targa, Félix; Reinberg, Danny

2014-01-01

The multifunctional CCCTC-binding factor (CTCF) protein exhibits a broad range of functions, including that of insulator and higher-order chromatin organizer. We found that CTCF comprises a previously unrecognized region that is necessary and sufficient to bind RNA (RNA-binding region [RBR]) and is distinct from its DNA-binding domain. Depletion of cellular CTCF led to a decrease in not only levels of p53 mRNA, as expected, but also those of Wrap53 RNA, an antisense transcript originated from the p53 locus. PAR-CLIP-seq (photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation [PAR-CLIP] combined with deep sequencing) analyses indicate that CTCF binds a multitude of transcripts genome-wide as well as to Wrap53 RNA. Apart from its established role at the p53 promoter, CTCF regulates p53 expression through its physical interaction with Wrap53 RNA. Cells harboring a CTCF mutant in its RBR exhibit a defective p53 response to DNA damage. Moreover, the RBR facilitates CTCF multimerization in an RNA-dependent manner, which may bear directly on its role in establishing higher-order chromatin structures in vivo. PMID:24696455

A spin transition mechanism for cooperative adsorption in metal-organic frameworks

NASA Astrophysics Data System (ADS)

Reed, Douglas A.; Keitz, Benjamin K.; Oktawiec, Julia; Mason, Jarad A.; Runčevski, Tomče; Xiao, Dianne J.; Darago, Lucy E.; Crocellà, Valentina; Bordiga, Silvia; Long, Jeffrey R.

2017-10-01

Cooperative binding, whereby an initial binding event facilitates the uptake of additional substrate molecules, is common in biological systems such as haemoglobin. It was recently shown that porous solids that exhibit cooperative binding have substantial energetic benefits over traditional adsorbents, but few guidelines currently exist for the design of such materials. In principle, metal-organic frameworks that contain coordinatively unsaturated metal centres could act as both selective and cooperative adsorbents if guest binding at one site were to trigger an electronic transformation that subsequently altered the binding properties at neighbouring metal sites. Here we illustrate this concept through the selective adsorption of carbon monoxide (CO) in a series of metal-organic frameworks featuring coordinatively unsaturated iron(II) sites. Functioning via a mechanism by which neighbouring iron(II) sites undergo a spin-state transition above a threshold CO pressure, these materials exhibit large CO separation capacities with only small changes in temperature. The very low regeneration energies that result may enable more efficient Fischer-Tropsch conversions and extraction of CO from industrial waste feeds, which currently underutilize this versatile carbon synthon. The electronic basis for the cooperative adsorption demonstrated here could provide a general strategy for designing efficient and selective adsorbents suitable for various separations.

Genetically Encoded Fragment-Based Discovery of Glycopeptide Ligands for Carbohydrate-Binding Proteins

DOE PAGES

Ng, Simon; Lin, Edith; Kitov, Pavel I.; ...

2015-04-10

Here we describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based-discovery (GE-FBD) uses selection of phagedisplayed glycopeptides to dock a glycan fragment at the CRD and guide selection of Synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 10 8 glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40 50-fold enhancement in affinity over methyl α-D-mannopyranoside (MeMan). Lectin array Suggested specificity: Man-WYD derivative bound only to 3more » out of 17 proteins-ConA, LcH, and PSA-that bind to Man. An X-ray structure of ConA.:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking; but their extra-CRD binding modes are significantly. different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry :of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins.« less

Genetically Encoded Fragment-Based Discovery of Glycopeptide Ligands for Carbohydrate-Binding Proteins

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

Ng, Simon; Lin, Edith; Kitov, Pavel I.

Here we describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based-discovery (GE-FBD) uses selection of phagedisplayed glycopeptides to dock a glycan fragment at the CRD and guide selection of Synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 10 8 glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40 50-fold enhancement in affinity over methyl α-D-mannopyranoside (MeMan). Lectin array Suggested specificity: Man-WYD derivative bound only to 3more » out of 17 proteins-ConA, LcH, and PSA-that bind to Man. An X-ray structure of ConA.:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking; but their extra-CRD binding modes are significantly. different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry :of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins.« less

Podocalyxin Is a Glycoprotein Ligand of the Human Pluripotent Stem Cell-Specific Probe rBC2LCN

PubMed Central

Tateno, Hiroaki; Matsushima, Asako; Hiemori, Keiko; Onuma, Yasuko; Ito, Yuzuru; Hasehira, Kayo; Nishimura, Ken; Ohtaka, Manami; Takayasu, Satoko; Nakanishi, Mahito; Ikehara, Yuzuru; Nakanishi, Mio; Ohnuma, Kiyoshi; Chan, Techuan; Toyoda, Masashi; Akutsu, Hidenori; Umezawa, Akihiro; Asashima, Makoto

2013-01-01

In comprehensive glycome analysis with a high-density lectin microarray, we have previously shown that the recombinant N-terminal domain of the lectin BC2L-C from Burkholderia cenocepacia (rBC2LCN) binds exclusively to undifferentiated human induced pluripotent stem (iPS) cells and embryonic stem (ES) cells but not to differentiated somatic cells. Here we demonstrate that podocalyxin, a heavily glycosylated type 1 transmembrane protein, is a glycoprotein ligand of rBC2LCN on human iPS cells and ES cells. When analyzed by DNA microarray, podocalyxin was found to be highly expressed in both iPS cells and ES cells. Western and lectin blotting revealed that rBC2LCN binds to podocalyxin with a high molecular weight of more than 240 kDa in undifferentiated iPS cells of six different origins and four ES cell lines, but no binding was observed in either differentiated mouse feeder cells or somatic cells. The specific binding of rBC2LCN to podocalyxin prepared from a large set of iPS cells (138 types) and ES cells (15 types) was also confirmed using a high-throughput antibody-overlay lectin microarray. Alkaline digestion greatly reduced the binding of rBC2LCN to podocalyxin, indicating that the major glycan ligands of rBC2LCN are presented on O-glycans. Furthermore, rBC2LCN was found to exhibit significant affinity to a branched O-glycan comprising an H type 3 structure (Ka, 2.5 × 104 M−1) prepared from human 201B7 iPS cells, indicating that H type 3 is a most probable potential pluripotency marker. We conclude that podocalyxin is a glycoprotein ligand of rBC2LCN on human iPS cells and ES cells. PMID:23526252

Chromatographic study of highly methoxylated lime pectins deesterified by different pectin methyl-esterases.

PubMed

Ralet, M C; Bonnin, E; Thibault, J F

2001-03-25

The inter-molecular distribution of free carboxyl groups of two highly methoxylated pectins enzymatically deesterified by plant and fungus pectin methyl-esterases were investigated by size-exclusion (SEC) and ion-exchange chromatography (IEC). "Homogeneous" populations with respect to molar mass or charge density were thereby obtained and their chemical composition and physico-chemical properties (transport parameter for monovalent cations and calcium, calcium activity coefficient) were studied. Chemical analysis showed that the composition varies from one SEC fraction to another, the highest molar mass fraction being richer in rhamnose and galactose and exhibiting a slightly higher degree of methylation. Separation of pectins by IEC revealed a quite homogeneous charge density distribution for F58 contrary to P60 which exhibited a large distribution of methoxyl groups. The free carboxyl groups distributions and calcium binding behaviours of SEC and IEC fractions were shown to differ widely for highly methoxylated pectins deesterified by plant and fungus pectin methyl-esterases.

A transcriptionally active estrogen receptor mutant is a novel type of dominant negative inhibitor of estrogen action.

PubMed

McInerney, E M; Ince, B A; Shapiro, D J; Katzenellenbogen, B S

1996-12-01

We have characterized a human estrogen receptor (ER) mutant, V364E, which has a single amino acid substitution in its hormone-binding domain. This ER mutant is fully active or even superactive at saturating levels of estradiol (10(-8) M E2) yet has the capacity to act as a strong dominant negative inhibitor of the wild type ER. In transient transfection assays using ER-negative Chinese hamster ovary (CHO) cells and two different estrogen response element (ERE)-containing promoter reporter genes, V364E treated with 10(-8) M E2 exhibited approximately 250% and 100% of the activity of the wild type ER with these two promoter contexts, respectively. Despite the high activity of V364E when present alone in cells, coexpression of both V364E and wild type ER causes a significant decrease in overall ER-mediated transcriptional activity. On the TATA promoter, where V364E was more inhibitory, estrogen-stimulated activity was reduced by approximately 50% at a 1:1 ratio of mutant to wild type ER expression vector, and at a 10:1 ratio, 75% of ER activity was inhibited. V364E was expressed at lower levels than wild type ER and has a approximately 40-fold lower affinity for E2 compared with wild type ER. In promoter interference assays, V364E exhibited a strict dependence upon E2 for binding to an ERE. Surprisingly, even when V364E was unable to bind to ERE DNA (i.e. either at low E2 concentration or by mutation of its DNA-binding domain), this mutant retained full dominant negative activity. This highly active ER mutant is, thus, able to repress ER-mediated transcription when the mutant and wild type ER are present together in cells, even without DNA binding. Since competition for ERE binding and the formation of inactive heterodimers cannot fully account for the dominant negative activity of V364E, it is probable that altered interactions with proteins important in ER-mediated transcription play a key role in the repression of transcription by V364E. The properties and probable mechanism of action of V364E distinguish it from other previously described dominant negative inhibitors, in which competition for cis-acting DNA elements by transcriptionally inactive receptors played a large role in the resultant dominant negative phenotype.

Novel styrylbenzothiazolium dye-based sensor for mercury, cyanide and hydroxide ions

NASA Astrophysics Data System (ADS)

Gwon, Seon-Young; Rao, Boddu Ananda; Kim, Hak-Soo; Son, Young-A.; Kim, Sung-Hoon

2015-06-01

We report the design and synthesis of a novel styrylbenzothiazolium (3) derivative developed as a fluorescent and colorimetric chemodosimeter with high selectivity toward Hg2+, CN- and OH- ions. An obvious loss of pink color in the presence of Hg2+ and CN- ions could make it a suitable "naked eye" indicator. We propose a sensing mechanism whereby the benzenoid form is changed to a quinoid form upon Hg2+ binding in a 1:1 stoichiometric ratio. More significantly, the styrylbenzothiazolium-Hg2+ and styrylbenzothiazolium-CN- complexes exhibited a dual-channel chromo-fluorogenic response. The sensors exhibit remarkable Hg2+-, CN--, and OH--selective red fluorescence but remain dark-green in the presence of a wide range of tested metal ions and anions.

Highly Sensitive and Selective Colorimetric and Off-On Fluorescent Reversible Chemosensors for Al3+ Based on the Rhodamine Fluorophore

PubMed Central

Mergu, Naveen; Singh, Ashok Kumar; Gupta, Vinod Kumar

2015-01-01

A series of rhodamine derivatives L1–L3 have been prepared and characterized by IR, 1H-NMR, 13C-NMR and ESI-MS. These compounds exhibited selective and sensitive “turn-on” fluorescent and colorimetric responses to Al3+ in methanol. Upon the addition of Al(III), the spiro ring was opened and a metal-probe complex was formed in a 1:1 stoichiometry, as was further confirmed by ESI-MS spectroscopy. The chemo-dosimeters L1–L3 exhibited good binding constants and low detection limits towards Al(III). We also successfully demonstrate the reversibility of the metal to ligand complexation (opened ring to spirolactam ring). PMID:25897498

Spectroscopic analysis and the excellent reusability of sphere-capped ferrocene in the oxidation of glucose oxidase.

PubMed

Antepli, Esin; Sarı, Nurşen

2016-08-01

Sphere-capped ferrocene nanospheres with Schiff base spacers have been prepared using a template, and used as carriers to immobilize glucose oxidase (GOx). GOx immobilized on spheres with one C-spacer (APS-Fc) exhibited high binding affinity to the substrate, which was attributed to appropriate position for the GOx conformation. When glucose oxidase was immobilized with spacers of different lengths, it was found that storage stability decreased with increasing the length of the spacer. It has been found that nanospheres, including capped ferrocene, exhibit good performance as the immobilized supporters of GOx. (APS-EtFc-GOx) retain more than 10% of the initial activity after forty-two successive cycles, which is a remarkable result.

Curcumin as an amyloid-indicator dye in E. coli.

PubMed

McCrate, Oscar A; Zhou, Xiaoxue; Cegelski, Lynette

2013-05-14

We have demonstrated that curcumin is an amyloid-specific dye in E. coli. Curcumin binds to curliated whole cells and to isolated curli amyloid fibers. Similar to Congo red, curcumin exhibits a red-shift in absorbance and a significant increase in fluorescence upon binding to isolated curli.

Curcumin as an Amyloid-indicator Dye in E. coli †

PubMed Central

McCrate, Oscar A.; Zhou, Xiaoxue; Cegelski, Lynette

2013-01-01

We have demonstrated that curcumin is an amyloid-specific dye in E. coli. Curcumin binds to curliated whole cells and to isolated curli amyloid fibers. Similar to Congo red, curcumin exhibits a red-shift in absorbance and a significant increase in fluorescence upon binding to isolated curli. PMID:23287899

A Receptor-targeted Fluorescent Radiopharmaceutical for Multireporter Sentinel Lymph Node Imaging

PubMed Central

Emerson, Derek K.; Limmer, Karl K.; Hall, David J.; Han, Sung-Ho; Eckelman, William C.; Kane, Christopher J.; Wallace, Anne M.

2012-01-01

Purpose: To determine the imaging and receptor-binding properties of a multireporter probe designed for sentinel lymph node (SLN) mapping via nuclear and fluorescence detection. Materials and Methods: The animal experiments were approved by the institutional animal care and use committee. A multireporter probe was synthesized by covalently attaching cyanine 7 (Cy7), a near-infrared cyanine dye, to tilmanocept, a radiopharmaceutical that binds to a receptor specific to recticuloendothelial cells. In vitro binding assays of technetium 99m (99mTc) -labeled Cy7 tilmanocept were conducted at 4°C by using receptor-bearing macrophages. Optical SLN imaging after foot pad administration was performed by using two molar doses of Cy7 tilmanocept. Six mice were injected with 0.11 nmol of 99mTc-labeled Cy7 tilmanocept (low-dose group); an additional six mice were injected with 31 nmol of 99mTc-labeled Cy7 tilmanocept (high-dose group) to saturate the receptor sites within the SLN. After 2.5 hours of imaging, the mice were euthanized, and the sentinel and distal lymph nodes were excised and assayed for radioactivity for calculation of SLN percentage of injected dose and extraction. Four mice were used as controls for autofluorescence. Standard optical imaging software was used to plot integrated fluorescence intensity against time for calculation of the SLN uptake rate constant and scaled peak intensity. Significance was calculated by using the Student t test. Results: In vitro binding assays showed subnanomolar affinity (mean dissociation constant, 0.25 nmol/L ± 0.10 [standard deviation]). Fluorescence imaging showed a detection sensitivity of 1.6 × 103 counts · sec−1 · μW−1 per picomole of Cy7. All four imaging metrics (percentage of injected dose, SLN extraction, SLN uptake rate constant, and expected peak fluorescence intensity) exhibited higher values (P = .005 to P = .042) in the low-dose group than in the high-dose group; this finding was consistent with receptor-mediated image formation. Conclusion: The multireporter probe 99mTc-labeled Cy7 tilmanocept exhibits in vitro and in vivo receptor-binding properties for successful receptor-targeted SLN mapping with nuclear and optical imaging. © RSNA, 2012 Supplemental material: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.12120638/-/DC1 PMID:22753678

A Label-Free Aptasensor for Ochratoxin a Detection Based on the Structure Switch of Aptamer.

