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Sample records for affinity binding component

  1. Prolactin-binding components in rabbit mammary gland: characterization by partial purification and affinity labeling

    SciTech Connect

    Katoh, M.; Djiane, J.; Kelly, P.A.

    1985-06-01

    The molecular characteristics of the PRL receptor isolated from rabbit mammary gland microsomes were investigated. Two approaches were employed: 1) affinity purification of PRL receptors and direct electrophoretic analysis, and 2) affinity cross-linking of microsomal receptors with (/sup 125/I)ovine PRL ((/sup 125/I)oPRL). PRL receptors were solubilized from mammary microsomes with 3-((3-cholamidopropyl)dimethylammonio)1-propane sulfonate and purified using an oPRL agarose affinity column. Sodium dodecylsulfate-polyacrylamide gel electrophoresis and silver staining of the gel revealed at least nine bands, including a 32,000 mol wt band which was most intensively labeled with /sup 125/I using the chloramine-T method. Covalent labeling of PRL receptors with (/sup 125/I)oPRL was performed using N-hydroxysuccinimidyl-4-azido benzoate, disuccinimidyl suberate, or ethylene glycol bis (succinimidyl succinate). A single band of 59,000 mol wt was produced by all three cross-linkers when sodium dodecylsulfate-polyacrylamide gel electrophoresis was performed under reducing conditions. Assuming 1:1 binding of hormone and binding subunit and by subtracting the mol wt of (/sup 125/I)oPRL, which was estimated from the migration distance on the gel, the mol wt of the binding subunit was calculated as 32,000. In the absence of dithiothreitol during electrophoresis, only one major hormone-receptor complex band was observed. The same mol wt binding components were also detected in microsomal fractions of rabbit kidney, ovary, and adrenal. A slightly higher mol wt binding subunit was observed in rat liver microsomes. Rabbit liver microsomes revealed five (/sup 125/I)oPRL-binding components, three of which were considered to be those of a GH receptor. Moreover, affinity labeling of detergent-solubilized and affinity purified mammary PRL receptors showed a similar major binding subunit.

  2. Structural determinants of enzyme binding affinity: the E1 component of pyruvate dehydrogenase from Escherichia coli in complex with the inhibitor thiamin thiazolone diphosphate.

    PubMed

    Arjunan, Palaniappa; Chandrasekhar, Krishnamoorthy; Sax, Martin; Brunskill, Andrew; Nemeria, Natalia; Jordan, Frank; Furey, William

    2004-03-01

    Thiamin thiazolone diphosphate (ThTDP), a potent inhibitor of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc), binds to the enzyme with greater affinity than does the cofactor thiamin diphosphate (ThDP). To identify what determines this difference, the crystal structure of the apo PDHc E1 component complex with ThTDP and Mg(2+) has been determined at 2.1 A and compared to the known structure of the native holoenzyme, PDHc E1-ThDP-Mg(2+) complex. When ThTDP replaces ThDP, reorganization occurs in the protein structure in the vicinity of the active site involving positional and conformational changes in some amino acid residues, a change in the V coenzyme conformation, addition of new hydration sites, and elimination of others. These changes culminate in an increase in the number of hydrogen bonds to the protein, explaining the greater affinity of the apoenzyme for ThTDP. The observed hydrogen bonding pattern is not an invariant feature of ThDP-dependent enzymes but rather specific to this enzyme since the extra hydrogen bonds are made with nonconserved residues. Accordingly, these sequence-related hydrogen bonding differences likewise explain the wide variation in the affinities of different thiamin-dependent enzymes for ThTDP and ThDP. The sequence of each enzyme determines its ability to form hydrogen bonds to the inhibitor or cofactor. Mechanistic roles are suggested for the aforementioned reorganization and its reversal in PDHc E1 catalysis: to promote substrate binding and product release. This study also provides additional insight into the role of water in enzyme inhibition and catalysis. PMID:14992577

  3. Methods for Improving Aptamer Binding Affinity.

    PubMed

    Hasegawa, Hijiri; Savory, Nasa; Abe, Koichi; Ikebukuro, Kazunori

    2016-01-01

    Aptamers are single stranded oligonucleotides that bind a wide range of biological targets. Although aptamers can be isolated from pools of random sequence oligonucleotides using affinity-based selection, aptamers with high affinities are not always obtained. Therefore, further refinement of aptamers is required to achieve desired binding affinities. The optimization of primary sequences and stabilization of aptamer conformations are the main approaches to refining the binding properties of aptamers. In particular, sequence optimization using combined in silico sequence recombinations and in vitro functional evaluations is effective for the improvement of binding affinities, however, the binding affinities of aptamers are limited by the low hydrophobicity of nucleic acids. Accordingly, introduction of hydrophobic moieties into aptamers expands the diversity of interactions between aptamers and targets. Moreover, construction of multivalent aptamers by connecting aptamers that recognize distinct epitopes is an attractive approach to substantial increases in binding affinity. In addition, binding affinities can be tuned by optimizing the scaffolds of multivalent constructs. In this review, we summarize the various techniques for improving the binding affinities of aptamers. PMID:27043498

  4. Identification of pheromone components and their binding affinity to the odorant binding protein CcapOBP83a-2 of the Mediterranean fruit fly, Ceratitis capitata

    PubMed Central

    Siciliano, P.; He, X.L.; Woodcock, C.; Pickett, J.A.; Field, L.M.; Birkett, M.A.; Kalinova, B.; Gomulski, L.M.; Scolari, F.; Gasperi, G.; Malacrida, A.R.; Zhou, J.J.

    2014-01-01

    The Mediterranean fruit fly (or medfly), Ceratitis capitata (Wiedemann; Diptera: Tephritidae), is a serious pest of agriculture worldwide, displaying a very wide larval host range with more than 250 different species of fruit and vegetables. Olfaction plays a key role in the invasive potential of this species. Unfortunately, the pheromone communication system of the medfly is complex and still not well established. In this study, we report the isolation of chemicals emitted by sexually mature individuals during the “calling” period and the electrophysiological responses that these compounds elicit on the antennae of male and female flies. Fifteen compounds with electrophysiological activity were isolated and identified in male emissions by gas chromatography coupled to electroantennography (GC–EAG). Within the group of 15 identified compounds, 11 elicited a response in antennae of both sexes, whilst 4 elicited a response only in female antennae. The binding affinity of these compounds, plus 4 additional compounds known to be behaviourally active from other studies, was measured using C. capitata OBP, CcapOBP83a-2. This OBP has a high homology to Drosophila melanogaster OBPs OS-E and OS-F, which are associated with trichoid sensilla and co-expressed with the well-studied Drosophila pheromone binding protein LUSH. The results provide evidence of involvement of CcapOBP83a-2 in the medfly's odorant perception and its wider specificity for (E,E)-α-farnesene, one of the five major compounds in medfly male pheromone emission. This represents the first step in the clarification of the C. capitata and pheromone reception pathway, and a starting point for further studies aimed towards the creation of new powerful attractants or repellents applicable in the actual control strategies. PMID:24607850

  5. Two bradykinin binding sites with picomolar affinities

    SciTech Connect

    Manning, D.C.; Vavrek, R.; Stewart, J.M.; Snyder, S.H.

    1986-05-01

    Bradykinin (BK) and related peptides exert a wide range of effects on several organ systems. We have attempted to sort out these effects by studying the binding interaction of (/sup 3/H)BK at the membrane level with in vitro receptor binding techniques. High specific activity (/sup 3/H)BK and an enzyme inhibitor cocktail has enabled us to label two BK binding sites with different affinity and peptide specificity in several guinea-pig tissues. In the guinea-pig ileum the high-affinity site has an equilibrium dissociation constant (Kd) for (/sup 3/H)BK of 13 pM and a maximal number of binding sites of 8.3 pmol/g of tissue wet weight. The low-affinity guinea-pig ileum site displays a Kd of 910 pM, a maximum number of binding sites of 14 pmol/g of tissue wet weight and shows a greater selectivity for BK analogs over Lysyl-BK analogs. Two similar sites can also be discriminated in kidney and heart. The potencies of a series of BK analogs at the high-affinity guinea-pig ileum site correlate well with their potencies in contracting ileal smooth muscle. The binding of (/sup 3/H)BK in the guinea-pig ileum is inhibited by physiological concentrations of monovalent and divalent cations.

  6. Search for Amyloid-Binding Proteins by Affinity Chromatography

    PubMed Central

    Calero, Miguel; Rostagno, Agueda; Ghiso, Jorge

    2013-01-01

    ‘Amyloid binging proteins’ is a generic term used to designate proteins that interact with different forms of amyloidogenic peptides or proteins and that, as a result, may modulate their physiological and pathological functions by altering solubility, transport, clearance, degradation, and fibril formation. We describe a simple affinity chromatography protocol to isolate and characterize amyloid-binding proteins based on the use of sequential elution steps that may provide further information on the type of binding interaction. As an example, we depict the application of this protocol to the study of Alzheimer’s amyloid β (Aβ) peptide-binding proteins derived from human plasma. Biochemical analysis of the proteins eluted under different conditions identified serum amyloid P component (SAP) and apolipoprotein J (clusterin) as the main plasma Aβ-binding proteins while various apolipoproteins (apoA-IV, apoE, and apoA-I), as well as albumin (HSA) and fibulin were identified as minor contributors. PMID:22528093

  7. High-affinity binding of fibronectin to cultured Kupffer cells

    SciTech Connect

    Cardarelli, P.M.; Blumenstock, F.A.; McKeown-Longo, P.J.; Saba, T.M.; Mazurkiewicz, J.E.; Dias, J.A. )

    1990-11-01

    Hepatic Kupffer cells are a major component of the reticuloendothelial or macrophage system. They were the first phagocytic cell type whose phagocytosis was shown to be influenced by plasma fibronectin, a dimeric opsonic glycoprotein. In the current study, the binding of soluble radioiodinated fibronectin purified from rat serum to isolated rat hepatic Kupffer cells was investigated using a cultured Kupffer cell monolayer technique. Binding was specific, since unlabeled purified fibronectin competed in a dose-dependent manner with the 125I-fibronectin for binding to the Kupffer cells. Addition of gelatin enhanced the binding of 125I-fibronectin to Kupffer cells. The phagocytosis of gelatinized-coated red cells by Kupffer cells was increased either by preopsonizing the target particles with purified fibronectin or by the addition of purified fibronectin to the culture medium. In contrast, exposure of the Kupffer cells to medium containing purified fibronectin followed by wash-removal of the fibronectin did not increase the uptake of gelatin-coated red blood cells, even though fibronectin was detected on the surface of the Kupffer cells by immunofluorescence. Trypsinized monolayers expressed decreased capacity to bind 125I-fibronectin as well as fibronectin-coated sheep erythrocytes. The binding of 125I-fibronectin-gelatin complexes was inhibited by excess unlabeled fibronectin. We calculated that specific high-affinity (Kd = 7.46 x 10(-9) M) binding sites for fibronectin exist on Kupffer cells. There are approximately 2,800-3,500 binding sites or putative fibronectin receptors per Kupffer cell. These sites appear to mediate the enhanced phagocytosis of gelatin-coated particles opsonized by fibronectin.

  8. Affinity Regulates Spatial Range of EGF Receptor Autocrine Ligand Binding

    SciTech Connect

    Dewitt, Ann; Iida, Tomoko; Lam, Ho-Yan; Hill, Virginia; Wiley, H S.; Lauffenburger, Douglas A.

    2002-08-08

    Proper spatial localization of EGFR signaling activated by autocrine ligands represents a critical factor in embryonic development as well as tissue organization and function, and ligand/receptor binding affinity is among the molecular and cellular properties suggested to play a role in governing this localization. The authors employ a computational model to predict how receptor-binding affinity affects local capture of autocrine ligand vis-a-vis escape to distal regions, and provide experimental test by constructing cell lines expressing EGFR along with either wild-type EGF or a low-affinity mutant, EGF{sup L47M}. The model predicts local capture of a lower affinity autocrine ligand to be less efficient when the ligand production rate is small relative to receptor appearance rate. The experimental data confirm this prediction, demonstrating that cells can use ligand/receptor binding affinity to regulate ligand spatial distribution when autocrine ligand production is limiting for receptor signaling.