PubMed

Liu, Feng; Ding, Ailing; Zheng, Jiushang; Chen, Jiucun; Wang, Bin

2018-06-01

A label-free sensing platform is developed based on switching the structure of aptamer for highly sensitive and selective fluorescence detection of ochratoxin A (OTA). OTA induces the structure of aptamer, transforms into G-quadruplex and produces strong fluorescence in the presence of zinc(II)-protoporphyrin IX probe due to the specific bind to G-quadruplex. The simple method exhibits high sensitivity towards OTA with a detection limit of 0.03 nM and excellent selectivity over other mycotoxins. In addition, the successful detection of OTA in real samples represents a promising application in food safety.

MMP Inhibitors: Past, present and future.

PubMed

Cathcart, Jillian M; Cao, Jian

2015-06-01

  Development of inhibitors of matrix metalloproteinases (MMPs) has been fraught with challenges. Early compounds largely failed due to poor selectivity and bioavailability. Dose-limiting side effects, off-target interactions, and improperly designed clinical trials significantly impeded clinical success. As information becomes available and technology evolves, tools to combat these obstacles have been developed. Improved methods for high throughput screening and drug design have led to identification of compounds exhibiting high potency, binding affinity, and favorable pharmacokinetic profiles. Current research into MMP inhibitors employs innovative approaches for drug delivery methods and allosteric inhibitors. Such innovation is key for development of clinically successful compounds.

High-Mannose Specific Lectin and Its Recombinants from a Carrageenophyta Kappaphycus alvarezii Represent a Potent Anti-HIV Activity Through High-Affinity Binding to the Viral Envelope Glycoprotein gp120.

PubMed

Hirayama, Makoto; Shibata, Hiromi; Imamura, Koji; Sakaguchi, Takemasa; Hori, Kanji

2016-02-01

We previously reported that a high-mannose binding lectin KAA-2 from the red alga Kappaphycus alvarezii, which is an economically important species and widely cultivated as a source of carrageenans, had a potent anti-influenza virus activity. In this study, the full-length sequences of two KAA isoforms, KAA-1 and KAA-2, were elucidated by a combination of peptide mapping and complementary DNA (cDNA) cloning. They consisted of four internal tandem-repeated domains, which are conserved in high-mannose specific lectins from lower organisms, including a cyanobacterium Oscillatoria agardhii and a red alga Eucheuma serra. Using an Escherichia coli expression system, an active recombinant form of KAA-1 (His-tagged rKAA-1) was successfully generated in the yield of 115 mg per liter of culture. In a detailed oligosaccharide binding analysis by a centrifugal ultrafiltration-HPLC method with 27 pyridylaminated oligosaccharides, His-tagged rKAA-1 and rKAA-1 specifically bound to high-mannose N-glycans with an exposed α1-3 mannose in the D2 arm as the native lectin did. Predicted from oligosaccharide binding specificity, a surface plasmon resonance analysis revealed that the recombinants exhibit strong interaction with gp120, a heavily glycosylated envelope glycoprotein of HIV with high association constants (1.48 - 1.61 × 10(9) M(-1)). Native KAAs and the recombinants inhibited the HIV-1 entry at IC50s of low nanomolar levels (7.3-12.9 nM). Thus, the recombinant proteins would be useful as antiviral reagents targeting the viral surface glycoproteins with high-mannose N-glycans, and the cultivated alga K. alvarezii could also be a good source of not only carrageenans but also this functional lectin(s).

High-Mannose Specific Lectin and Its Recombinants from a Carrageenophyta Kappaphycus alvarezii Represent a Potent Anti-HIV Activity Through High-Affinity Binding to the Viral Envelope Glycoprotein gp120.

PubMed

Hirayama, Makoto; Shibata, Hiromi; Imamura, Koji; Sakaguchi, Takemasa; Hori, Kanji

2016-04-01

We previously reported that a high-mannose binding lectin KAA-2 from the red alga Kappaphycus alvarezii, which is an economically important species and widely cultivated as a source of carrageenans, had a potent anti-influenza virus activity. In this study, the full-length sequences of two KAA isoforms, KAA-1 and KAA-2, were elucidated by a combination of peptide mapping and cDNA cloning. They consisted of four internal tandem-repeated domains, which are conserved in high-mannose specific lectins from lower organisms, including a cyanobacterium Oscillatoria agardhii and a red alga Eucheuma serra. Using an Escherichia coli expression system, an active recombinant form of KAA-1 (His-tagged rKAA-1) was successfully generated in the yield of 115 mg per a litter of culture. In a detailed oligosaccharide binding analysis by a centrifugal ultrafiltration-HPLC method with 27 pyridylaminated oligosaccharides, His-tagged rKAA-1 and rKAA-1 specifically bound to high-mannose N-glycans with an exposed α1-3 mannose in the D2 arm as the native lectin did. Predicted from oligosaccharide-binding specificity, a surface plasmon resonance analysis revealed that the recombinants exhibit strong interaction with gp120, a heavily glycosylated envelope glycoprotein of HIV with high association constants (1.48-1.61 × 10(9) M(-1)). Native KAAs and the recombinants inhibited the HIV-1 entry at IC50s of low nanomolar levels (7.3-12.9 nM). Thus, the recombinant proteins would be useful as antiviral reagents targeting the viral surface glycoproteins with high-mannose N-glycans, and the cultivated alga K. alvarezii could also be a good source of not only carrageenans but also this functional lectin(s).

Boosting Affinity by Correct Ligand Preorganization for the S2 Pocket of Thrombin: A Study by Isothermal Titration Calorimetry, Molecular Dynamics, and High-Resolution Crystal Structures.

PubMed

Rühmann, Eggert H; Rupp, Melinda; Betz, Michael; Heine, Andreas; Klebe, Gerhard

2016-02-04

Structural preorganization to fix bioactive conformations at protein binding sites is a popular strategy to enhance binding affinity during late-stage optimization. The rationale for this enhancement relates to entropic advantages assigned to rigidified versus flexible ligands. We analyzed a narrow series of peptidomimetics binding to thrombin. The individual ligands exhibit at P2 a conformationally flexible glycine, more restricted alanine, N-methylglycine, N-methylhomoalanine, and largely rigidified proline moiety. Overall, affinity was found to increase by a factor of 1000, explained partly by an entropic advantage. All ligands adopt the same binding mode with small deviations. The residual mobility of the bound ligands is decreased across the series, and a protein side chain differs in its order/disorder behavior along with changes in the surface-water network pattern established across the newly generated protein-ligand surfaces. The enthalpy/entropy inventory displays a rather complex picture and emphasizes that thermodynamics can only be compared in terms of relative differences within a structurally similar ligand series. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pathway Analysis Revealed Potential Diverse Health Impacts of Flavonoids that Bind Estrogen Receptors

PubMed Central

Ye, Hao; Ng, Hui Wen; Sakkiah, Sugunadevi; Ge, Weigong; Perkins, Roger; Tong, Weida; Hong, Huixiao

2016-01-01

Flavonoids are frequently used as dietary supplements in the absence of research evidence regarding health benefits or toxicity. Furthermore, ingested doses could far exceed those received from diet in the course of normal living. Some flavonoids exhibit binding to estrogen receptors (ERs) with consequential vigilance by regulatory authorities at the U.S. EPA and FDA. Regulatory authorities must consider both beneficial claims and potential adverse effects, warranting the increases in research that has spanned almost two decades. Here, we report pathway enrichment of 14 targets from the Comparative Toxicogenomics Database (CTD) and the Herbal Ingredients’ Targets (HIT) database for 22 flavonoids that bind ERs. The selected flavonoids are confirmed ER binders from our earlier studies, and were here found in mainly involved in three types of biological processes, ER regulation, estrogen metabolism and synthesis, and apoptosis. Besides cancers, we conjecture that the flavonoids may affect several diseases via apoptosis pathways. Diseases such as amyotrophic lateral sclerosis, viral myocarditis and non-alcoholic fatty liver disease could be implicated. More generally, apoptosis processes may be importantly evolved biological functions of flavonoids that bind ERs and high dose ingestion of those flavonoids could adversely disrupt the cellular apoptosis process. PMID:27023590

Dimensionality of Carbon Nanomaterials Determines the Binding and Dynamics of Amyloidogenic Peptides: Multiscale Theoretical Simulations

PubMed Central

Hine, Nicholas D. M.; Mostofi, Arash A.; Yarovsky, Irene

2013-01-01

Experimental studies have demonstrated that nanoparticles can affect the rate of protein self-assembly, possibly interfering with the development of protein misfolding diseases such as Alzheimer's, Parkinson's and prion disease caused by aggregation and fibril formation of amyloid-prone proteins. We employ classical molecular dynamics simulations and large-scale density functional theory calculations to investigate the effects of nanomaterials on the structure, dynamics and binding of an amyloidogenic peptide apoC-II(60-70). We show that the binding affinity of this peptide to carbonaceous nanomaterials such as C60, nanotubes and graphene decreases with increasing nanoparticle curvature. Strong binding is facilitated by the large contact area available for π-stacking between the aromatic residues of the peptide and the extended surfaces of graphene and the nanotube. The highly curved fullerene surface exhibits reduced efficiency for π-stacking but promotes increased peptide dynamics. We postulate that the increase in conformational dynamics of the amyloid peptide can be unfavorable for the formation of fibril competent structures. In contrast, extended fibril forming peptide conformations are promoted by the nanotube and graphene surfaces which can provide a template for fibril-growth. PMID:24339760

Factors influencing the antifolate activity of synthetic tea-derived catechins.

PubMed

Sáez-Ayala, Magalí; Fernández-Pérez, María Piedad; Chazarra, Soledad; Mchedlishvili, Nani; Tárraga-Tomás, Alberto; Rodríguez-López, José Neptuno

2013-07-16

Novel tea catechin derivatives have been synthesized, and a structure-activity study, related to the capacity of these and other polyphenols to bind dihydrofolate reductase (DHFR), has been performed. The data showed an effective binding between all molecules and the free enzyme, and the dissociation constants of the synthetic compounds and of the natural analogues were on the same order. Polyphenols with a catechin configuration were better DHFR inhibitors than those with an epicatechin configuration. Antiproliferative activity was also studied in cultured tumour cells, and the data showed that the activity of the novel derivatives was higher in catechin isomers. Derivatives with a hydroxyl group para on the ester-bonded gallate moiety presented a high in vitro binding to DHFR, but exhibited transport problems in cell culture due to ionization at physiologic pHs. The impact of the binding of catechins to serum albumin on their biological activity was also evaluated. The information provided in this study could be important for the design of novel medicinal active compounds derived from tea catechins. The data suggest that changes in their structure to avoid serum albumin interactions and to facilitate plasmatic membrane transport are essential for the intracellular functions of catechins.

Biophysical Characterization of Nucleophosmin Interactions with Human Immunodeficiency Virus Rev and Herpes Simplex Virus US11

PubMed Central

Nouri, Kazem; Moll, Jens M.; Milroy, Lech-Gustav; Hain, Anika; Dvorsky, Radovan; Amin, Ehsan; Lenders, Michael; Nagel-Steger, Luitgard; Howe, Sebastian; Smits, Sander H. J.; Hengel, Hartmut; Schmitt, Lutz; Münk, Carsten; Brunsveld, Luc; Ahmadian, Mohammad R.

2015-01-01

Nucleophosmin (NPM1, also known as B23, numatrin or NO38) is a pentameric RNA-binding protein with RNA and protein chaperon functions. NPM1 has increasingly emerged as a potential cellular factor that directly associates with viral proteins; however, the significance of these interactions in each case is still not clear. In this study, we have investigated the physical interaction of NPM1 with both human immunodeficiency virus type 1 (HIV-1) Rev and Herpes Simplex virus type 1 (HSV-1) US11, two functionally homologous proteins. Both viral proteins show, in mechanistically different modes, high affinity for a binding site on the N-terminal oligomerization domain of NPM1. Rev, additionally, exhibits low-affinity for the central histone-binding domain of NPM1. We also showed that the proapoptotic cyclic peptide CIGB-300 specifically binds to NPM1 oligomerization domain and blocks its association with Rev and US11. Moreover, HIV-1 virus production was significantly reduced in the cells treated with CIGB-300. Results of this study suggest that targeting NPM1 may represent a useful approach for antiviral intervention. PMID:26624888

Identification of STAT1 and STAT3 Specific Inhibitors Using Comparative Virtual Screening and Docking Validation

PubMed Central

Szelag, Malgorzata; Czerwoniec, Anna; Wesoly, Joanna; Bluyssen, Hans A. R.

2015-01-01

Signal transducers and activators of transcription (STATs) facilitate action of cytokines, growth factors and pathogens. STAT activation is mediated by a highly conserved SH2 domain, which interacts with phosphotyrosine motifs for specific STAT-receptor contacts and STAT dimerization. The active dimers induce gene transcription in the nucleus by binding to a specific DNA-response element in the promoter of target genes. Abnormal activation of STAT signaling pathways is implicated in many human diseases, like cancer, inflammation and auto-immunity. Searches for STAT-targeting compounds, exploring the phosphotyrosine (pTyr)-SH2 interaction site, yielded many small molecules for STAT3 but sparsely for other STATs. However, many of these inhibitors seem not STAT3-specific, thereby questioning the present modeling and selection strategies of SH2 domain-based STAT inhibitors. We generated new 3D structure models for all human (h)STATs and developed a comparative in silico docking strategy to obtain further insight into STAT-SH2 cross-binding specificity of a selection of previously identified STAT3 inhibitors. Indeed, by primarily targeting the highly conserved pTyr-SH2 binding pocket the majority of these compounds exhibited similar binding affinity and tendency scores for all STATs. By comparative screening of a natural product library we provided initial proof for the possibility to identify STAT1 as well as STAT3-specific inhibitors, introducing the ‘STAT-comparative binding affinity value’ and ‘ligand binding pose variation’ as selection criteria. In silico screening of a multi-million clean leads (CL) compound library for binding of all STATs, likewise identified potential specific inhibitors for STAT1 and STAT3 after docking validation. Based on comparative virtual screening and docking validation, we developed a novel STAT inhibitor screening tool that allows identification of specific STAT1 and STAT3 inhibitory compounds. This could increase our understanding of the functional role of these STATs in different diseases and benefit the clinical need for more drugable STAT inhibitors with high specificity, potency and excellent bioavailability. PMID:25710482

IGF2BP2/IMP2-Deficient mice resist obesity through enhanced translation of Ucp1 mRNA and Other mRNAs encoding mitochondrial proteins.

PubMed

Dai, Ning; Zhao, Liping; Wrighting, Diedra; Krämer, Dana; Majithia, Amit; Wang, Yanqun; Cracan, Valentin; Borges-Rivera, Diego; Mootha, Vamsi K; Nahrendorf, Matthias; Thorburn, David R; Minichiello, Liliana; Altshuler, David; Avruch, Joseph

2015-04-07

Although variants in the IGF2BP2/IMP2 gene confer risk for type 2 diabetes, IMP2, an RNA binding protein, is not known to regulate metabolism. Imp2(-/-) mice gain less lean mass after weaning and have increased lifespan. Imp2(-/-) mice are highly resistant to diet-induced obesity and fatty liver and display superior glucose tolerance and insulin sensitivity, increased energy expenditure, and better defense of core temperature on cold exposure. Imp2(-/-) brown fat and Imp2(-/-) brown adipocytes differentiated in vitro contain more UCP1 polypeptide than Imp2(+/+) despite similar levels of Ucp1 mRNA; the Imp2(-/-)adipocytes also exhibit greater uncoupled oxygen consumption. IMP2 binds the mRNAs encoding Ucp1 and other mitochondrial components, and most exhibit increased translational efficiency in the absence of IMP2. In vitro IMP2 inhibits translation of mRNAs bearing the Ucp1 untranslated segments. Thus IMP2 limits longevity and regulates nutrient and energy metabolism in the mouse by controlling the translation of its client mRNAs. Copyright © 2015 Elsevier Inc. All rights reserved.

Smart Fluid System Dually Responsive to Light and Electric Fields: An Electrophotorheological Fluid.

PubMed

Yoon, Chang-Min; Jang, Yoonsun; Noh, Jungchul; Kim, Jungwon; Jang, Jyongsik

2017-10-24

Electrophotorheological (EPR) fluids, whose rheological activity is dually responsive to light and electric fields (E fields), is formulated by mixing photosensitive spiropyran-decorated silica (SP-sSiO 2 ) nanoparticles with zwitterionic lecithin and mineral oil. A reversible photorheological (PR) activity of the EPR fluid is developed via the binding and releasing mechanism of lecithin and merocyanine (MC, a photoisomerized form of SP) under ultraviolet (UV) and visible (VIS) light applications. Moreover, the EPR fluid exhibits an 8-fold higher electrorheological (ER) performance compared to the SP-sSiO 2 nanoparticle-based ER fluid (without lecithin) under an E field, which is attributed to the enhanced dielectric properties facilitated by the binding of the lecithin and SP molecules. Upon dual application of UV light and an E field, the EPR fluid exhibits high EPR performance (ca. 115.3 Pa) that far exceeds its separate PR (ca. 0.8 Pa) and ER (ca. 57.5 Pa) activities, because of the synergistic contributions of the PR and ER effects through rigid and fully connected fibril-like structures. Consequently, this study offers a strategy on formulation of dual-stimuli responsive smart fluid systems.

Bioconversion of Lignocellulosic Biomass to Fermentable Sugars by Immobilized Magnetic Cellulolytic Enzyme Cocktails.

PubMed

Periyasamy, Karthik; Santhalembi, Laishram; Mortha, Gérard; Aurousseau, Marc; Boyer, Agnès; Subramanian, Sivanesan

2018-06-05

Enzyme cocktails of reusable, highly stable cellulolytic enzymes play an inevitable role in bioconversion of biomass to biofuels economically. Cellulase, xylanase and β-1,3-glucanase bound silica-amine functionalized iron oxide magnetic nanoparticles (ISN-CLEAs) were prepared and used as the biocatalyst for the depolymerization of cellulosic biomass into monomeric sugar in the present study. The Fe 3 O 4 -NPs and Fe 3 O 4 @SiO 2 -NH 2 -NPs and ISN-CLEAs had an average hydrodynamic size of 82.2, 86.4, and 976.9 nm, respectively, which was confirmed by dynamic light scattering (DLS). About 97% of protein binding was achieved with 135 mM glutaraldehyde at 10 h of cross-linking time and successful binding was confirmed by Fourier transform infrared spectroscopy (FTIR). The ISN-CLEAs exhibited the highest thermal stability of 95% at 50 °C for 2 h and retained extended storage stability of 97% compared to 60% of its free counterpart. Besides, cross-linking allowed ISN-CLEAs reuse for at least eight consecutive cycles retaining over 70% of its initial activity. ISN-CLEAs exhibited approximately 15% increase in carbohydrate digestibility on sugar cane bagasse and eucalyptus pulp than the free enzyme.

Molecular basis for CPAP-tubulin interaction in controlling centriolar and ciliary length

PubMed Central

Zheng, Xiangdong; Ramani, Anand; Soni, Komal; Gottardo, Marco; Zheng, Shuangping; Ming Gooi, Li; Li, Wenjing; Feng, Shan; Mariappan, Aruljothi; Wason, Arpit; Widlund, Per; Pozniakovsky, Andrei; Poser, Ina; Deng, Haiteng; Ou, Guangshuo; Riparbelli, Maria; Giuliano, Callaini; Hyman, Anthony A.; Sattler, Michael; Gopalakrishnan, Jay; Li, Haitao

2016-01-01

Centrioles and cilia are microtubule-based structures, whose precise formation requires controlled cytoplasmic tubulin incorporation. How cytoplasmic tubulin is recognized for centriolar/ciliary-microtubule construction remains poorly understood. Centrosomal-P4.1-associated-protein (CPAP) binds tubulin via its PN2-3 domain. Here, we show that a C-terminal loop-helix in PN2-3 targets β-tubulin at the microtubule outer surface, while an N-terminal helical motif caps microtubule's α-β surface of β-tubulin. Through this, PN2-3 forms a high-affinity complex with GTP-tubulin, crucial for defining numbers and lengths of centriolar/ciliary-microtubules. Surprisingly, two distinct mutations in PN2-3 exhibit opposite effects on centriolar/ciliary-microtubule lengths. CPAPF375A, with strongly reduced tubulin interaction, causes shorter centrioles and cilia exhibiting doublet- instead of triplet-microtubules. CPAPEE343RR that unmasks the β-tubulin polymerization surface displays slightly reduced tubulin-binding affinity inducing over-elongation of newly forming centriolar/ciliary-microtubules by enhanced dynamic release of its bound tubulin. Thus CPAP regulates delivery of its bound-tubulin to define the size of microtubule-based cellular structures using a ‘clutch-like' mechanism. PMID:27306797

Hints for Metal-Preference Protein Sequence Determinants: Different Metal Binding Features of the Five Tetrahymena thermophila Metallothioneins

PubMed Central

Espart, Anna; Marín, Maribel; Gil-Moreno, Selene; Palacios, Òscar; Amaro, Francisco; Martín-González, Ana; Gutiérrez, Juan C.; Capdevila, Mercè; Atrian, Sílvia

2015-01-01

The metal binding preference of metallothioneins (MTs) groups them in two extreme subsets, the Zn/Cd- and the Cu-thioneins. Ciliates harbor the largest MT gene/protein family reported so far, including 5 paralogs that exhibit relatively low sequence similarity, excepting MTT2 and MTT4. In Tetrahymena thermophila, three MTs (MTT1, MTT3 and MTT5) were considered Cd-thioneins and two (MTT2 and MTT4) Cu-thioneins, according to gene expression inducibility and phylogenetic analysis. In this study, the metal-binding abilities of the five MTT proteins were characterized, to obtain information about the folding and stability of their cognate- and non-cognate metal complexes, and to characterize the T. thermophila MT system at protein level. Hence, the five MTTs were recombinantly synthesized as Zn2+-, Cd2+- or Cu+-complexes, which were analyzed by electrospray mass spectrometry (ESI-MS), circular dichroism (CD), and UV-vis spectrophotometry. Among the Cd-thioneins, MTT1 and MTT5 were optimal for Cd2+ coordination, yielding unique Cd17- and Cd8- complexes, respectively. When binding Zn2+, they rendered a mixture of Zn-species. Only MTT5 was capable to coordinate Cu+, although yielding heteronuclear Zn-, Cu-species or highly unstable Cu-homometallic species. MTT3 exhibited poor binding abilities both for Cd2+ and for Cu+, and although not optimally, it yielded the best result when coordinating Zn2+. The two Cu-thioneins, MTT2 and MTT4 isoforms formed homometallic Cu-complexes (major Cu20-MTT) upon synthesis in Cu-supplemented hosts. Contrarily, they were unable to fold into stable Cd-complexes, while Zn-MTT species were only recovered for MTT4 (major Zn10-MTT4). Thus, the metal binding preferences of the five T. thermophila MTs correlate well with their previous classification as Cd- and Cu-thioneins, and globally, they can be classified from Zn/Cd- to Cu-thioneins according to the gradation: MTT1>MTT5>MTT3>MTT4>MTT2. The main mechanisms underlying the evolution and specialization of the MTT metal binding preferences may have been internal tandem duplications, presence of doublet and triplet Cys patterns in Zn/Cd-thioneins, and optimization of site specific amino acid determinants (Lys for Zn/Cd- and Asn for Cu-coordination). PMID:25798065

Nicotinic α4β2 receptor imaging agents. Part IV. Synthesis and biological evaluation of 3-(2-(S)-3,4-dehydropyrrolinyl methoxy)-5-(3'-¹⁸F-fluoropropyl)pyridine (¹⁸F-Nifrolene) using PET.

PubMed

Pichika, Rama; Kuruvilla, Sharon A; Patel, Narmisha; Vu, Kenny; Sinha, Sangamitra; Easwaramoorthy, Balu; Narayanan, Tanjore K; Shi, Bingzhi; Christian, Bradley; Mukherjee, Jogeshwar

2013-01-01

Imaging agents for nicotinic α4β2 receptors in the brain have been under way for studying various CNS disorders. Previous studies from our laboratories have reported the successful development of agonist, ¹⁸F-nifene. In attempts to develop potential antagonists, ¹⁸F-nifrolidine and ¹⁸F-nifzetidine were previously reported. Further optimization of these fluoropropyl derivatives has now been carried out resulting in 3-(2-(S)-3,4-dehydropyrrolinylmethoxy)-5-(3'-Fluoropropyl)pyridine (nifrolene) as a new high affinity agent for nicotinic α4β2 receptors. Nifrolene in rat brain homogenate assays--labeled with ³H-cytisine--exhibited a binding affinity of 0.36 nM. The fluorine-18 analog, ¹⁸F-nifrolene, was synthesized in approximately 10%-20% yield and specific activity was estimated to be >2000 Ci/mmol. Rat brain slices indicated selective binding to anterior thalamic nuclei, thalamus, subiculum, striata, cortex and other regions consistent with α4β2 receptor distribution. This selective binding was displaced >90% by 300 μM nicotine. Thalamus to cerebellum ratio (>10) was the highest for ¹⁸F-nifrolene with several other regions showing selective binding. In vivo rat PET studies exhibited rapid uptake of ¹⁸F-nifrolene in the brain with specific retention in the thalamus and other brain regions while clearing out from the cerebellum. Thalamus to cerebellum ratio value in the rat was >4. Administration of nicotine caused a rapid decline in the thalamic ¹⁸F-nifrolene suggesting reversible binding to nicotinic receptors. PET imaging studies of ¹⁸F-nifrolene in anesthetized rhesus monkey revealed highest binding in the thalamus followed by regions of the lateral cingulated and temporal cortex. Cerebellum showed the least binding. Thalamus to cerebellum ratio in the monkey brain was >3 at 120 min. These ratios of ¹⁸F-nifrolene are higher than measured for ¹⁸F-nifrolidine and ¹⁸F-nifzetidine. ¹⁸F-Nifrolene thus shows promise as a new PET imaging agent for α4β2 nAChR. Copyright © 2013 Elsevier Inc. All rights reserved.

An integrated computational approach of molecular dynamics simulations, receptor binding studies and pharmacophore mapping analysis in search of potent inhibitors against tuberculosis.

PubMed

Agarwal, Shivangi; Verma, Ekta; Kumar, Vivek; Lall, Namrita; Sau, Samaresh; Iyer, Arun K; Kashaw, Sushil K

2018-05-03

Tuberculosis is an infectious chronic disease caused by obligate pathogen Mycobacterium tuberculosis that affects millions of people worldwide. Although many first and second line drugs are available for its treatment, but their irrational use has adversely lead to the emerging cases of multiple drug resistant and extensively drug-resistant tuberculosis. Therefore, there is an intense need to develop novel potent analogues for its treatment. This has prompted us to develop potent analogues against TB. The Mycobacterium tuberculosis genome provides us with number of validated targets to combat against TB. Study of Mtb genome disclosed six epoxide hydrolases (A to F) which convert harmful epoxide into diols and act as a potential drug target for rational drug design. Our current strategy is to develop such analogues which inhibits epoxide hydrolase enzyme present in Mtb genome. To achieve this, we adopted an integrated computational approach involving QSAR, pharmacophore mapping, molecular docking and molecular dynamics simulation studies. The approach envisaged vital information about the role of molecular descriptors, essential pharmacophoric features and binding energy for compounds to bind into the active site of epoxide hydrolase. Molecular docking analysis revealed that analogues exhibited significant binding to Mtb epoxide hydrolase. Further, three docked complexes 2s, 37s and 15s with high, moderate and low docking scores respectively were selected for molecular dynamics simulation studies. RMSD analysis revealed that all complexes are stable with average RMSD below 2 Å throughout the 10 ns simulations. The B-factor analysis showed that the active site residues of epoxide hydrolase are flexible enough to interact with inhibitor. Moreover, to confirm the binding of these urea derivatives, MM-GBSA binding energy analysis were performed. The calculations showed that 37s has more binding affinity (ΔGtotal = -52.24 kcal/mol) towards epoxide hydrolase compared to 2s (ΔGtotal = -51.70 kcal/mol) and 15s (ΔGtotal = -49.97 kcal/mol). The structural features inferred in our study may provide the future directions to the scientists towards the discovery of new chemical entity exhibiting anti-TB property. Copyright © 2018 Elsevier Inc. All rights reserved.

Gel shift analysis of the empA promoter region in Vibrio anguillarum.

PubMed

Denkin, Steven M; Sekaric, Pedja; Nelson, David R

2004-10-29

The induction of metalloprotease encoded by empA in Vibrio anguillarum occurs at high cell density in salmon intestinal mucus. Previously we have shown that there are significant differences in empA expression in two strains of V. anguillarum, M93Sm and NB10. It is hypothesized that differences in empA regulation are due to differences in binding of regulatory elements. Two strains of V. anguillarum, M93Sm and NB10, were examined and compared for the presence of DNA regulatory proteins that bind to and control the empA promoter region. Gel mobility shift assays, using a digoxigenin (DIG)-labeled oligomer containing a lux box-like element and the promoter for empA, were done to demonstrate the presence of a DNA-binding protein. Protein extracts from NB10 cells incubated in Luria Bertani broth + 2% NaCl (LB20), nine salts solution + 200 microg/ml mucus (NSSM), 3M (marine minimal medium), or NSS resulted in a gel mobility shift. No gel mobility shift was seen when protein extracts from either LB20- or NSSM-grown M93Sm cells were mixed with the DIG-labeled empA oligomer. The azocasein assay detected protease activity in all incubation conditions for NB10 culture supernatants. In contrast, protease activity was detected in M93Sm culture supernatants only when incubated in NSSM. Since the luxR homologue in V. anguillarum, vanT, has been cloned, sequenced, and shown to be required for protease activity, we wanted to determine if vanT mutants of NB10 exhibit the same gel shift observed in the wild-type. Site-directed mutagenesis was used to create vanT mutants in V. anguillarum M93Sm and NB10 to test whether VanT is involved with the gel mobility shift. Both vanT mutants, M02 and NB02, did not produce protease activity in any conditions. However, protein extracts from NB02 incubated in each condition still exhibited a gel shift when mixed with the DIG-labeled empA oligomer. The data demonstrate that protein extracts of V. anguillarum NB10 cells contain a protein that binds to a 50 bp oligomer containing the empA promoter-lux box-like region. NB10 cells express empA during stationary phase in all growth conditions. The DNA binding protein is not present in M93Sm extracts. M93Sm cells express protease activity only when incubated at high cell density in fish gastrointestinal mucus. The gel shift observed with NB10 cells is not due to VanT binding. The data also suggest that the DNA binding protein is responsible for the less restrictive expression of empA in NB10 compared to M93Sm.

High-affinity, noninhibitory pathogenic C1 domain antibodies are present in patients with hemophilia A and inhibitors.