  9. Affinity Purification of Sequence-Specific DNA Binding Proteins

    NASA Astrophysics Data System (ADS)

    Kadonaga, James T.; Tjian, Robert

    1986-08-01

    We describe a method for affinity purification of sequence-specific DNA binding proteins that is fast and effective. Complementary chemically synthesized oligodeoxynucleotides that contain a recognition site for a sequence-specific DNA binding protein are annealed and ligated to give oligomers. This DNA is then covalently coupled to Sepharose CL-2B with cyanogen bromide to yield the affinity resin. A partially purified protein fraction is combined with competitor DNA and subsequently passed through the DNA-Sepharose resin. The desired sequence-specific DNA binding protein is purified because it preferentially binds to the recognition sites in the affinity resin rather than to the nonspecific competitor DNA in solution. For example, a protein fraction that is enriched for transcription factor Sp1 can be further purified 500- to 1000-fold by two sequential affinity chromatography steps to give Sp1 of an estimated 90% homogeneity with 30% yield. In addition, the use of tandem affinity columns containing different protein binding sites allows the simultaneous purification of multiple DNA binding proteins from the same extract. This method provides a means for the purification of rare sequence-specific DNA binding proteins, such as Sp1 and CAAT-binding transcription factor.

  10. Flexible Linker Modulates Glycosaminoglycan Affinity of Decorin Binding Protein A.

    PubMed

    Morgan, Ashli; Sepuru, Krishna Mohan; Feng, Wei; Rajarathnam, Krishna; Wang, Xu

    2015-08-18

    Decorin binding protein A (DBPA) is a glycosaminoglycan (GAG)-binding adhesin found on the surface of the bacterium Borrelia burgdorferi (B. burgdorferi), the causative agent of Lyme disease. DBPA facilitates bacterial adherence to extracellular matrices of human tissues and is crucial during the early stage of the infection process. Interestingly, DBPA from different strains (B31, N40, and PBr) show significant differences in GAG affinities, but the structural basis for the differences is not clear. In this study, we show that GAG affinity of N40 DBPA is modulated in part by flexible segments that control access to the GAG binding site, such that shortening of the linker leads to higher GAG affinity when analyzed using ELISA, gel mobility shift assay, solution NMR, and isothermal titration calorimetry. Our observation that GAG affinity differences among different B. burgdorferi strains can be attributed to a flexible linker domain regulating access to the GAG-binding domain is novel. It also provides a rare example of how neutral amino acids and dynamic segments in GAG binding proteins can have a large influence on GAG affinity and provides insights into why the number of basic amino acids in the GAG-binding site may not be the only factor determining GAG affinity of proteins. PMID:26223367

  11. 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

  12. Structural insights into the affinity of Cel7A carbohydrate-binding module for lignin.

    PubMed

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

    2015-09-11

    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

  13. Structural insights into the affinity of Cel7A carbohydrate-binding module for lignin.

    PubMed

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

    2015-09-11

    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.

  14. AB-Bind: Antibody binding mutational database for computational affinity predictions.

    PubMed

    Sirin, Sarah; Apgar, James R; Bennett, Eric M; Keating, Amy E

    2016-02-01

    Antibodies (Abs) are a crucial component of the immune system and are often used as diagnostic and therapeutic agents. The need for high-affinity and high-specificity antibodies in research and medicine is driving the development of computational tools for accelerating antibody design and discovery. We report a diverse set of antibody binding data with accompanying structures that can be used to evaluate methods for modeling antibody interactions. Our Antibody-Bind (AB-Bind) database includes 1101 mutants with experimentally determined changes in binding free energies (ΔΔG) across 32 complexes. Using the AB-Bind data set, we evaluated the performance of protein scoring potentials in their ability to predict changes in binding free energies upon mutagenesis. Numerical correlations between computed and observed ΔΔG values were low (r = 0.16-0.45), but the potentials exhibited predictive power for classifying variants as improved vs weakened binders. Performance was evaluated using the area under the curve (AUC) for receiver operator characteristic (ROC) curves; the highest AUC values for 527 mutants with |ΔΔG| > 1.0 kcal/mol were 0.81, 0.87, and 0.88 using STATIUM, FoldX, and Discovery Studio scoring potentials, respectively. Some methods could also enrich for variants with improved binding affinity; FoldX and Discovery Studio were able to correctly rank 42% and 30%, respectively, of the 80 most improved binders (those with ΔΔG < -1.0 kcal/mol) in the top 5% of the database. This modest predictive performance has value but demonstrates the continuing need to develop and improve protein energy functions for affinity prediction. PMID:26473627

  15. Ethylene binding site affinity in ripening apples

    SciTech Connect

    Blankenship, S.M. . Dept. of Horticultural Science); Sisler, E.C. )

    1993-09-01

    Scatchard plots for ethylene binding in apples (Malus domestica Borkh.), which were harvested weekly for 5 weeks to include the ethylene climacteric rise, showed C[sub 50] values (concentration of ethylene needed to occupy 50% of the ethylene binding sites) of 0.10, 0.11, 0.34, 0.40, and 0.57 [mu]l ethylene/liter[sup [minus]1], respectively, for each of the 5 weeks. Higher ethylene concentrations were required to saturate the binding sites during the climacteric rise than at other times. Diffusion of [sup 14]C-ethylene from the binding sites was curvilinear and did not show any indication of multiple binding sites. Ethylene was not metabolized by apple tissue.

  16. Accurate Evaluation Method of Molecular Binding Affinity from Fluctuation Frequency

    NASA Astrophysics Data System (ADS)

    Hoshino, Tyuji; Iwamoto, Koji; Ode, Hirotaka; Ohdomari, Iwao

    2008-05-01

    Exact estimation of the molecular binding affinity is significantly important for drug discovery. The energy calculation is a direct method to compute the strength of the interaction between two molecules. This energetic approach is, however, not accurate enough to evaluate a slight difference in binding affinity when distinguishing a prospective substance from dozens of candidates for medicine. Hence more accurate estimation of drug efficacy in a computer is currently demanded. Previously we proposed a concept of estimating molecular binding affinity, focusing on the fluctuation at an interface between two molecules. The aim of this paper is to demonstrate the compatibility between the proposed computational technique and experimental measurements, through several examples for computer simulations of an association of human immunodeficiency virus type-1 (HIV-1) protease and its inhibitor (an example for a drug-enzyme binding), a complexation of an antigen and its antibody (an example for a protein-protein binding), and a combination of estrogen receptor and its ligand chemicals (an example for a ligand-receptor binding). The proposed affinity estimation has proven to be a promising technique in the advanced stage of the discovery and the design of drugs.