PubMed

Batsuli, Glaivy; Deng, Wei; Healey, John F; Parker, Ernest T; Baldwin, W Hunter; Cox, Courtney; Nguyen, Brenda; Kahle, Joerg; Königs, Christoph; Li, Renhao; Lollar, Pete; Meeks, Shannon L

2016-10-20

Inhibitor formation in hemophilia A is the most feared treatment-related complication of factor VIII (fVIII) therapy. Most inhibitor patients with hemophilia A develop antibodies against the fVIII A2 and C2 domains. Recent evidence demonstrates that the C1 domain contributes to the inhibitor response. Inhibitory anti-C1 monoclonal antibodies (mAbs) have been identified that bind to putative phospholipid and von Willebrand factor (VWF) binding epitopes and block endocytosis of fVIII by antigen presenting cells. We now demonstrate by competitive enzyme-linked immunosorbent assay and hydrogen-deuterium exchange mass spectrometry that 7 of 9 anti-human C1 mAbs tested recognize an epitope distinct from the C1 phospholipid binding site. These mAbs, designated group A, display high binding affinities for fVIII, weakly inhibit fVIII procoagulant activity, poorly inhibit fVIII binding to phospholipid, and exhibit heterogeneity with respect to blocking fVIII binding to VWF. Another mAb, designated group B, inhibits fVIII procoagulant activity, fVIII binding to VWF and phospholipid, fVIIIa incorporation into the intrinsic Xase complex, thrombin generation in plasma, and fVIII uptake by dendritic cells. Group A and B epitopes are distinct from the epitope recognized by the canonical, human-derived inhibitory anti-C1 mAb, KM33, whose epitope overlaps both groups A and B. Antibodies recognizing group A and B epitopes are present in inhibitor plasmas from patients with hemophilia A. Additionally, group A and B mAbs increase fVIII clearance and are pathogenic in a hemophilia A mouse tail snip bleeding model. Group A anti-C1 mAbs represent the first identification of pathogenic, weakly inhibitory antibodies that increase fVIII clearance. © 2016 by The American Society of Hematology.

Alzheimer's Therapeutics Targeting Amyloid Beta 1–42 Oligomers I: Abeta 42 Oligomer Binding to Specific Neuronal Receptors Is Displaced by Drug Candidates That Improve Cognitive Deficits

PubMed Central

Izzo, Nicholas J.; Staniszewski, Agnes; To, Lillian; Fa, Mauro; Teich, Andrew F.; Saeed, Faisal; Wostein, Harrison; Walko, Thomas; Vaswani, Anisha; Wardius, Meghan; Syed, Zanobia; Ravenscroft, Jessica; Mozzoni, Kelsie; Silky, Colleen; Rehak, Courtney; Yurko, Raymond; Finn, Patricia; Look, Gary; Rishton, Gilbert; Safferstein, Hank; Miller, Miles; Johanson, Conrad; Stopa, Edward; Windisch, Manfred; Hutter-Paier, Birgit; Shamloo, Mehrdad; Arancio, Ottavio; LeVine, Harry; Catalano, Susan M.

2014-01-01

Synaptic dysfunction and loss caused by age-dependent accumulation of synaptotoxic beta amyloid (Abeta) 1–42 oligomers is proposed to underlie cognitive decline in Alzheimer's disease (AD). Alterations in membrane trafficking induced by Abeta oligomers mediates reduction in neuronal surface receptor expression that is the basis for inhibition of electrophysiological measures of synaptic plasticity and thus learning and memory. We have utilized phenotypic screens in mature, in vitro cultures of rat brain cells to identify small molecules which block or prevent the binding and effects of Abeta oligomers. Synthetic Abeta oligomers bind saturably to a single site on neuronal synapses and induce deficits in membrane trafficking in neuronal cultures with an EC50 that corresponds to its binding affinity. The therapeutic lead compounds we have found are pharmacological antagonists of Abeta oligomers, reducing the binding of Abeta oligomers to neurons in vitro, preventing spine loss in neurons and preventing and treating oligomer-induced deficits in membrane trafficking. These molecules are highly brain penetrant and prevent and restore cognitive deficits in mouse models of Alzheimer's disease. Counter-screening these compounds against a broad panel of potential CNS targets revealed they are highly potent and specific ligands of the sigma-2/PGRMC1 receptor. Brain concentrations of the compounds corresponding to greater than 80% receptor occupancy at the sigma-2/PGRMC1 receptor restore cognitive function in transgenic hAPP Swe/Ldn mice. These studies demonstrate that synthetic and human-derived Abeta oligomers act as pharmacologically-behaved ligands at neuronal receptors - i.e. they exhibit saturable binding to a target, they exert a functional effect related to their binding and their displacement by small molecule antagonists blocks their functional effect. The first-in-class small molecule receptor antagonists described here restore memory to normal in multiple AD models and sustain improvement long-term, representing a novel mechanism of action for disease-modifying Alzheimer's therapeutics. PMID:25390368

Alzheimer's therapeutics targeting amyloid beta 1-42 oligomers I: Abeta 42 oligomer binding to specific neuronal receptors is displaced by drug candidates that improve cognitive deficits.

PubMed

Izzo, Nicholas J; Staniszewski, Agnes; To, Lillian; Fa, Mauro; Teich, Andrew F; Saeed, Faisal; Wostein, Harrison; Walko, Thomas; Vaswani, Anisha; Wardius, Meghan; Syed, Zanobia; Ravenscroft, Jessica; Mozzoni, Kelsie; Silky, Colleen; Rehak, Courtney; Yurko, Raymond; Finn, Patricia; Look, Gary; Rishton, Gilbert; Safferstein, Hank; Miller, Miles; Johanson, Conrad; Stopa, Edward; Windisch, Manfred; Hutter-Paier, Birgit; Shamloo, Mehrdad; Arancio, Ottavio; LeVine, Harry; Catalano, Susan M

2014-01-01

Synaptic dysfunction and loss caused by age-dependent accumulation of synaptotoxic beta amyloid (Abeta) 1-42 oligomers is proposed to underlie cognitive decline in Alzheimer's disease (AD). Alterations in membrane trafficking induced by Abeta oligomers mediates reduction in neuronal surface receptor expression that is the basis for inhibition of electrophysiological measures of synaptic plasticity and thus learning and memory. We have utilized phenotypic screens in mature, in vitro cultures of rat brain cells to identify small molecules which block or prevent the binding and effects of Abeta oligomers. Synthetic Abeta oligomers bind saturably to a single site on neuronal synapses and induce deficits in membrane trafficking in neuronal cultures with an EC50 that corresponds to its binding affinity. The therapeutic lead compounds we have found are pharmacological antagonists of Abeta oligomers, reducing the binding of Abeta oligomers to neurons in vitro, preventing spine loss in neurons and preventing and treating oligomer-induced deficits in membrane trafficking. These molecules are highly brain penetrant and prevent and restore cognitive deficits in mouse models of Alzheimer's disease. Counter-screening these compounds against a broad panel of potential CNS targets revealed they are highly potent and specific ligands of the sigma-2/PGRMC1 receptor. Brain concentrations of the compounds corresponding to greater than 80% receptor occupancy at the sigma-2/PGRMC1 receptor restore cognitive function in transgenic hAPP Swe/Ldn mice. These studies demonstrate that synthetic and human-derived Abeta oligomers act as pharmacologically-behaved ligands at neuronal receptors--i.e. they exhibit saturable binding to a target, they exert a functional effect related to their binding and their displacement by small molecule antagonists blocks their functional effect. The first-in-class small molecule receptor antagonists described here restore memory to normal in multiple AD models and sustain improvement long-term, representing a novel mechanism of action for disease-modifying Alzheimer's therapeutics.

Antigenicity analysis of human parvovirus B19-VP1u protein in the induction of anti-phospholipid syndrome.

PubMed

Lin, Chun-Yu; Chiu, Chun-Ching; Cheng, Ju; Lin, Chia-Yun; Shi, Ya-Fang; Tsai, Chun-Chou; Tzang, Bor-Show; Hsu, Tsai-Ching

2018-01-01

Mounting evidence suggests a connection between human parvovirus B19 (B19) and autoimmune diseases, and especially an association between the B19-VP1 unique region (VP1u) and anti-phospholipid syndrome (APS). However, little is known about the antigenicity of B19-VP1u in the induction of APS-like syndrome. To elucidate the antigenicity of B19-VP1u in the induction of APS, N-terminal truncated B19-VP1u (tVP1u) proteins were prepared to immunize Balb/c mice to generate antibodies against B19-tVP1u proteins. The secreted phospholipase A2 (sPLA2) activities and binding specificity of mice anti-B19-tVP1u antibodies with cardiolipin (CL) and beta-2-glycoprotein I (β2GPI) were evaluated by performing immunoblot, ELISA and absorption experiments. A mice model of passively induced APS was adopted. Although sPLA2 activities were identified in all B19-tVP1u proteins, only amino acid residues 61-227 B19-tVP1u exhibited a higher sPLA2 activity. Autoantibodies against CL and β2GPI exhibited binding activities with all B19-tVP1u proteins. IgG that was purified from mice that had been immunized with amino acid residues 21-227 to 121-227 B19-tVP1u proteins exhibited significantly higher binding activity with CL. IgG that was purified from mice that had been immunized with amino acid residues 21-227, 31-227, 82-227 and 91-227 B19-tVP1u proteins exhibited significantly higher binding activity with β2GPI. Accordingly, significantly higher binding inhibition of CL was detected in the presence of amino acid residues 61-227 and 101-227 B19-tVP1u. Significantly higher binding inhibition of β2GPI was detected in the presence of amino acid residues 21-227, 31-227, 82-227 and 91-227 B19-tVP1u. The mice that received amino acid residues 31-227 or 61-227 anti-tB19-VP1u IgG revealed significant thrombocytopenia and those that received amino acid residues 21-227, 31-227, 61-227, 71-227, 82-227, 91-227, 101-227 or 114-227 anti-tB19-VP1u IgG exhibited significantly prolonged aPTT. These findings provide further information concerning the role of B19-VP1u antigenicity in APS-like autoimmunity.

Linear scaffolds for multivalent targeting of melanocortin receptors.

PubMed

Dehigaspitiya, Dilani Chathurika; Anglin, Bobbi L; Smith, Kara R; Weber, Craig S; Lynch, Ronald M; Mash, Eugene A

2015-12-21

Molecules bearing one, two, three, or four copies of the tetrapeptide His-dPhe-Arg-Trp were attached to scaffolds based on ethylene glycol, glycerol, and d-mannitol by means of the copper-assisted azide-alkyne cyclization. The abilities of these compounds to block binding of a probe at the melanocortin 4 receptor were evaluated using a competitive binding assay. All of the multivalent molecules studied exhibited 30- to 40-fold higher apparent affinites when compared to a monovalent control. These results are consistent with divalent binding to receptor dimers. No evidence for tri- or tetravalent binding was obtained. Differences in the interligand spacing required for divalent binding, as opposed to tri- or tetravalent binding, may be responsible for these results.

Peptide-based antibody alternatives for biological sensing in austere environments

NASA Astrophysics Data System (ADS)

Coppock, Matthew B.; Sarkes, Deborah A.; Hurley, Margaret M.; Stratis-Cullum, Dimitra N.

2017-02-01

The most critical component of a biosensor, the biorecognition element, must exhibit high selectivity and strong affinity for a target of interest in operational sensing. Monoclonal antibodies are the current standard reagents for such devices, but their adaptability, manufacturability, and stability greatly limit their effectiveness in fieldable sensors. Peptides have emerged as potential antibody replacements in such applications due to their similar binding performance, extreme chemical and thermal stabilities, and on-demand scalability. In conjunction with modeling capabilities, work at the Army Research Lab focuses on protein catalyzed capture (PCC) agent technology and bacterial display for the discovery of these novel peptide binding reagents. The synthetic, bottom-up PCC agent technology uses an iterative, in situ "click chemistry" approach to produce high performing peptides against specific epitopes translatable to the protein target. Bacterial display allows rapid reagent discovery due to the combination of fast bacterial growth and effective peptide sequence enrichment through multiple rounds of biopanning. Recent advances in both methods are highlighted in regards to the discovery of reagents against Army high priority protein targets for soldier safety, performance, and diagnostics.

Influence of structural features of carrageenan on the formation of polyelectrolyte complexes with chitosan.

PubMed

Volod'ko, A V; Davydova, V N; Glazunov, V P; Likhatskaya, G N; Yermak, I M

2016-03-01

The polyelectrolyte complexes (PEC) of carrageenans (CG)-κ-, κ/β-, λ-and x-CG with chitosan were obtained. The formation of PEC was detected by Fourier-transform infrared (FTIR) spectroscopy and by centrifugation in a Percoll gradient. The influence of the structural peculiarities of CG on its interaction with chitosan was studied. The results of centrifugation showed that x-CG with a high degree of sulphation (SD) was completely bound to chitosan, unlike low SD κ-CG and κ/β-CG. Binding constant values showed there was a high affinity of CG for chitosan. CG with flexible macromolecule conformation and high SD exhibited the greatest binding affinity for chitosan. The full-atomic 3D-structures of the PEC κ-CG: chitosan in solution have been obtained by the experiments in silico for the first time. The amino groups of chitosan make the largest contribution to the energy of the complex formation by means of hydrogen and ionic bonds. The most probable complexes have stoichiometries of 1:1 and 1:1.5. Copyright © 2015 Elsevier B.V. All rights reserved.

Molecularly imprinted composite cryogel for albumin depletion from human serum.

PubMed

Andaç, Müge; Baydemir, Gözde; Yavuz, Handan; Denizli, Adil

2012-11-01

A new composite protein-imprinted macroporous cryogel was prepared for depletion of albumin from human serum prior to use in proteom applications. Polyhydroxyethyl-methacylate-based molecularly imprinted polymer (MIP) composite cryogel was prepared with high gel fraction yields up to 83%, and its morphology and porosity were characterized by Fourier transform infrared, scanning electron microscopy, swelling studies, flow dynamics, and surface area measurements. Selective binding experiments were performed in the presence of competitive proteins human transferrin (HTR) and myoglobin (MYB). MIP composite cryogel exhibited a high binding capacity and selectivity for human serum albumin (HSA) in the presence of HTR and MYB. The competitive adsorption amount for HSA in MIP composite cryogel is 722.1 mg/dL in the presence of competitive proteins (HTR and MYB). MIP composite cryogel column was successfully applied in the fast protein liquid chromatography system for selective depletion of albumin in human serum. The depletion ratio was highly increased by embedding beads into cryogel (85%). Finally, MIP composite cryogel can be reused many times with no apparent decrease in HSA adsorption capacity. Copyright © 2012 John Wiley & Sons, Ltd.

Rapid detection of methicillin resistance in coagulase-negative staphylococci by a penicillin-binding protein 2a-specific latex agglutination test.

PubMed

Horstkotte, M A; Knobloch, J K; Rohde, H; Mack, D

2001-10-01

The detection of PBP 2a by the MRSA-Screen latex agglutination test with 201 clinical coagulase-negative staphylococci had an initial sensitivity of 98% and a high degree of specificity for Staphylococcus epidermidis strains compared to PCR for mecA. Determination of oxacillin MICs evaluated according to the new breakpoint (0.5 microg/ml) of the National Committee for Clinical Laboratory Standards exhibited an extremely low specificity for this population.

Electroabsorption Spectroscopy Measurements of the Exciton Binding Energy, ElectronHole Reduced Effective Mass, and Band Gap in the Perovskite CHsub3NHsub3PbIsub3

DTIC Science & Technology

2016-07-28

can essentially be described in terms of free carrier generation . KEYWORDS: solar cell, photovoltaic, Franz−Keldysh effect, Wannier exciton, Stark...optoelectronic devices such as lasers,1,2 LEDs,3 and solar cells,4−6 despite requiring only inexpensive and relatively crude processing conditions...compared to current high-performance crystalline semiconductors. The archetypal material, CH3NH3PbI3, has exhibited excellent power con- version

T-state inhibitors of E. coli aspartate transcarbamoylase that prevent the allosteric transition.

PubMed

Heng, Sabrina; Stieglitz, Kimberly A; Eldo, Joby; Xia, Jiarong; Cardia, James P; Kantrowitz, Evan R

2006-08-22

Escherichia coli aspartate transcarbamoylase (ATCase) catalyzes the committed step in pyrimidine nucleotide biosynthesis, the reaction between carbamoyl phosphate (CP) and l-aspartate to form N-carbamoyl-l-aspartate and inorganic phosphate. The enzyme exhibits homotropic cooperativity and is allosterically regulated. Upon binding l-aspartate in the presence of a saturating concentration of CP, the enzyme is converted from the low-activity low-affinity T state to the high-activity high-affinity R state. The potent inhibitor N-phosphonacetyl-l-aspartate (PALA), which combines the binding features of Asp and CP into one molecule, has been shown to induce the allosteric transition to the R state. In the presence of only CP, the enzyme is the T structure with the active site primed for the binding of aspartate. In a structure of the enzyme-CP complex (T(CP)), two CP molecules were observed in the active site approximately 7A apart, one with high occupancy and one with low occupancy. The high occupancy site corresponds to the position for CP observed in the structure of the enzyme with CP and the aspartate analogue succinate bound. The position of the second CP is in a unique site and does not overlap with the aspartate binding site. As a means to generate a new class of inhibitors for ATCase, the domain-open T state of the enzyme was targeted. We designed, synthesized, and characterized three inhibitors that were composed of two phosphonacetamide groups linked together. These two phosphonacetamide groups mimic the positions of the two CP molecules in the T(CP) structure. X-ray crystal structures of ATCase-inhibitor complexes revealed that each of these inhibitors bind to the T state of the enzyme and occupy the active site area. As opposed to the binding of Asp in the presence of CP or PALA, these inhibitors are unable to initiate the global T to R conformational change. Although the best of these T-state inhibitors only has a K(i) value in the micromolar range, the structural information with respect to their mode of binding provides important information for the design of second generation inhibitors that will have even higher affinity for the active site of the T state of the enzyme.

Post-ExSELEX stabilization of an unnatural-base DNA aptamer targeting VEGF165 toward pharmaceutical applications.

PubMed

Kimoto, Michiko; Nakamura, Mana; Hirao, Ichiro

2016-09-06

A new technology, genetic alphabet expansion using artificial bases (unnatural bases), has created high-affinity DNA ligands (aptamers) that specifically bind to target proteins by ExSELEX (genetic alphabet Expansion for Systematic Evolution of Ligands by EXponential enrichment). We recently found that the unnatural-base DNA aptamers can be stabilized against nucleases, by introducing an extraordinarily stable, unique hairpin DNA (mini-hairpin DNA) and by reinforcing the stem region with G-C pairs. Here, to establish this aptamer generation method, we examined the stabilization of a high-affinity anti-VEGF165 unnatural-base DNA aptamer. The stabilized aptamers displayed significantly increased thermal and nuclease stabilities, and furthermore, exhibited higher affinity to the target. As compared to the well-known anti-VEGF165 RNA aptamer, pegaptanib (Macugen), our aptamers did not require calcium ions for binding to VEGF165 Biological experiments using cultured cells revealed that our stabilized aptamers efficiently inhibited the interaction between VEGF165 and its receptor, with the same or slightly higher efficiency than that of the pegaptanib RNA aptamer. The development of cost-effective and calcium ion-independent high-affinity anti-VEGF165 DNA aptamers encourages further progress in diagnostic and therapeutic applications. In addition, the stabilization process provided additional information about the key elements required for aptamer binding to VEGF165. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Stability of local secondary structure determines selectivity of viral RNA chaperones.

PubMed

Bravo, Jack P K; Borodavka, Alexander; Barth, Anders; Calabrese, Antonio N; Mojzes, Peter; Cockburn, Joseph J B; Lamb, Don C; Tuma, Roman

2018-05-18

To maintain genome integrity, segmented double-stranded RNA viruses of the Reoviridae family must accurately select and package a complete set of up to a dozen distinct genomic RNAs. It is thought that the high fidelity segmented genome assembly involves multiple sequence-specific RNA-RNA interactions between single-stranded RNA segment precursors. These are mediated by virus-encoded non-structural proteins with RNA chaperone-like activities, such as rotavirus (RV) NSP2 and avian reovirus σNS. Here, we compared the abilities of NSP2 and σNS to mediate sequence-specific interactions between RV genomic segment precursors. Despite their similar activities, NSP2 successfully promotes inter-segment association, while σNS fails to do so. To understand the mechanisms underlying such selectivity in promoting inter-molecular duplex formation, we compared RNA-binding and helix-unwinding activities of both proteins. We demonstrate that octameric NSP2 binds structured RNAs with high affinity, resulting in efficient intramolecular RNA helix disruption. Hexameric σNS oligomerizes into an octamer that binds two RNAs, yet it exhibits only limited RNA-unwinding activity compared to NSP2. Thus, the formation of intersegment RNA-RNA interactions is governed by both helix-unwinding capacity of the chaperones and stability of RNA structure. We propose that this protein-mediated RNA selection mechanism may underpin the high fidelity assembly of multi-segmented RNA genomes in Reoviridae.

Clinical isolates of Enterococcus faecium exhibit strain-specific collagen binding mediated by Acm, a new member of the MSCRAMM family.

PubMed

Nallapareddy, Sreedhar R; Weinstock, George M; Murray, Barbara E

2003-03-01

A collagen-binding adhesin of Enterococcus faecium, Acm, was identified. Acm shows 62% similarity to the Staphylococcus aureus collagen adhesin Cna over the entire protein and is more similar to Cna (60% and 75% similarity with Cna A and B domains respectively) than to the Enterococcus faecalis collagen-binding adhesin, Ace, which shares homology with Acm only in the A domain. Despite the detection of acm in 32 out of 32 E. faecium isolates, only 11 of these (all clinical isolates, including four vancomycin-resistant endocarditis isolates and seven other isolates) exhibited binding to collagen type I (CI). Although acm from three CI-binding vancomycin-resistant E. faecium clinical isolates showed 100% identity, analysis of acm genes and their promoter regions from six non-CI-binding strains identified deletions or mutations that introduced stop codons and/or IS elements within the gene or the promoter region in five out of six strains, suggesting that the presence of an intact functional acm gene is necessary for binding of E. faecium strains to CI. Recombinant Acm A domain showed specific and concentration-dependent binding to collagen, and this protein competed with E. faecium binding to immobilized CI. Consistent with the adherence phenotype and sequence data, probing with Acm-specific IgGs purified from anti-recombinant Acm A polyclonal rabbit serum confirmed the surface expression of Acm in three out of three collagen-binding clinical isolates of E. faecium tested, but in none of the strains with a non-functional pseudo acm gene. Introduction of a functional acm gene into two non-CI-binding natural acm mutant strains conferred a CI-binding phenotype, further confirming that native Acm is sufficient for the binding of E. faecium to CI. These results demonstrate that acm, which encodes a potential virulence factor, is functional only in certain infection-derived clinical isolates of E. faecium, and suggest that Acm is the primary adhesin responsible for the ability of E. faecium to bind collagen.

Rearranged diterpenoids from the biotransformation of ent-trachyloban-18-oic acid by Rhizopus arrhizus.

PubMed

Leverrier, Aurélie; Martin, Marie-Thérèse; Servy, Claudine; Ouazzani, Jamal; Retailleau, Pascal; Awang, Khalijah; Mukhtar, Mat Ropi; Guéritte, Françoise; Litaudon, Marc

2010-06-25

In our search for inhibitors of the antiapoptotic protein Bcl-xL, investigation of Xylopia caudata afforded a new diterpenoid, ent-trachyloban-4beta-ol (2), and five known ent-trachylobane or ent-atisane compounds. Only ent-trachyloban-18-oic acid (1) exhibited weak binding activity to Bcl-xL. These compounds exhibited cytotoxicity against KB and HCT-116 cell lines with IC(50) values between 10 and 30 microM. Bioconversion of compound 1 by Rhizopus arrhizus afforded new hydroxylated metabolites (3-7) of the ent-trachylobane and ent-kaurene type and compound 8, with a rearranged pentacyclic carbon framework that was named rhizopene. Compounds 3-8 were noncytotoxic to the two cancer cell lines, and compounds 3 and 5 exhibited only weak binding affinity for Bcl-xL.

Crystal structure correlations with the intrinsic thermodynamics of human carbonic anhydrase inhibitor binding

PubMed Central

Smirnov, Alexey; Zubrienė, Asta; Manakova, Elena; Gražulis, Saulius

2018-01-01

The structure-thermodynamics correlation analysis was performed for a series of fluorine- and chlorine-substituted benzenesulfonamide inhibitors binding to several human carbonic anhydrase (CA) isoforms. The total of 24 crystal structures of 16 inhibitors bound to isoforms CA I, CA II, CA XII, and CA XIII provided the structural information of selective recognition between a compound and CA isoform. The binding thermodynamics of all structures was determined by the analysis of binding-linked protonation events, yielding the intrinsic parameters, i.e., the enthalpy, entropy, and Gibbs energy of binding. Inhibitor binding was compared within structurally similar pairs that differ by para- or meta-substituents enabling to obtain the contributing energies of ligand fragments. The pairs were divided into two groups. First, similar binders—the pairs that keep the same orientation of the benzene ring exhibited classical hydrophobic effect, a less exothermic enthalpy and a more favorable entropy upon addition of the hydrophobic fragments. Second, dissimilar binders—the pairs of binders that demonstrated altered positions of the benzene rings exhibited the non-classical hydrophobic effect, a more favorable enthalpy and variable entropy contribution. A deeper understanding of the energies contributing to the protein-ligand recognition should lead toward the eventual goal of rational drug design where chemical structures of ligands could be designed based on the target protein structure. PMID:29503769

Liver Fatty acid binding protein (L-Fabp) modulates murine stellate cell activation and diet induced nonalcoholic fatty liver disease

PubMed Central

Chen, Anping; Tang, Youcai; Davis, Victoria; Hsu, Fong-Fu; Kennedy, Susan M.; Song, Haowei; Turk, John; Brunt, Elizabeth M.; Newberry, Elizabeth P.; Davidson, Nicholas O.

2013-01-01

Activation of hepatic stellate cells (HSCs) is crucial to the development of fibrosis in nonalcoholic fatty liver disease. Quiescent HSCs contain lipid droplets (LDs), whose depletion upon activation induces a fibrogenic gene program. Here we show that liver fatty acid-binding protein (L-Fabp), an abundant cytosolic protein that modulates fatty acid (FA) metabolism in enterocytes and hepatocytes also modulates HSC FA utilization and in turn regulates the fibrogenic program. L-Fabp expression decreased 10-fold following HSC activation, concomitant with depletion of LDs. Primary HSCs isolated from L-FABP−/− mice contain fewer LDs than wild type (WT) HSCs, and exhibit upregulated expression of genes involved in HSC activation. Adenoviral L-Fabp transduction inhibited activation of passaged WT HSCs and increased both the expression of prolipogenic genes and also augmented intracellular lipid accumulation, including triglyceride and FA, predominantly palmitate. Freshly isolated HSCs from L-FABP−/− mice correspondingly exhibited decreased palmitate in the free FA pool. To investigate whether L-FABP deletion promotes HSC activation in vivo, we fed L-FABP−/− and WT mice a high fat diet supplemented with trans-fatty acids and fructose (TFF). TFF-fed L-FABP−/− mice exhibited reduced hepatic steatosis along with decreased LD abundance and size compared to WT mice. In addition, TFF-fed L-FABP−/− mice exhibited decreased hepatic fibrosis, with reduced expression of fibrogenic genes, compared to WT mice. Conclusion L-FABP deletion attenuates both diet-induced hepatic steatosis and fibrogenesis, despite the observation that L-Fabp paradoxically promotes FA and LD accumulation and inhibits HSC activation in vitro. These findings highlight the importance of cell-specific modulation of hepatic lipid metabolism in promoting fibrogenesis in nonalcoholic fatty liver disease. PMID:23401290

Direct labelling of the human P2X7 receptor and identification of positive and negative cooperativity of binding.

PubMed

Michel, A D; Chambers, L J; Clay, W C; Condreay, J P; Walter, D S; Chessell, I P

2007-05-01

The P2X(7) receptor exhibits complex pharmacological properties. In this study, binding of a [(3)H]-labelled P2X(7) receptor antagonist to human P2X(7) receptors has been examined to further understand ligand interactions with this receptor. The P2X(7) receptor antagonist, N-[2-({2-[(2-hydroxyethyl)amino]ethyl}amino)-5-quinolinyl]-2-tricyclo[3.3.1.1(3,7)]dec-1-ylacetamide (compound-17), was radiolabelled with tritium and binding studies were performed using membranes prepared from U-2 OS or HEK293 cells expressing human recombinant P2X(7) receptors. Binding of [(3)H]-compound-17 was higher in membranes prepared from cells expressing P2X(7) receptors than from control cells and was inhibited by ATP suggesting labelled sites represented human P2X(7) receptors. Binding was reversible, saturable and modulated by P2X(7) receptor ligands (Brilliant Blue G, KN62, ATP, decavanadate). Furthermore, ATP potency was reduced in the presence of divalent cations or NaCl. Radioligand binding exhibited both positive and negative cooperativity. Positive cooperativity was evident from bell shaped Scatchard plots, reduction in radioligand dissociation rate by unlabelled compound-17 and enhancement of radioligand binding by KN62 and unlabelled compound-17. ATP and decavanadate inhibited binding in a negative cooperative manner as they enhanced radioligand dissociation. These data demonstrate that human P2X(7) receptors can be directly labelled and provide novel insights into receptor function. The positive cooperativity observed suggests that binding of compound-17 to one subunit in the P2X(7) receptor complex enhances subsequent binding to other P2X(7) subunits in the same complex. The negative cooperative effects of ATP suggest that ATP and compound-17 bind at separate, interacting, sites on the P2X(7) receptor.

Direct labelling of the human P2X7 receptor and identification of positive and negative cooperativity of binding

PubMed Central

Michel, A D; Chambers, L J; Clay, W C; Condreay, J P; Walter, D S; Chessell, I P

2007-01-01

Background and Purpose: The P2X7 receptor exhibits complex pharmacological properties. In this study, binding of a [3H]-labelled P2X7 receptor antagonist to human P2X7 receptors has been examined to further understand ligand interactions with this receptor. Experimental Approach: The P2X7 receptor antagonist, N-[2-({2-[(2-hydroxyethyl)amino]ethyl}amino)-5-quinolinyl]-2-tricyclo[3.3.1.13,7]dec-1-ylacetamide (compound-17), was radiolabelled with tritium and binding studies were performed using membranes prepared from U-2 OS or HEK293 cells expressing human recombinant P2X7 receptors. Key Results: Binding of [3H]-compound-17 was higher in membranes prepared from cells expressing P2X7 receptors than from control cells and was inhibited by ATP suggesting labelled sites represented human P2X7 receptors. Binding was reversible, saturable and modulated by P2X7 receptor ligands (Brilliant Blue G, KN62, ATP, decavanadate). Furthermore, ATP potency was reduced in the presence of divalent cations or NaCl. Radioligand binding exhibited both positive and negative cooperativity. Positive cooperativity was evident from bell shaped Scatchard plots, reduction in radioligand dissociation rate by unlabelled compound-17 and enhancement of radioligand binding by KN62 and unlabelled compound-17. ATP and decavanadate inhibited binding in a negative cooperative manner as they enhanced radioligand dissociation. Conclusions: These data demonstrate that human P2X7 receptors can be directly labelled and provide novel insights into receptor function. The positive cooperativity observed suggests that binding of compound-17 to one subunit in the P2X7 receptor complex enhances subsequent binding to other P2X7 subunits in the same complex. The negative cooperative effects of ATP suggest that ATP and compound-17 bind at separate, interacting, sites on the P2X7 receptor. PMID:17339830

Development of QSAR models for predicting the binding affinity of endocrine disrupting chemicals to eight fish estrogen receptor.