  17. Improving antibody binding affinity and specificity for therapeutic development.

    PubMed

    Bostrom, Jenny; Lee, Chingwei V; Haber, Lauric; Fuh, Germaine

    2009-01-01

    Affinity maturation is an important part of the therapeutic antibody development process as in vivo activity often requires high binding affinity. Here, we describe a targeted approach for affinity improvement of therapeutic antibodies. Sets of CDR residues that are solvent accessible and relatively diverse in natural antibodies are targeted for diversification. Degenerate oligonucleotides are used to generate combinatorial phage-displayed antibody libraries with varying degree of diversity at randomized positions from which high-affinity antibodies can be selected. An advantage of using antibodies for therapy is their exquisite target specificity, which enables selective antigen binding and reduces off-target effects. However, it can be useful, and often it is necessary, to generate cross-reactive antibodies binding to not only the human antigen but also the corresponding non-human primate or rodent orthologs. Such cross-reactive antibodies can be used to validate the therapeutic targeting and examine the safety profile in preclinical animal models before committing to a costly development track. We show how affinity improvement and cross-species binding can be achieved in a one-step process.

  18. Improving Binding Affinity and Selectivity of Computationally Designed Ligand-Binding Proteins Using Experiments.

    PubMed

    Tinberg, Christine E; Khare, Sagar D

    2016-01-01

    The ability to de novo design proteins that can bind small molecules has wide implications for synthetic biology and medicine. Combining computational protein design with the high-throughput screening of mutagenic libraries of computationally designed proteins is emerging as a general approach for creating binding proteins with programmable binding modes, affinities, and selectivities. The computational step enables the creation of a binding site in a protein that otherwise does not (measurably) bind the intended ligand, and targeted mutagenic screening allows for validation and refinement of the computational model as well as provides orders-of-magnitude increases in the binding affinity. Deep sequencing of mutagenic libraries can provide insights into the mutagenic binding landscape and enable further affinity improvements. Moreover, in such a combined computational-experimental approach where the binding mode is preprogrammed and iteratively refined, selectivity can be achieved (and modulated) by the placement of specified amino acid side chain groups around the ligand in defined orientations. Here, we describe the experimental aspects of a combined computational-experimental approach for designing-using the software suite Rosetta-proteins that bind a small molecule of choice and engineering, using fluorescence-activated cell sorting and high-throughput yeast surface display, high affinity and ligand selectivity. We illustrated the utility of this approach by performing the design of a selective digoxigenin (DIG)-binding protein that, after affinity maturation, binds DIG with picomolar affinity and high selectivity over structurally related steroids. PMID:27094290

  19. Affinity Chromatography Purification of Cytochrome c Binding Enzymes

    NASA Astrophysics Data System (ADS)

    Azzi, Angelo; Bill, Kurt; Broger, Clemens

    1982-04-01

    An efficient affinity chromatography procedure for the isolation of mitochondrial cytochrome c oxidase and reductase is described. Saccharomyces cerevisiae cytochrome c was used as a ligand, bound to a thiol-Sepharose 4B gel through cysteine-107. In this way, the site of interaction of cytochrome c with cytochrome oxidase and reductase remained unmodified and available for binding to a number of partner enzymes. The procedure is adequate for the purification of all those proteins having in common the property of binding with high affinity to cytochrome c--e.g., cytochrome c oxidase, reductase, and peroxidase, sulfite oxidase, and reaction centers of photosynthetic bacteria.

  20. Relative binding affinities of monolignols to horseradish peroxidase

    DOE PAGES

    Sangha, Amandeep K.; Petridis, Loukas; Cheng, Xiaolin; Smith, Jeremy C.

    2016-07-22

    Monolignol binding to the peroxidase active site is the first step in lignin polymerization in plant cell walls. Using molecular dynamics, docking, and free energy perturbation calculations, we investigate the binding of monolignols to horseradish peroxidase C. Our results suggest that p-coumaryl alcohol has the strongest binding affinity followed by sinapyl and coniferyl alcohol. Stacking interactions between the monolignol aromatic rings and nearby phenylalanine residues play an important role in determining the calculated relative binding affinities. p-Coumaryl and coniferyl alcohols bind in a pose productive for reaction in which a direct H-bond is formed between the phenolic –OH group andmore » a water molecule (W2) that may facilitate proton transfer during oxidation. In contrast, in the case of sinapyl alcohol there is no such direct interaction, the phenolic –OH group instead interacting with Pro139. Furthermore, since proton and electron transfer is the rate-limiting step in monolignol oxidation by peroxidase, the binding pose (and thus the formation of near attack conformation) appears to play a more important role than the overall binding affinity in determining the oxidation rate.« less

  1. Relative Binding Affinities of Monolignols to Horseradish Peroxidase.

    PubMed

    Sangha, Amandeep K; Petridis, Loukas; Cheng, Xiaolin; Smith, Jeremy C

    2016-08-11

    Monolignol binding to the peroxidase active site is the first step in lignin polymerization in plant cell walls. Using molecular dynamics, docking, and free energy perturbation calculations, we investigate the binding of monolignols to horseradish peroxidase C. Our results suggest that p-coumaryl alcohol has the strongest binding affinity followed by sinapyl and coniferyl alcohol. Stacking interactions between the monolignol aromatic rings and nearby phenylalanine residues play an important role in determining the calculated relative binding affinities. p-Coumaryl and coniferyl alcohols bind in a pose productive for reaction in which a direct H-bond is formed between the phenolic -OH group and a water molecule (W2) that may facilitate proton transfer during oxidation. In contrast, in the case of sinapyl alcohol there is no such direct interaction, the phenolic -OH group instead interacting with Pro139. Since proton and electron transfer is the rate-limiting step in monolignol oxidation by peroxidase, the binding pose (and thus the formation of near attack conformation) appears to play a more important role than the overall binding affinity in determining the oxidation rate. PMID:27447548

  2. Affinity screening using competitive binding with fluorine-19 hyperpolarized ligands.

    PubMed

    Kim, Yaewon; Hilty, Christian

    2015-04-13

    Fluorine-19 NMR and hyperpolarization form a powerful combination for drug screening. Under a competitive equilibrium with a selected fluorinated reporter ligand, the dissociation constant (K(D)) of other ligands of interest is measurable using a single-scan Carr-Purcell-Meiboom-Gill (CPMG) experiment, without the need for a titration. This method is demonstrated by characterizing the binding of three ligands with different affinities for the serine protease trypsin. Monte Carlo simulations show that the highest accuracy is obtained when about one-half of the bound reporter ligand is displaced in the binding competition. Such conditions can be achieved over a wide range of affinities, allowing for rapid screening of non-fluorinated compounds when a single fluorinated ligand for the binding pocket of interest is known.