PubMed

He, Junyi; Peng, Tao; Yang, Xianhai; Liu, Huihui

2018-02-01

Endocrine disrupting effect has become a central point of concern, and various biological mechanisms involve in the disruption of endocrine system. Recently, we have explored the mechanism of disrupting hormonal transport protein, through the binding affinity of sex hormone-binding globulin in different fish species. This study, serving as a companion article, focused on the mechanism of activating/inhibiting hormone receptor, by investigating the binding interaction of chemicals with the estrogen receptor (ER) of different fish species. We collected the relative binding affinity (RBA) of chemicals with 17β-estradiol binding to the ER of eight fish species. With this parameter as the endpoints, quantitative structure-activity relationship (QSAR) models were established using DRAGON descriptors. Statistical results indicated that the developed models had satisfactory goodness of fit, robustness and predictive ability. The Euclidean distance and Williams plot verified that these models had wide application domains, which covered a large number of structurally diverse chemicals. Based on the screened descriptors, we proposed an appropriate mechanism interpretation for the binding potency. Additionally, even though the same chemical had different affinities for ER from different fish species, the affinity of ER exhibited a high correlation for fish species within the same Order (i.e., Salmoniformes, Cypriniformes, Perciformes), which consistent with that in our previous study. Hence, when performing the endocrine disrupting effect assessment, the species diversity should be taken into account, but maybe the fish species in the same Order can be grouped together. Copyright © 2017 Elsevier Inc. All rights reserved.

Solution structure of the DNA-binding domain of RPA from Saccharomyces cerevisiae and its interaction with single-stranded DNA and SV40 T antigen

PubMed Central

Park, Chin-Ju; Lee, Joon-Hwa; Choi, Byong-Seok

2005-01-01

Replication protein A (RPA) is a three-subunit complex with multiple roles in DNA metabolism. DNA-binding domain A in the large subunit of human RPA (hRPA70A) binds to single-stranded DNA (ssDNA) and is responsible for the species-specific RPA–T antigen (T-ag) interaction required for Simian virus 40 replication. Although Saccharomyces cerevisiae RPA70A (scRPA70A) shares high sequence homology with hRPA70A, the two are not functionally equivalent. To elucidate the similarities and differences between these two homologous proteins, we determined the solution structure of scRPA70A, which closely resembled the structure of hRPA70A. The structure of ssDNA-bound scRPA70A, as simulated by residual dipolar coupling-based homology modeling, suggested that the positioning of the ssDNA is the same for scRPA70A and hRPA70A, although the conformational changes that occur in the two proteins upon ssDNA binding are not identical. NMR titrations of hRPA70A with T-ag showed that the T-ag binding surface is separate from the ssDNA-binding region and is more neutral than the corresponding part of scRPA70A. These differences might account for the species-specific nature of the hRPA70A–T-ag interaction. Our results provide insight into how these two homologous RPA proteins can exhibit functional differences, but still both retain their ability to bind ssDNA. PMID:16043636

Chitin binding proteins act synergistically with chitinases in Serratia proteamaculans 568.

PubMed

Purushotham, Pallinti; Arun, P V Parvati Sai; Prakash, Jogadhenu S S; Podile, Appa Rao

2012-01-01

Genome sequence of Serratia proteamaculans 568 revealed the presence of three family 33 chitin binding proteins (CBPs). The three Sp CBPs (Sp CBP21, Sp CBP28 and Sp CBP50) were heterologously expressed and purified. Sp CBP21 and Sp CBP50 showed binding preference to β-chitin, while Sp CBP28 did not bind to chitin and cellulose substrates. Both Sp CBP21 and Sp CBP50 were synergistic with four chitinases from S. proteamaculans 568 (Sp ChiA, Sp ChiB, Sp ChiC and Sp ChiD) in degradation of α- and β-chitin, especially in the presence of external electron donor (reduced glutathione). Sp ChiD benefited most from Sp CBP21 or Sp CBP50 on α-chitin, while Sp ChiB and Sp ChiD had major advantage with these Sp CBPs on β-chitin. Dose responsive studies indicated that both the Sp CBPs exhibit synergism ≥ 0.2 µM. The addition of both Sp CBP21 and Sp CBP50 in different ratios to a synergistic mixture did not significantly increase the activity. Highly conserved polar residues, important in binding and activity of CBP21 from S. marcescens (Sm CBP21), were present in Sp CBP21 and Sp CBP50, while Sp CBP28 had only one such polar residue. The inability of Sp CBP28 to bind to the test substrates could be attributed to the absence of important polar residues.

Chitin Binding Proteins Act Synergistically with Chitinases in Serratia proteamaculans 568

PubMed Central

Purushotham, Pallinti; Arun, P. V. Parvati Sai; Prakash, Jogadhenu S. S.; Podile, Appa Rao

2012-01-01

Genome sequence of Serratia proteamaculans 568 revealed the presence of three family 33 chitin binding proteins (CBPs). The three Sp CBPs (Sp CBP21, Sp CBP28 and Sp CBP50) were heterologously expressed and purified. Sp CBP21 and Sp CBP50 showed binding preference to β-chitin, while Sp CBP28 did not bind to chitin and cellulose substrates. Both Sp CBP21 and Sp CBP50 were synergistic with four chitinases from S. proteamaculans 568 (Sp ChiA, Sp ChiB, Sp ChiC and Sp ChiD) in degradation of α- and β-chitin, especially in the presence of external electron donor (reduced glutathione). Sp ChiD benefited most from Sp CBP21 or Sp CBP50 on α-chitin, while Sp ChiB and Sp ChiD had major advantage with these Sp CBPs on β-chitin. Dose responsive studies indicated that both the Sp CBPs exhibit synergism ≥0.2 µM. The addition of both Sp CBP21 and Sp CBP50 in different ratios to a synergistic mixture did not significantly increase the activity. Highly conserved polar residues, important in binding and activity of CBP21 from S. marcescens (Sm CBP21), were present in Sp CBP21 and Sp CBP50, while Sp CBP28 had only one such polar residue. The inability of Sp CBP28 to bind to the test substrates could be attributed to the absence of important polar residues. PMID:22590591

The Role of Aging in Intra-Item and Item-Context Binding Processes in Visual Working Memory

ERIC Educational Resources Information Center

Peterson, Dwight J.; Naveh-Benjamin, Moshe

2016-01-01

Aging is accompanied by declines in both working memory and long-term episodic memory processes. Specifically, important age-related memory deficits are characterized by performance impairments exhibited by older relative to younger adults when binding distinct components into a single integrated representation, despite relatively intact memory…

A dominant conformational role for amino acid diversity in minimalist protein–protein interfaces

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

Gilbreth, Ryan N.; Esaki, Kaori; Koide, Akiko

Recent studies have shown that highly simplified interaction surfaces consisting of combinations of just two amino acids, Tyr and Ser, exhibit high affinity and specificity. The high functional levels of such minimalist interfaces might thus indicate small contributions of greater amino acid diversity seen in natural interfaces. Toward addressing this issue, we have produced a pair of binding proteins built on the fibronectin type III scaffold, termed “monobodies.” One monobody contains the Tyr/Ser binary-code interface (termed YS) and the other contains an expanded amino acid diversity interface (YSX), but both bind to an identical target, maltose-binding protein. The YSX monobodymore » bound with higher affinity, a slower off rate and a more favorable enthalpic contribution than the YS monobody. High-resolution X-ray crystal structures revealed that both proteins bound to an essentially identical epitope, providing a unique opportunity to directly investigate the role of amino acid diversity in a protein interaction interface. Surprisingly, Tyr still dominates the YSX paratope and the additional amino acid types are primarily used to conformationally optimize contacts made by tyrosines. Scanning mutagenesis showed that while all contacting Tyr side chains are essential in the YS monobody, the YSX interface was more tolerant to mutations. These results suggest that the conformational, not chemical, diversity of additional types of amino acids provided higher functionality and evolutionary robustness, supporting the dominant role of Tyr and the importance of conformational diversity in forming protein interaction interfaces.« less

A Dominant Conformational Role for Amino Acid Diversity in Minimalist Protein-Protein Interfaces

PubMed Central

Gilbreth, Ryan N.; Esaki, Kaori; Koide, Akiko; Sidhu, Sachdev S.; Koide, Shohei

2008-01-01

Recent studies have shown that highly simplified interaction surfaces consisting of combinations of just two amino acids, Tyr and Ser, exhibit high affinity and specificity. The high functional levels of such minimalist interfaces might thus indicate small contributions of greater amino acid diversity seen in natural interfaces. Toward addressing this issue, we have produced a pair of binding proteins built on the fibronectin type III scaffold, termed “monobodies”. One monobody contains the Tyr/Ser binary-code interface (termed YS) and the other contains an expanded amino acid diversity interface (YSX), but both bind to an identical target, maltose binding protein (MBP). The YSX monobody bound with higher affinity, a slower off rate and a more favorable enthalpic contribution than the YS monobody. High-resolution x-ray crystal structures revealed that both proteins bound to an essentially identical epitope, providing a unique opportunity to directly investigate the role of amino acid diversity in a protein interaction interface. Surprisingly, Tyr still dominates the YSX paratope and the additional amino acid types are primarily used to conformationally optimize contacts made by tyrosines. Scanning mutagenesis showed that while all contacting Tyr side-chains are essential in the YS monobody, the YSX interface was more tolerant to mutations. These results suggest that the conformational, not chemical, diversity of additional types of amino acids provided higher functionality and evolutionary robustness, supporting the dominant role of Tyr and the importance of conformational diversity in forming protein interaction interfaces. PMID:18602117

A 10-aa-long sequence in SLP-76 upstream of the Gads binding site is essential for T cell development and function.

PubMed

Kumar, Lalit; Feske, Stefan; Rao, Anjana; Geha, Raif S

2005-12-27

The adapter SLP-76 is essential for T cell development and function. SLP-76 binds to the src homology 3 domain of Lck in vitro. This interaction depends on amino acids 185-194 of SLP-76. To examine the role of the Lck-binding region of SLP-76 in T cell development and function, SLP-76(-/-) mice were reconstituted with an SLP-76 mutant that lacks amino acids 185-194. Double and single positive thymocytes from reconstituted mice were severely reduced in numbers and exhibited impaired positive selection and increased apoptosis. Peripheral T cells were also reduced in numbers, exhibited impaired phospholipase C-gamma1 and Erk phosphorylation, and failed to flux calcium, secrete IL-2, and proliferate in response to T cell antigen receptor ligation. Delayed cutaneous hypersensitivity responses and Ab responses to T cell-dependent antigen were severely impaired. These results indicate that the Lck binding region of SLP-76 is essential for T cell antigen receptor signaling and normal T cell development and function.

C-Terminal carbohydrate-binding module 9_2 fused to the N-terminus of GH11 xylanase from Aspergillus niger.

PubMed

Xu, Wenxuan; Liu, Yajuan; Ye, Yanxin; Liu, Meng; Han, Laichuang; Song, Andong; Liu, Liangwei

2016-10-01

The 9_2 carbohydrate-binding module (C2) locates natively at the C-terminus of the GH10 thermophilic xylanase from Thermotoga marimita. When fused to the C-terminus, C2 improved thermostability of a GH11 xylanase (Xyn) from Aspergillus niger. However, a question is whether the C-terminal C2 would have a thermostabilizing effect when fused to the N-terminus of a catalytic module. A chimeric enzyme, C2-Xyn, was created by step-extension PCR, cloned in pET21a(+), and expressed in E. coli BL21(DE3). The C2-Xyn exhibited a 2 °C higher optimal temperature, a 2.8-fold longer thermostability, and a 4.5-fold higher catalytic efficiency on beechwood xylan than the Xyn. The C2-Xyn exhibited a similar affinity for binding to beechwood xylan and a higher affinity for oat-spelt xylan than Xyn. C2 is a thermostabilizing carbohydrate-binding module and provides a model of fusion at an enzymatic terminus inconsistent with the modular natural terminal location.

Region-specific associations between sex, social status, and oxytocin receptor density in the brains of eusocial rodents.

PubMed

Mooney, S J; Coen, C W; Holmes, M M; Beery, A K

2015-09-10

Naturally occurring variations in neuropeptide receptor distributions in the brain contribute to numerous mammalian social behaviors. In naked mole-rats, which live in large social groups and exhibit remarkable reproductive skew, colony-related social behaviors vary with reproductive status. Here we examined whether variation in social status is associated with variations in the location and/or density of oxytocin binding in this species. Autoradiography was performed to assess forebrain oxytocin receptor (OTR) densities in breeding and non-breeding naked mole-rats of both sexes. Overall, males exhibited higher OTR binding in the medial amygdala in comparison to females. While there were no main effects of reproductive status in any region, a sex difference in OTR binding in the nucleus accumbens was mediated by status. Specifically, breeding males tended to have more OTR binding than breeding females in the nucleus accumbens, while no sex difference was observed in subordinates. These effects suggest that oxytocin may act in a sex- and region-specific way that corresponds to reproductive status and associated social behaviors. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Toxoplasma gondii peptide ligands open the gate of the HLA class I binding groove

PubMed Central

McMurtrey, Curtis; Trolle, Thomas; Sansom, Tiffany; Remesh, Soumya G; Kaever, Thomas; Bardet, Wilfried; Jackson, Kenneth; McLeod, Rima; Sette, Alessandro; Nielsen, Morten; Zajonc, Dirk M; Blader, Ira J; Peters, Bjoern; Hildebrand, William

2016-01-01

HLA class I presentation of pathogen-derived peptide ligands is essential for CD8+ T-cell recognition of Toxoplasma gondii infected cells. Currently, little data exist pertaining to peptides that are presented after T. gondii infection. Herein we purify HLA-A*02:01 complexes from T. gondii infected cells and characterize the peptide ligands using LCMS. We identify 195 T. gondii encoded ligands originating from both secreted and cytoplasmic proteins. Surprisingly, T. gondii ligands are significantly longer than uninfected host ligands, and these longer pathogen-derived peptides maintain a canonical N-terminal binding core yet exhibit a C-terminal extension of 1–30 amino acids. Structural analysis demonstrates that binding of extended peptides opens the HLA class I F’ pocket, allowing the C-terminal extension to protrude through one end of the binding groove. In summary, we demonstrate that unrealized structural flexibility makes MHC class I receptive to parasite-derived ligands that exhibit unique C-terminal peptide extensions. DOI: http://dx.doi.org/10.7554/eLife.12556.001 PMID:26824387

Solution structure and backbone dynamics of the N-terminal region of the calcium regulatory domain from soybean calcium-dependent protein kinase alpha.

PubMed

Weljie, Aalim M; Gagné, Stéphane M; Vogel, Hans J

2004-12-07

Ca(2+)-dependent protein kinases (CDPKs) are vital Ca(2+)-signaling proteins in plants and protists which have both a kinase domain and a self-contained calcium regulatory calmodulin-like domain (CLD). Despite being very similar to CaM (>40% identity) and sharing the same fold, recent biochemical and structural evidence suggests that the behavior of CLD is distinct from its namesake, calmodulin. In this study, NMR spectroscopy is employed to examine the structure and backbone dynamics of a 168 amino acid Ca(2+)-saturated construct of the CLD (NtH-CLD) in which almost the entire C-terminal domain is exchange broadened and not visible in the NMR spectra. Structural characterization of the N-terminal domain indicates that the first Ca(2+)-binding loop is significantly more open than in a recently reported structure of the CLD complexed with a putative intramolecular binding region (JD) in the CDPK. Backbone dynamics suggest that parts of the third helix exhibit unusually high mobility, and significant exchange, consistent with previous findings that this helix interacts with the C-terminal domain. Dynamics data also show that the "tether" region, consisting of the first 11 amino acids of CLD, is highly mobile and these residues exhibit distinctive beta-type secondary structure, which may help to position the JD and CLD. Finally, the unusual global dynamic behavior of the protein is rationalized on the basis of possible interdomain rearrangements and the highly variable environments of the C- and N-terminal domains.