  3. Assessment of Solvated Interaction Energy Function for Ranking Antibody-Antigen Binding Affinities.

    PubMed

    Sulea, Traian; Vivcharuk, Victor; Corbeil, Christopher R; Deprez, Christophe; Purisima, Enrico O

    2016-07-25

    Affinity modulation of antibodies and antibody fragments of therapeutic value is often required in order to improve their clinical efficacies. Virtual affinity maturation has the potential to quickly focus on the critical hotspot residues without the combinatorial explosion problem of conventional display and library approaches. However, this requires a binding affinity scoring function that is capable of ranking single-point mutations of a starting antibody. We focus here on assessing the solvated interaction energy (SIE) function that was originally developed for and is widely applied to scoring of protein-ligand binding affinities. To this end, we assembled a structure-function data set called Single-Point Mutant Antibody Binding (SiPMAB) comprising several antibody-antigen systems suitable for this assessment, i.e., based on high-resolution crystal structures for the parent antibodies and coupled with high-quality binding affinity measurements for sets of single-point antibody mutants in each system. Using this data set, we tested the SIE function with several mutation protocols based on the popular methods SCWRL, Rosetta, and FoldX. We found that the SIE function coupled with a protocol limited to sampling only the mutated side chain can reasonably predict relative binding affinities with a Spearman rank-order correlation coefficient of about 0.6, outperforming more aggressive sampling protocols. Importantly, this performance is maintained for each of the seven system-specific component subsets as well as for other relevant subsets including non-alanine and charge-altering mutations. The transferability and enrichment in affinity-improving mutants can be further enhanced using consensus ranking over multiple methods, including the SIE, Talaris, and FOLDEF energy functions. The knowledge gained from this study can lead to successful prospective applications of virtual affinity maturation. PMID:27367467

  4. Assessment of Solvated Interaction Energy Function for Ranking Antibody-Antigen Binding Affinities.

    PubMed

    Sulea, Traian; Vivcharuk, Victor; Corbeil, Christopher R; Deprez, Christophe; Purisima, Enrico O

    2016-07-25

    Affinity modulation of antibodies and antibody fragments of therapeutic value is often required in order to improve their clinical efficacies. Virtual affinity maturation has the potential to quickly focus on the critical hotspot residues without the combinatorial explosion problem of conventional display and library approaches. However, this requires a binding affinity scoring function that is capable of ranking single-point mutations of a starting antibody. We focus here on assessing the solvated interaction energy (SIE) function that was originally developed for and is widely applied to scoring of protein-ligand binding affinities. To this end, we assembled a structure-function data set called Single-Point Mutant Antibody Binding (SiPMAB) comprising several antibody-antigen systems suitable for this assessment, i.e., based on high-resolution crystal structures for the parent antibodies and coupled with high-quality binding affinity measurements for sets of single-point antibody mutants in each system. Using this data set, we tested the SIE function with several mutation protocols based on the popular methods SCWRL, Rosetta, and FoldX. We found that the SIE function coupled with a protocol limited to sampling only the mutated side chain can reasonably predict relative binding affinities with a Spearman rank-order correlation coefficient of about 0.6, outperforming more aggressive sampling protocols. Importantly, this performance is maintained for each of the seven system-specific component subsets as well as for other relevant subsets including non-alanine and charge-altering mutations. The transferability and enrichment in affinity-improving mutants can be further enhanced using consensus ranking over multiple methods, including the SIE, Talaris, and FOLDEF energy functions. The knowledge gained from this study can lead to successful prospective applications of virtual affinity maturation.

  5. Exploration of dimensions of estrogen potency: parsing ligand binding and coactivator binding affinities.

    PubMed

    Jeyakumar, M; Carlson, Kathryn E; Gunther, Jillian R; Katzenellenbogen, John A

    2011-04-15

    The estrogen receptors, ERα and ERβ, are ligand-regulated transcription factors that control gene expression programs in target tissues. The molecular events underlying estrogen action involve minimally two steps, hormone binding to the ER ligand-binding domain followed by coactivator recruitment to the ER·ligand complex; this ligand·receptor·coactivator triple complex then alters gene expression. Conceptually, the potency of an estrogen in activating a cellular response should reflect the affinities that characterize both steps involved in the assembly of the active ligand·receptor·coactivator complex. Thus, to better understand the molecular basis of estrogen potency, we developed a completely in vitro system (using radiometric and time-resolved FRET assays) to quantify independently three parameters: (a) the affinity of ligand binding to ER, (b) the affinity of coactivator binding to the ER·ligand complex, and (c) the potency of ligand recruitment of coactivator. We used this system to characterize the binding and potency of 12 estrogens with both ERα and ERβ. Some ligands showed good correlations between ligand binding affinity, coactivator binding affinity, and coactivator recruitment potency with both ERs, whereas others showed correlations with only one ER subtype or displayed discordant coactivator recruitment potencies. When ligands with low receptor binding affinity but high coactivator recruitment potencies to ERβ were evaluated in cell-based assays, elevation of cellular coactivator levels significantly and selectively improved their potency. Collectively, our results indicate that some low affinity estrogens may elicit greater cellular responses in those target cells that express higher levels of specific coactivators capable of binding to their ER complexes with high affinity. PMID:21321128

  6. Biochemical characterization of high-affinity 3H-opioid binding. Further evidence for Mu1 sites

    SciTech Connect

    Nishimura, S.L.; Recht, L.D.; Pasternak, G.W.