Mechanism and Substrate Recognition of Human Holo ACP Synthase

PubMed Central

Bunkoczi, Gabor; Pasta, Saloni; Joshi, Anil; Wu, Xiaoqiu; Kavanagh, Kathryn L.; Smith, Stuart; Oppermann, Udo

2007-01-01

Summary Mammals utilize a single phosphopantetheinyl transferase for the posttranslational modification of at least three different apoproteins: the carrier protein components of cytosolic and mitochondrial fatty acid synthases and the aminoadipate semialdehyde reductase involved in lysine degradation. We determined the crystal structure of the human phosphopantetheinyl transferase, a eukaryotic phosphopantetheinyl transferase characterized, complexed with CoA and Mg2+, and in ternary complex with CoA and ACP. The involvement of key residues in ligand binding and catalysis was confirmed by mutagenesis and kinetic analysis. Human phosphopantetheinyl transferase exhibits an α/β fold and 2-fold pseudosymmetry similar to the Sfp phosphopantetheinyl transferase from Bacillus subtilis. Although the bound ACP exhibits a typical four-helix structure, its binding is unusual in that it is facilitated predominantly by hydrophobic interactions. A detailed mechanism is proposed describing the substrate binding and catalytic process. PMID:18022563

Comparative studies on the human serum albumin binding of the clinically approved EGFR inhibitors gefitinib, erlotinib, afatinib, osimertinib and the investigational inhibitor KP2187.

PubMed

Dömötör, Orsolya; Pelivan, Karla; Borics, Attila; Keppler, Bernhard K; Kowol, Christian R; Enyedy, Éva A

2018-05-30

Binding interactions between human serum albumin (HSA) and four approved epidermal growth factor receptor (EGFR) inhibitors gefitinib (GEF), erlotinib (ERL), afatinib (AFA), osimertinib (OSI), as well as the experimental drug KP2187, were investigated by means of spectrofluorometric and molecular modelling methods. Steady-state and time resolved spectrofluorometric techniques were carried out, including direct quenching of protein fluorescence and site marker displacement measurements. Proton dissociation processes and solvent dependent fluorescence properties were investigated as well. The EGFR inhibitors were predominantly presented in their single protonated form (HL + ) at physiological pH except ERL, which is charge-neutral. Significant solvent dependent fluorescence properties were found for GEF, ERL and KP2187, namely their emission spectra show strong dependence on the polarity and the hydrogen bonding ability of the solvents. The inhibitors proved to be bound at site I of HSA (in subdomain IIA) in a weak-to-moderate fashion (logK' 3.9-4.9) using spectrofluorometry. OSI (logK' 4.3) and KP2187 can additionally bind in site II (in subdomain IIIA), while GEF, ERL and AFA clearly show no interaction here. Docking methods qualitatively confirmed binding site preferences of compounds GEF and KP2187, and indicated that they probably bind to HSA in their neutral forms. Binding constants calculated on the basis of the various experimental data indicate a weak-to-moderate binding on HSA, only OSI exhibits somewhat higher affinity towards this protein. However, model calculations performed at physiological blood concentrations of HSA resulted in high (ca. 90%) bound fractions for the inhibitors, highlighting the importance of plasma protein binding. Copyright © 2018 Elsevier B.V. All rights reserved.

The oxygen-binding properties of hemocyanin from the mollusk Concholepas concholepas.

PubMed

González, Andrea; Nova, Esteban; Del Campo, Miguel; Manubens, Augusto; De Ioannes, Alfredo; Ferreira, Jorge; Becker, María Inés

2017-12-01

Hemocyanins have highly conserved copper-containing active sites that bind oxygen. However, structural differences among the hemocyanins of various mollusks may affect their physicochemical properties. Here, we studied the oxygen-binding cooperativity and affinity of Concholepas concholepas hemocyanin (CCH) and its two isolated subunits over a wide range of temperatures and pH values. Considering the differences in the quaternary structures of CCH and keyhole limpet hemocyanin (KLH), we hypothesized that the heterodidecameric CCH has different oxygen-binding parameters than the homodidecameric KLH. A novel modification of the polarographic method was applied in which rat liver submitochondrial particles containing cytochrome c oxidase were introduced to totally deplete oxygen of the test solution using ascorbate as the electron donor. This method was both sensitive and reproducible. The results showed that CCH, like other hemocyanins, exhibits cooperativity, showing an inverse relationship between the oxygen-binding parameters and temperature. According to their Hill coefficients, KLH has greater cooperativity than CCH at physiological pH; however, CCH is less sensitive to pH changes than KLH. Appreciable differences in binding behavior were found between the CCH subunits: the cooperativity of CCH-A was not only almost double that of CCH-B, but it was also slightly superior to that of CCH, thus suggesting that the oxygen-binding domains of the CCH subunits are different in their primary structure. Collectively, these data suggest that CCH-A is the main oxygen-binding domain in CCH; CCH-B may play a more structural role, perhaps utilizing its surprising predisposition to form tubular polymers, unlike CCH-A, as demonstrated here using electron microscopy. Copyright © 2017 Elsevier B.V. All rights reserved.

Structure of the Mycobacterium tuberculosis D-Alanine:D-Alanine Ligase, a Target of the Antituberculosis Drug D-Cycloserine

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

Bruning, John B.; Murillo, Ana C.; Chacon, Ofelia

D-Alanine:D-alanine ligase (EC 6.3.2.4; Ddl) catalyzes the ATP-driven ligation of two D-alanine (D-Ala) molecules to form the D-alanyl:D-alanine dipeptide. This molecule is a key building block in peptidoglycan biosynthesis, making Ddl an attractive target for drug development. D-Cycloserine (DCS), an analog of D-Ala and a prototype Ddl inhibitor, has shown promise for the treatment of tuberculosis. Here, we report the crystal structure of Mycobacterium tuberculosis Ddl at a resolution of 2.1 {angstrom}. This structure indicates that Ddl is a dimer and consists of three discrete domains; the ligand binding cavity is at the intersection of all three domains and conjoinedmore » by several loop regions. The M. tuberculosis apo Ddl structure shows a novel conformation that has not yet been observed in Ddl enzymes from other species. The nucleotide and D-alanine binding pockets are flexible, requiring significant structural rearrangement of the bordering regions for entry and binding of both ATP and D-Ala molecules. Solution affinity and kinetic studies showed that DCS interacts with Ddl in a manner similar to that observed for D-Ala. Each ligand binds to two binding sites that have significant differences in affinity, with the first binding site exhibiting high affinity. DCS inhibits the enzyme, with a 50% inhibitory concentration (IC{sub 50}) of 0.37 mM under standard assay conditions, implicating a preferential and weak inhibition at the second, lower-affinity binding site. Moreover, DCS binding is tighter at higher ATP concentrations. The crystal structure illustrates potential drugable sites that may result in the development of more-effective Ddl inhibitors.« less

Functional and probiotic attributes of an indigenous isolate of Lactobacillus plantarum.

PubMed

Kaushik, Jai K; Kumar, Ashutosh; Duary, Raj K; Mohanty, Ashok K; Grover, Sunita; Batish, Virender K

2009-12-01

Probiotic microorganisms favorably alter the intestinal microflora balance, promote intestinal integrity and mobility, inhibit the growth of harmful bacteria and increase resistance to infection. Probiotics are increasingly used in nutraceuticals, functional foods or in microbial interference treatment. However, the effectiveness of probiotic organism is considered to be population-specific due to variation in gut microflora, food habits and specific host-microbial interactions. Most of the probiotic strains available in the market are of western or European origin, and a strong need for exploring new indigenous probiotic organisms is felt. An indigenous isolate Lp9 identified as Lactobacillus plantarum by molecular-typing methods was studied extensively for its functional and probiotic attributes, viz., acid and bile salt tolerance, cell surface hydrophobicity, autoaggregation and Caco-2 cell-binding as well as antibacterial and antioxidative activities. Lp9 isolate could survive 2 h incubation at pH 1.5-2.0 and toxicity of 1.5-2.0% oxgall bile. Lp9 could deconjugate major bile salts like glycocholate and deoxytaurocholate, indicating its potential to cause hypocholesterolemia. The isolate exhibited cell-surface hydrophobicity of approximately 37% and autoaggregation of approximately 31%. Presence of putative probiotic marker genes like mucus-binding protein (mub), fibronectin-binding protein (fbp) and bile salt hydrolase (bsh) were confirmed by PCR. Presence of these genes suggested the possibility of specific interaction and colonization potential of Lp9 isolate in the gut, which was also suggested by a good adhesion ratio of 7.4+/-1.3% with Caco-2 cell line. The isolate demonstrated higher free radical scavenging activity than standard probiotics L. johnsonii LA1 and L. acidophilus LA7. Lp9 also exhibited antibacterial activity against E. coli, L. monocytogenes, S. typhi, S. aureus and B. cereus. The indigenous Lactobacillus plantarum Lp9 exhibited high resistance against low pH and bile and possessed antibacterial, antioxidative and cholesterol lowering properties with a potential for exploitation in the development of indigenous functional food or nutraceuticals.

Mechanism of preferential packaging of negative sense genomic RNA by viral nucleoproteins in Crimean-Congo hemorrhagic Fever virus.

PubMed

Dayer, Mohammad Reza; Dayer, Mohammad Saaid; Rezatofighi, Seyedeh Elham

2015-04-01

The Crimean-Congo Hemorrhagic Fever (CCHF) is an infectious disease of high virulence and mortality caused by a negative sense RNA nairovirus. The genomic RNA of CCHFV is enwrapped by its nucleoprotein. Positively charged residues on CCHFV nucleoprotein provide multiple binding sites to facilitate genomic RNA encapsidation. In the present work, we investigated the mechanism underlying preferential packaging of the negative sense genomic RNA by CCHFV nucleoprotein in the presence of host cell RNAs during viral assembly. The work included genome sequence analyses for different families of negative and positive sense RNA viruses, using serial docking experiments and molecular dynamic simulations. Our results indicated that the main determinant parameter of the nucleoprotein binding affinity for negative sense RNA is the ratio of purine/pyrimidine in the RNA molecule. A negative sense RNA with a purine/pyrimidine ratio (>1) higher than that of a positive sense RNA (<1) exhibits higher affinity for the nucleoprotein. Our calculations revealed that a negative sense RNA expresses about 0.5 kJ/mol higher binding energy per nucleotide compared to a positive sense RNA. This energy difference produces a binding energy high enough to make the negative sense RNA, the preferred substrate for packaging by CCHFV nucleoprotein in the presence of cellular or complementary positive sense RNAs. The outcome of this study may contribute to ongoing researches on other viral diseases caused by negative sense RNA viruses such as Ebola virus which poses a security threat to all humanity.

Ligand binding induces a sharp decrease in hydrophobicity of folate binding protein assessed by 1-anilinonaphthalene-8-sulphonate which suppresses self-association of the hydrophobic apo-protein.

PubMed

Holm, Jan; Lawaetz, Anders J; Hansen, Steen I

2012-08-17

High affinity folate binding protein (FBP) regulates as a soluble protein and as a cellular receptor intracellular trafficking of folic acid, a vitamin of great importance to cell growth and division. We addressed two issues of potential importance to the biological function of FBP, a possible decrease of the surface hydrophobicity associated with the ligand-induced conformation change of FBP, and protein-inter-protein interactions involved in self-association of hydrophobic apo-FBP. The extrinsic fluorescent apolar dye 1-anilinonaphthalene-8-sulphonate (ANS) exhibited enhanced fluorescence intensity and a blueshift of emission maximum from 510-520 nm to 460-470 nm upon addition of apo-FBP indicating binding to a strongly hydrophobic environment. Neither enhancement of fluorescence nor blueshift of ANS emission maximum occurred when folate-ligated holo-FBP replaced apo-FBP. The drastic decrease in surface hydrophobicity of holo-FBP could have bearings on the biological function of FBP since changes in surface hydrophobicity have critical effects on the biological function of receptors and transport proteins. ANS interacts with exposed hydrophobic surfaces on proteins and may thereby block and prevent aggregation of proteins (chaperone-like effect). Hence, hydrophobic interactions seemed to participate in the concentration-dependent self-association of apo-FBP which was suppressed by high ANS concentrations in light scatter measurements. Copyright © 2012 Elsevier Inc. All rights reserved.

Characterization of the APLF FHA-XRCC1 phosphopeptide interaction and its structural and functional implications.

PubMed

Kim, Kyungmin; Pedersen, Lars C; Kirby, Thomas W; DeRose, Eugene F; London, Robert E

2017-12-01

Aprataxin and PNKP-like factor (APLF) is a DNA repair factor containing a forkhead-associated (FHA) domain that supports binding to the phosphorylated FHA domain binding motifs (FBMs) in XRCC1 and XRCC4. We have characterized the interaction of the APLF FHA domain with phosphorylated XRCC1 peptides using crystallographic, NMR, and fluorescence polarization studies. The FHA-FBM interactions exhibit significant pH dependence in the physiological range as a consequence of the atypically high pK values of the phosphoserine and phosphothreonine residues and the preference for a dianionic charge state of FHA-bound pThr. These high pK values are characteristic of the polyanionic peptides typically produced by CK2 phosphorylation. Binding affinity is greatly enhanced by residues flanking the crystallographically-defined recognition motif, apparently as a consequence of non-specific electrostatic interactions, supporting the role of XRCC1 in nuclear cotransport of APLF. The FHA domain-dependent interaction of XRCC1 with APLF joins repair scaffolds that support single-strand break repair and non-homologous end joining (NHEJ). It is suggested that for double-strand DNA breaks that have initially formed a complex with PARP1 and its binding partner XRCC1, this interaction acts as a backup attempt to intercept the more error-prone alternative NHEJ repair pathway by recruiting Ku and associated NHEJ factors. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

A dimer of the lymphoid protein RAG1 recognizes the recombination signal sequence and the complex stably incorporates the high mobility group protein HMG2.

PubMed

Rodgers, K K; Villey, I J; Ptaszek, L; Corbett, E; Schatz, D G; Coleman, J E

1999-07-15

RAG1 and RAG2 are the two lymphoid-specific proteins required for the cleavage of DNA sequences known as the recombination signal sequences (RSSs) flanking V, D or J regions of the antigen-binding genes. Previous studies have shown that RAG1 alone is capable of binding to the RSS, whereas RAG2 only binds as a RAG1/RAG2 complex. We have expressed recombinant core RAG1 (amino acids 384-1008) in Escherichia coli and demonstrated catalytic activity when combined with RAG2. This protein was then used to determine its oligomeric forms and the dissociation constant of binding to the RSS. Electrophoretic mobility shift assays show that up to three oligomeric complexes of core RAG1 form with a single RSS. Core RAG1 was found to exist as a dimer both when free in solution and as the minimal species bound to the RSS. Competition assays show that RAG1 recognizes both the conserved nonamer and heptamer sequences of the RSS. Zinc analysis shows the core to contain two zinc ions. The purified RAG1 protein overexpressed in E.coli exhibited the expected cleavage activity when combined with RAG2 purified from transfected 293T cells. The high mobility group protein HMG2 is stably incorporated into the recombinant RAG1/RSS complex and can increase the affinity of RAG1 for the RSS in the absence of RAG2.