    1984-01-01

    In saturation studies with (/sup 3/H)dihydromorphine, unlabeled D-Ala2-D-Leu5-enkephalin (1 nM) inhibited the high-affinity binding component far more potently than the lower-affinity one. Similarly, morphine (1 nM) inhibited the higher-affinity binding of /sup 3/H-D-Ala2-D-Leu5-enkephalin to a greater extent than its lower-affinity binding component, consistent with a common high-affinity binding site for opiates and enkephalins. Treatment of tissue with either trypsin (1 microgram/ml) or N-ethylmaleimide (25 microM) effectively eliminated the high-affinity binding component of a series of /sup 3/H-opiates and opioid peptides. Competition studies following both treatments were consistent with a common high-affinity binding site. Both treatments also eliminated the ability of low morphine concentrations (less than 1 nM) to inhibit /sup 3/H-D-Ala2-D-Leu5-enkephalin binding and of low D-Ala2-D-Leu5-enkephalin concentrations (less than 1 nM) to inhibit (/sup 3/H)dihydromorphine binding. Protection experiments examining N-ethylmaleimide (25 microM) inhibition of (/sup 3/H)dihydromorphine binding showed significant protection (p less than 0.002) by both unlabeled D-Ala2-D-Leu5-enkephalin and morphine (both at 1 nM). When studied together, both naloxonazine and N-ethylmaleimide inhibited (/sup 3/H)dihydromorphine binding to a similar extent. Equally important, tissue previously treated with naloxonazine was far less sensitive to N-ethylmaleimide than was untreated control tissue, consistent with the possibility that both treatments affected the same site. Together, these results support the concept of a common high-affinity binding site for opiates and opioid peptides.

  7. Blind prediction of charged ligand binding affinities in a model binding site

    PubMed Central

    Rocklin, Gabriel J.; Boyce, Sarah E.; Fischer, Marcus; Fish, Inbar; Mobley, David L.; Shoichet, Brian K.; Dill, Ken A.

    2013-01-01

    Predicting absolute protein-ligand binding affinities remains a frontier challenge in ligand discovery and design. This becomes more difficult when ionic interactions are involved, because of the large opposing solvation and electrostatic attraction energies. In a blind test, we examined whether alchemical free energy calculations could predict binding affinities of 14 charged and 5 neutral compounds previously untested as ligands for a cavity binding site in Cytochrome C Peroxidase. In this simplified site, polar and cationic ligands compete with solvent to interact with a buried aspartate. Predictions were tested by calorimetry, spectroscopy, and crystallography. Of the 15 compounds predicted to bind, 13 were experimentally confirmed, while four compounds were false negative predictions. Predictions had an RMSE of 1.95 kcal/mol to the experimental affinities, and predicted poses had an average RMSD of 1.7 Å to the crystallographic poses. This test serves as a benchmark for these thermodynamically rigorous calculations at predicting binding affinities for charged compounds, and gives insights into the existing sources of error, which are primarily electrostatic interactions inside proteins. Our experiments also provide a useful set of ionic binding affinities in a simplified system for testing new affinity prediction methods. PMID:23896298

  8. Affinity labeling and binding of nitrobenzylthionosine (NBTI) to a membrane fraction (MF) of cultured cell lines

    SciTech Connect

    Woffendin, C.; Plagemann, P.G.W.

    1986-05-01

    Equilibrium binding identified high affinity NBTI binding sites (K/sub D/ = 1-3 nM) on the MF's of L929, L1210, P388, S49 and CHO cells. High affinity NBTI binding sites are associated with the nucleoside transporter since none were present in a MF of a transport-deficient mutant of S49 cells (AE1). MF's of Novikoff cells, like intact Novikoff cells, also lacked high affinity NBTI binding sites. MF's of the cell lines were equilibrium labeled with (/sup 3/H)NBTI using photoaffinity conditions and analyzed by SDS-polyacrylamide gel electrophoresis. Radioactivity was specifically incorporated covalently into a 50-70 Kd protein fraction, but the labeled proteins from CHO and L929 cells had a higher apparent molecular weight than those from S49 and P388 cells. In addition, in MF's from some cell lines lower molecular weight components became photoaffinity labeled. Maximum photoaffinity labeling of the MF proteins was observed with much higher (/sup 3/H)NBTI concentrations (100-200 nM) than those saturating the nucleoside transporter. This finding is explained by a reduced affinity of the photoactivated NBTI intermediate(s) for the transporter. When detergent solubilized MF's from cultured cells were chromotographed on a DEAE cellulose column, only 5-10% of the protein, but practically all high affinity NBTI sites, were recovered in the flow through fraction.

  9. Binding Affinity of Glycoconjugates to BACILLUS Spores and Toxins

    NASA Astrophysics Data System (ADS)

    Rasol, Aveen; Eassa, Souzan; Tarasenko, Olga

    2010-04-01

    Early recognition of Bacillus cereus group species is important since they can cause food-borne illnesses and deadly diseases in humans. Glycoconjugates (GCs) are carbohydrates covalently linked to non-sugar moieties including lipids, proteins or other entities. GCs are involved in recognition and signaling processes intrinsic to biochemical functions in cells. They also stimulate cell-cell adhesion and subsequent recognition and activation of receptors. We have demonstrated that GCs are involved in Bacillus cereus spore recognition. In the present study, we have investigated whether GCs possess the ability to bind and recognize B. cereus spores and Bacillus anthracis recombinant single toxins (sTX) and complex toxins (cTX). The affinity of GCs to spores + sTX and spores + cTX toxins was studied in the binding essay. Our results demonstrated that GC9 and GC10 were able to selectively bind to B. cereus spores and B. anthracis toxins. Different binding affinities for GCs were found toward Bacillus cereus spores + sTX and spores + cTX. Dilution of GCs does not impede the recognition and binding. Developed method provides a tool for simultaneous recognition and targeting of spores, bacteria toxins, and/or other entities.

  10. Affinity labeling of GTP-binding proteins in cellular extracts.

    PubMed

    Löw, A; Faulhammer, H G; Sprinzl, M

    1992-05-25

    GTP-binding proteins in cellular extracts from Escherichia coli, Thermus thermophilus, yeast, wheat germ or calf thymus were identified using in situ periodate-oxidized [alpha-32P]GTP as affinity label. Site-specific reaction of individual GTP-binding proteins was achieved by cross-linking the protein-bound 2',3'-dialdehyde derivative of GTP with the single lysine residue of the conserved NKXD sequence through Schiff's base formation and subsequent cyanoborohydride reduction. Labeled GTP-binding proteins from prokaryotic or eukaryotic cell homogenates were separated by polyacrylamide gel electrophoresis and visualized by autoradiography. In addition cross-linking of [alpha-32P]GTP with GTP-binding proteins was demonstrated in model systems using different purified GTPases, human c-H-ras p21, transducin from bovine retina, polypeptide elongation factor Tu (EF-Tu) from T. thermophilus and initiation factor 2 (IF2) from T. thermophilus. The described affinity labeling technique can serve as an analytical method for the identification of GTPases belonging to the classes of ras-proteins, elongation and initiation factors, and heterotrimeric signal transducing G-proteins. PMID:1592117

  11. Myo1e binds anionic phospholipids with high affinity.

    PubMed

    Feeser, Elizabeth A; Ignacio, Cherry Mae G; Krendel, Mira; Ostap, E Michael

    2010-11-01

    Myo1e is a single-headed motor protein that has been shown to play roles in clathrin-mediated endocytosis in HeLa cells and podocyte function in the kidney. The myo1e C-terminal tail domain includes a basic region that is required for localization to clathrin-coated vesicles and contains a putative pleckstrin-homology (PH) domain that has been shown to play a role in phospholipid binding in other myosin-I proteins. We used sedimentation assays, stopped-flow fluorescence, and fluorescence microscopy to determine the membrane binding affinities, kinetics, and in vivo localization of fluorescently labeled recombinant myo1e-tail constructs. We found that the myo1e tail binds tightly to large unilamellar vesicles (LUVs) containing physiological concentrations of the anionic phospholipids phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) or phosphatidylserine. The rate of myo1e attachment to LUVs nears the diffusion limit while the calculated rate of detachment from LUVs is slow (k(diss) ≤ 0.4 s(-1)). Mutation of conserved residues in the myo1e PH domain has little effect on lipid binding in vitro or membrane localization in vivo. Soluble inositol phosphate headgroups, such as inositol 1,4,5-trisphosphate, can compete with PtdIns(4,5)P(2) for binding, but the apparent affinity for the soluble inositol phosphate is substantially lower than that for PtdIns(4,5)P(2). These results suggest that myo1e binds lipids through nonspecific electrostatic interactions rather than a stereospecific protein-phosphoinositide interaction. PMID:20860408

  12. Binding of ionic species: a general approach to measuring binding constants and assessing affinities.

    PubMed

    Roelens, Stefano; Vacca, Alberto; Venturi, Chiara

    2009-03-01

    Bound together: The association of receptors with ionic species cannot be assimilated to the binding of neutral guests. When dealing with salts, both ion pairing and binding to the free and the ion-paired ionic guest determine the actual association pattern (see figure). The general issue of measuring association constants and assessing affinities for ions is addressed and validated in two cases of anion binding.A general approach to the largely underestimated issue of measuring binding constants and assessing affinities in the binding of ionic species is described. The approach is based on a rigorous, nongraphical determination of binding constants in multiequilibrium systems by nonlinear regression of chemical shift data from NMR titrations and on the use of the BC(50) descriptor for assessing affinities and ranking the binding ability of receptors on a common scale. The approach has been validated with two tripodal anion-binding receptors, namely, a ureidic (1) and a pyrrolic (2) receptor, binding to tetramethylammonium chloride in CDCl(3)/CD(3)CN (80:20). A set of five and six formation constants could be measured for 1 and 2, respectively, including, in addition to the ion pair, complexes of the free and the ion-paired anion. The BC(50) values calculated from the measured constants allowed a quantitative assessment of each receptor's binding affinity towards the chloride anion, the pyrrolic receptor showing a 15-fold larger affinity over the ureidic receptor, a figure that quantifies the improvement obtained by replacing the amido-pyrrolic for ureidic binding groups on the tripodal scaffold of the receptor. The results have shown that, in contrast to common practice, neither of the two systems could be appropriately described by a 1:1 association with the anion only, but required the ion-pairing and ion-pair binding equilibria to be taken into account because these contribute substantially to the complexation process. The BC(50) descriptor has also been shown

  13. SOD1 exhibits allosteric frustration to facilitate metal binding affinity.

    PubMed

    Das, Atanu; Plotkin, Steven S

    2013-03-01

    Superoxide dismutase-1 (SOD1) is a ubiquitous, Cu and Zn binding, free-radical defense enzyme whose misfolding and aggregation play a potential key role in amyotrophic lateral sclerosis, an invariably fatal neurodegenerative disease. Over 150 mutations in SOD1 have been identified with a familial form of the disease, but it is presently not clear what unifying features, if any, these mutants share to make them pathogenic. Here, we develop several unique computational assays for probing the thermo-mechanical properties of both ALS-associated and rationally designed SOD1 variants. Allosteric interaction-free energies between residues and metals are calculated, and a series of atomic force microscopy experiments are simulated with variable tether positions to quantify mechanical rigidity "fingerprints" for SOD1 variants. Mechanical fingerprinting studies of a series of C-terminally truncated mutants, along with an analysis of equilibrium dynamic fluctuations while varying native constraints, potential energy change upon mutation, frustratometer analysis, and analysis of the coupling between local frustration and metal binding interactions for a glycine scan of 90 residues together, reveal that the apo protein is internally frustrated, that these internal stresses are partially relieved by mutation but at the expense of metal-binding affinity, and that the frustration of a residue is directly related to its role in binding metals. This evidence points to apo SOD1 as a strained intermediate with "self-allostery" for high metal-binding affinity. Thus, the prerequisites for the function of SOD1 as an antioxidant compete with apo state thermo-mechanical stability, increasing the susceptibility of the protein to misfold in the apo state.

  14. Multiple lectin detection by cell membrane affinity binding.

    PubMed

    Ribeiro, Ana; Catarino, Sofia; Ferreira, Ricardo Boavida

    2012-05-01

    Assuming that lectins evolved to recognise relatively complex and branched oligosaccharides or parts of them, rather than simple sugars, a procedure based on lectin affinity binding to isolated erythrocyte (or any other cell type) membranes is proposed. This methodology was validated using six pure commercial lectins, as well as lectins from total protein extracts of Arbutus unedo leaves. All commercial lectins, as well as five polypeptides from A. unedo leaves bound to the glycosylated membrane receptors and were eluted by the corresponding sugars. When compared to the standard affinity chromatography procedure involving an individual sugar bound to a solid matrix, the new method provides a single-step, effective detection method for lectins and allows the rapid screening of their profile present in any unknown protein solution, indicates their biological carbohydrate affinities as well as their sugar specificities (if any), enables the simultaneous analysis of a large number of samples, does not require any pre-purification steps, permits detection of additional lectins and provides data which are more relevant from the physiological point of view. PMID:22381939