Structural basis for a hand-like site in the calcium sensor CatchER with fast kinetics

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

Zhang, Ying; Reddish, Florence; Tang, Shen

2013-12-01

High-resolution crystal structures of the designed calcium sensor CatchER revealed snapshots of calcium and gadolinium ions binding within the designed site in agreement with its fast kinetics. Calcium ions, which are important signaling molecules, can be detected in the endoplasmic reticulum by an engineered mutant of green fluorescent protein (GFP) designated CatchER with a fast off-rate. High resolution (1.78–1.20 Å) crystal structures were analyzed for CatchER in the apo form and in complexes with calcium or gadolinium to probe the binding site for metal ions. While CatchER exhibits a 1:1 binding stoichiometry in solution, two positions were observed for eachmore » of the metal ions bound within the hand-like site formed by the carboxylate side chains of the mutated residues S147E, S202D, Q204E, F223E and T225E that may be responsible for its fast kinetic properties. Comparison of the structures of CatchER, wild-type GFP and enhanced GFP confirmed that different conformations of Thr203 and Glu222 are associated with the two forms of Tyr66 of the chromophore which are responsible for the absorbance wavelengths of the different proteins. Calcium binding to CatchER may shift the equilibrium for conformational population of the Glu222 side chain and lead to further changes in its optical properties.« less

Computational Approaches for Decoding Select Odorant-Olfactory Receptor Interactions Using Mini-Virtual Screening

PubMed Central

Harini, K.; Sowdhamini, Ramanathan

2015-01-01

Olfactory receptors (ORs) belong to the class A G-Protein Coupled Receptor superfamily of proteins. Unlike G-Protein Coupled Receptors, ORs exhibit a combinatorial response to odors/ligands. ORs display an affinity towards a range of odor molecules rather than binding to a specific set of ligands and conversely a single odorant molecule may bind to a number of olfactory receptors with varying affinities. The diversity in odor recognition is linked to the highly variable transmembrane domains of these receptors. The purpose of this study is to decode the odor-olfactory receptor interactions using in silico docking studies. In this study, a ligand (odor molecules) dataset of 125 molecules was used to carry out in silico docking using the GLIDE docking tool (SCHRODINGER Inc Pvt LTD). Previous studies, with smaller datasets of ligands, have shown that orthologous olfactory receptors respond to similarly-tuned ligands, but are dramatically different in their efficacy and potency. Ligand docking results were applied on homologous pairs (with varying sequence identity) of ORs from human and mouse genomes and ligand binding residues and the ligand profile differed among such related olfactory receptor sequences. This study revealed that homologous sequences with high sequence identity need not bind to the same/ similar ligand with a given affinity. A ligand profile has been obtained for each of the 20 receptors in this analysis which will be useful for expression and mutation studies on these receptors. PMID:26221959

Differential Receptor Binding and Regulatory Mechanisms for the Lymphangiogenic Growth Factors Vascular Endothelial Growth Factor (VEGF)-C and -D*

PubMed Central

Davydova, Natalia; Harris, Nicole C.; Roufail, Sally; Paquet-Fifield, Sophie; Ishaq, Musarat; Streltsov, Victor A.; Williams, Steven P.; Karnezis, Tara; Stacker, Steven A.; Achen, Marc G.

2016-01-01

VEGF-C and VEGF-D are secreted glycoproteins that induce angiogenesis and lymphangiogenesis in cancer, thereby promoting tumor growth and spread. They exhibit structural homology and activate VEGFR-2 and VEGFR-3, receptors on endothelial cells that signal for growth of blood vessels and lymphatics. VEGF-C and VEGF-D were thought to exhibit similar bioactivities, yet recent studies indicated distinct signaling mechanisms (e.g. tumor-derived VEGF-C promoted expression of the prostaglandin biosynthetic enzyme COX-2 in lymphatics, a response thought to facilitate metastasis via the lymphatic vasculature, whereas VEGF-D did not). Here we explore the basis of the distinct bioactivities of VEGF-D using a neutralizing antibody, peptide mapping, and mutagenesis to demonstrate that the N-terminal α-helix of mature VEGF-D (Phe93–Arg108) is critical for binding VEGFR-2 and VEGFR-3. Importantly, the N-terminal part of this α-helix, from Phe93 to Thr98, is required for binding VEGFR-3 but not VEGFR-2. Surprisingly, the corresponding part of the α-helix in mature VEGF-C did not influence binding to either VEGFR-2 or VEGFR-3, indicating distinct determinants of receptor binding by these growth factors. A variant of mature VEGF-D harboring a mutation in the N-terminal α-helix, D103A, exhibited enhanced potency for activating VEGFR-3, was able to promote increased COX-2 mRNA levels in lymphatic endothelial cells, and had enhanced capacity to induce lymphatic sprouting in vivo. This mutant may be useful for developing protein-based therapeutics to drive lymphangiogenesis in clinical settings, such as lymphedema. Our studies shed light on the VEGF-D structure/function relationship and provide a basis for understanding functional differences compared with VEGF-C. PMID:27852824

Involvement of ectodomain Leu 214 in ATP binding and channel desensitization of the P2X4 receptor.

PubMed

Zhang, Longmei; Xu, Huijuan; Jie, Yanling; Gao, Chao; Chen, Wanjuan; Yin, Shikui; Samways, Damien S K; Li, Zhiyuan

2014-05-13

P2X receptors are trimeric ATP-gated cation permeable ion channels. When ATP binds, the extracellular head and dorsal fin domains are predicted to move closer to each other. However, there are scant functional data corroborating the role of the dorsal fin in ligand binding. Here using site-directed mutagenesis and electrophysiology, we show that a dorsal fin leucine, L214, contributes to ATP binding. Mutant receptors containing a single substitution of alanine, serine, glutamic acid, or phenylalanine at L214 of the rat P2X4 receptor exhibited markedly reduced sensitivities to ATP. Mutation of other dorsal fin side chains, S216, T223, and D224, did not significantly alter ATP sensitivity. Exposure of L214C to sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) or (2-aminoethyl) methanethiosulfonate hydrobromide in the absence of ATP blocked responses evoked by subsequent ATP application. In contrast, when MTSES(-) was applied in the presence of ATP, no current inhibition was observed. Furthermore, L214A also slightly reduced the inhibitory effect of the antagonist 2',3'-O-(2,4,6-trinitrophenyl)-ATP, and the blockade was more rapidly reversible after washout. Certain L214 mutants also showed effects on current desensitization in the continued presence of ATP. L214I exhibited an accelerated current decline, whereas L214M exhibited a slower rate. Taken together, these data reveal that position L214 participates in both ATP binding and conformational changes accompanying channel opening and desensitization, providing compelling evidence that the dorsal fin domain indeed has functional properties that are similar to those previously reported for the body domains.

Structure-Function Analysis of Friedreich's Ataxia Mutants Reveals Determinants of Frataxin Binding and Activation of the Fe-S Assembly Complex

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

Bridwell-Rabb, Jennifer; Winn, Andrew M; Barondeau, David P

2012-08-01

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease associated with the loss of function of the protein frataxin (FXN) that results from low FXN levels due to a GAA triplet repeat expansion or, occasionally, from missense mutations in the FXN gene. Here biochemical and structural properties of FXN variants, including three FRDA missense mutations (N146K, Q148R, and R165C) and three related mutants (N146A, Q148G, and Q153A), were determined in an effort to understand the structural basis for the loss of function. In vitro assays revealed that although the three FRDA missense mutations exhibited similar losses of cysteine desulfurase and Fe-Smore » cluster assembly activities, the causes for these activation defects were distinct. The R165C variant exhibited a k cat/K M higher than that of native FXN but weak binding to the NFS1, ISD11, and ISCU2 (SDU) complex, whereas the Q148R variant exhibited the lowest k cat/K M of the six tested FXN variants and only a modest binding deficiency. The order of the FXN binding affinities for the SDU Fe-S assembly complex was as follows: FXN > Q148R > N146A > Q148G > N146K > Q153A > R165C. Four different classes of FXN variants were identified on the basis of their biochemical properties. Together, these structure-function studies reveal determinants for the binding and allosteric activation of the Fe-S assembly complex and provide insight into how FRDA missense mutations are functionally compromised.« less

Mannose-binding lectin binds to a range of clinically relevant microorganisms and promotes complement deposition.

PubMed

Neth, O; Jack, D L; Dodds, A W; Holzel, H; Klein, N J; Turner, M W

2000-02-01

Mannose-binding lectin (MBL) is a collagenous serum lectin believed to be of importance in innate immunity. Genetically determined low levels of the protein are known to predispose to infections. In this study the binding of purified MBL to pathogens isolated from immunocompromised children was investigated by flow cytometry. Diverse Candida species, Aspergillus fumigatus, Staphylococcus aureus, and beta-hemolytic group A streptococci exhibited strong binding of MBL, whereas Escherichia coli, Klebsiella species, and Haemophilus influenzae type b were characterized by heterogeneous binding patterns. In contrast, beta-hemolytic group B streptococci, Streptococcus pneumoniae, and Staphylococcus epidermidis showed low levels of binding. Bound MBL was able to promote C4 deposition in a concentration-dependent manner. We conclude that MBL may be of importance in first-line immune defense against several important pathogens.

Method for Developing Optical Sensors Using a Synthetic Dye-Fluorescent Protein FRET Pair and Computational Modeling and Assessment.

PubMed

Mitchell, Joshua A; Zhang, William H; Herde, Michel K; Henneberger, Christian; Janovjak, Harald; O'Mara, Megan L; Jackson, Colin J

2017-01-01

Biosensors that exploit Förster resonance energy transfer (FRET) can be used to visualize biological and physiological processes and are capable of providing detailed information in both spatial and temporal dimensions. In a FRET-based biosensor, substrate binding is associated with a change in the relative positions of two fluorophores, leading to a change in FRET efficiency that may be observed in the fluorescence spectrum. As a result, their design requires a ligand-binding protein that exhibits a conformational change upon binding. However, not all ligand-binding proteins produce responsive sensors upon conjugation to fluorescent proteins or dyes, and identifying the optimum locations for the fluorophores often involves labor-intensive iterative design or high-throughput screening. Combining the genetic fusion of a fluorescent protein to the ligand-binding protein with site-specific covalent attachment of a fluorescent dye can allow fine control over the positions of the two fluorophores, allowing the construction of very sensitive sensors. This relies upon the accurate prediction of the locations of the two fluorophores in bound and unbound states. In this chapter, we describe a method for computational identification of dye-attachment sites that allows the use of cysteine modification to attach synthetic dyes that can be paired with a fluorescent protein for the purposes of creating FRET sensors.

Structural and binding studies of SAP-1 protein with heparin.

PubMed

Yadav, Vikash K; Mandal, Rahul S; Puniya, Bhanwar L; Kumar, Rahul; Dey, Sharmistha; Singh, Sarman; Yadav, Savita

2015-03-01

SAP-1 is a low molecular weight cysteine protease inhibitor (CPI) which belongs to type-2 cystatins family. SAP-1 protein purified from human seminal plasma (HuSP) has been shown to inhibit cysteine and serine proteases and exhibit interesting biological properties, including high temperature and pH stability. Heparin is a naturally occurring glycosaminoglycan (with varied chain length) which interacts with a number of proteins and regulates multiple steps in different biological processes. As an anticoagulant, heparin enhances inhibition of thrombin by the serpin antithrombin III. Therefore, we have employed surface plasmon resonance (SPR) to improve our understanding of the binding interaction between heparin and SAP-1 (protease inhibitor). SPR data suggest that SAP-1 binds to heparin with a significant affinity (KD = 158 nm). SPR solution competition studies using heparin oligosaccharides showed that the binding of SAP-1 to heparin is dependent on chain length. Large oligosaccharides show strong binding affinity for SAP-1. Further to get insight into the structural aspect of interactions between SAP-1 and heparin, we used modelled structure of the SAP-1 and docked with heparin and heparin-derived polysaccharides. The results suggest that a positively charged residue lysine plays important role in these interactions. Such information should improve our understanding of how heparin, present in the reproductive tract, regulates cystatins activity. © 2014 John Wiley & Sons A/S.

Aptamer-based liposomes improve specific drug loading and release.

PubMed

Plourde, Kevin; Derbali, Rabeb Mouna; Desrosiers, Arnaud; Dubath, Céline; Vallée-Bélisle, Alexis; Leblond, Jeanne

2017-04-10

Aptamer technology has shown much promise in cancer therapeutics for its targeting abilities. However, its potential to improve drug loading and release from nanocarriers has not been thoroughly explored. In this study, we employed drug-binding aptamers to actively load drugs into liposomes. We designed a series of DNA aptamer sequences specific to doxorubicin, displaying multiple binding sites and various binding affinities. The binding ability of aptamers was preserved when incorporated into cationic liposomes, binding up to 15equivalents of doxorubicin per aptamer, therefore drawing the drug into liposomes. Optimization of the charge and drug/aptamer ratios resulted in ≥80% encapsulation efficiency of doxorubicin, ten times higher than classical passively-encapsulating liposomal formulations and similar to a pH-gradient active loading strategy. In addition, kinetic release profiles and cytotoxicity assay on HeLa cells demonstrated that the release and therapeutic efficacy of liposomal doxorubicin could be controlled by the aptamer's structure. Our results suggest that the aptamer exhibiting a specific intermediate affinity is the best suited to achieve high drug loading while maintaining efficient drug release and therapeutic activity. This strategy was successfully applied to tobramycin, a hydrophilic drug suffering from low encapsulation into liposomes, where its loading was improved six-fold using aptamers. Overall, we demonstrate that aptamers could act, in addition to their targeting properties, as multifunctional excipients for liposomal formulations. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification and characterization of Cu(2)O- and ZnO-binding polypeptides by Escherichia coli cell surface display: toward an understanding of metal oxide binding.

PubMed

Thai, Corrine K; Dai, Haixia; Sastry, M S R; Sarikaya, Mehmet; Schwartz, Daniel T; Baneyx, François

2004-07-20

We have used the FliTrx cell surface display system to identify disulfide-constrained dodecapeptides binding to the semiconducting metal oxides Cu(2)O and ZnO. Sequence analysis of the inserts revealed that the two populations exhibit similar, yet subtly different patterns of amino acid usage. Both sets of binders were enriched in arginine, tryptophan, and glycine with a higher degree of positional preference in the case of Cu(2)O binders. Tyrosine, proline, and serine were underrepresented in both populations. Peptides binding electrodeposited Cu(2)O or ZnO with high avidity could be subdivided into two classes based on pI and hydrophilicity. In the hydrophilic and positively charged Class I binders, the Arg-X-X-Arg tetrapeptide appears to be implicated in metal oxide binding, whereas Arg-Arg and Arg-Lys pairs allow for discrimination between Cu(2)O and ZnO. Molecular dynamics simulations of the disulfide-constrained peptides suggest that the aforementioned motifs are important to properly orient two basic residues that are likely to contact the metal oxides. The implications of our results in understanding the rules governing the interaction between peptides and inorganic compounds and in their use for the design of hybrid nanoarchitectures are discussed. Copyright 2004 Wiley Periodicals, Inc.

Biasing hydrogen bond donating host systems towards chemical warfare agent recognition.

PubMed

Hiscock, Jennifer R; Wells, Neil J; Ede, Jayne A; Gale, Philip A; Sambrook, Mark R

2016-10-12

A series of neutral ditopic and negatively charged, monotopic host molecules have been evaluated for their ability to bind chloride and dihydrogen phosphate anions, and neutral organophosphorus species dimethyl methylphosphonate (DMMP), pinacolyl methylphosphonate (PMP) and the chemical warfare agent (CWA) pinacolyl methylphosphonofluoridate (GD, soman) in organic solvent via hydrogen bonding. Urea, thiourea and boronic acid groups are shown to bind anions and neutral guests through the formation of hydrogen bonds, with the urea and thiourea groups typically exhibiting higher affinity interactions. The introduction of a negative charge on the host structure is shown to decrease anion affinity, whilst still allowing for high stability host-GD complex formation. Importantly, the affinity of the host for the neutral CWA GD is greater than for anionic guests, thus demonstrating the potential for selectivity reversal based on charge repulsion.