  15. Binding Kinetics versus Affinities in BRD4 Inhibition.

    PubMed

    Kuang, Ming; Zhou, Jingwei; Wang, Laiyou; Liu, Zhihong; Guo, Jiao; Wu, Ruibo

    2015-09-28

    Bromodomains (BRDs) are protein modules that selectively recognize histones as a "reader" by binding to an acetylated lysine substrate. The human BRD4 has emerged as a promising drug target for a number of disease pathways, and several potent BRD inhibitors have been discovered experimentally recently. However, the detailed inhibition mechanism especially for the inhibitor binding kinetics is not clear. Herein, by employing classical molecular dynamics (MD) and state-of-the-art density functional QM/MM MD simulations, the dynamic characteristics of ZA-loop in BRD4 are revealed. And then the correlation between binding pocket size and ZA-loop motion is elucidated. Moreover, our simulations found that the compound (-)-JQ1 could be accommodated reasonably in thermodynamics whereas it is infeasible in binding kinetics against BRD4. Its racemate (+)-JQ1 proved to be both thermodynamically reasonable and kinetically achievable against BRD4, which could explain the previous experimental results that (+)-JQ1 shows a high inhibitory effect toward BRD4 (IC50 is 77 nM) while (-)-JQ1 is inactive (>10 μM). Furthermore, the L92/L94/Y97 in the ZA-loop and Asn140 in the BC-loop are identified to be critical residues in (+)-JQ1 binding/releasing kinetics. All these findings shed light on further selective inhibitor design toward BRD family, by exploiting the non-negligible ligand binding kinetics features and flexible ZA-loop motions of BRD, instead of only the static ligand-protein binding affinity. PMID:26263125

  16. Binding Kinetics versus Affinities in BRD4 Inhibition.

    PubMed

    Kuang, Ming; Zhou, Jingwei; Wang, Laiyou; Liu, Zhihong; Guo, Jiao; Wu, Ruibo

    2015-09-28

    Bromodomains (BRDs) are protein modules that selectively recognize histones as a "reader" by binding to an acetylated lysine substrate. The human BRD4 has emerged as a promising drug target for a number of disease pathways, and several potent BRD inhibitors have been discovered experimentally recently. However, the detailed inhibition mechanism especially for the inhibitor binding kinetics is not clear. Herein, by employing classical molecular dynamics (MD) and state-of-the-art density functional QM/MM MD simulations, the dynamic characteristics of ZA-loop in BRD4 are revealed. And then the correlation between binding pocket size and ZA-loop motion is elucidated. Moreover, our simulations found that the compound (-)-JQ1 could be accommodated reasonably in thermodynamics whereas it is infeasible in binding kinetics against BRD4. Its racemate (+)-JQ1 proved to be both thermodynamically reasonable and kinetically achievable against BRD4, which could explain the previous experimental results that (+)-JQ1 shows a high inhibitory effect toward BRD4 (IC50 is 77 nM) while (-)-JQ1 is inactive (>10 μM). Furthermore, the L92/L94/Y97 in the ZA-loop and Asn140 in the BC-loop are identified to be critical residues in (+)-JQ1 binding/releasing kinetics. All these findings shed light on further selective inhibitor design toward BRD family, by exploiting the non-negligible ligand binding kinetics features and flexible ZA-loop motions of BRD, instead of only the static ligand-protein binding affinity.

  17. Lipid A-based affinity biosensor for screening anti-sepsis components from herbs.

    PubMed

    Yao, Jie; Chen, Yiguo; Wang, Ning; Jiang, Dongneng; Zheng, Jiang

    2014-01-01

    LPS (lipopolysaccharide), an outer membrane component of Gram-negative bacteria, plays an important role in the pathogenesis of sepsis and lipid A is known to be essential for its toxicity. Therefore it could be an effective measure to prevent sepsis by neutralizing or destroying LPS. Numerous studies have indicated that many traditional Chinese medicines are natural antagonists of LPS in vitro and in vivo. The goal of this study is to develop a rapid method to screen anti-sepsis components from Chinese herbs by use of a direct lipid A-based affinity biosensor technology based on a resonant mirror. The detergent OG (n-octyl β-D-glucopyranoside) was immobilized on a planar non-derivatized cuvette which provided an alternative surface to bind the terminal hydrophilic group of lipid A. A total of 78 herbs were screened based on the affinity biosensor with a target of lipid A. The aqueous extract of PSA (Paeonia suffruticosa Andr) was found to possess the highest capability of binding lipid A. Therefore an aqueous extraction from this plant was investigated further by our affinity biosensor, polyamide chromatography and IEC-HPLC. Finally, we obtained a component (PSA-I-3) from Paeonia suffruticosa Andr that was evaluated with the affinity biosensor. We also studied the biological activities of PSA-I-3 against sepsis in vitro and in vivo to further confirm the component we screened with the biosensor. In vitro, we found that PSA-I-3 could decrease TNFα (tumour necrosis factor α) release from RAW264.7 cells induced by LPS in a dose-dependent manner. In vivo, it increased remarkably the survival of KM (KunMing) mice by challenging both lethal-dose LPS and heat-killed Escherichia coli compared with control groups. Our results suggest that the constructed affinity biosensor can successfully screen the anti-sepsis component from Chinese herbs. PMID:24654965

  18. Compensating Enthalpic and Entropic Changes Hinder Binding Affinity Optimization

    SciTech Connect

    Lafont,V.; Armstrong, A.; Ohtaka, H.; Kiso, Y.; Amzel, L.; Freire, E.

    2007-01-01

    A common strategy to improve the potency of drug candidates is to introduce chemical functionalities, like hydrogen bond donors or acceptors, at positions where they are able to establish strong interactions with the target. However, it is often observed that the added functionalities do not necessarily improve potency even if they form strong hydrogen bonds. Here, we explore the thermodynamic and structural basis for those observations. KNI-10033 is a potent experimental HIV-1 protease inhibitor with picomolar affinity against the wild-type enzyme (Kd = 13 pm). The potency of the inhibitor is the result of favorable enthalpic (?H = -8.2 kcal/mol) and entropic (-T?S = -6.7 kcal/mol) interactions. The replacement of the thioether group in KNI-10033 by a sulfonyl group (KNI-10075) results in a strong hydrogen bond with the amide of Asp 30B of the HIV-1 protease. This additional hydrogen bond improves the binding enthalpy by 3.9 kcal/mol; however, the enthalpy gain is completely compensated by an entropy loss, resulting in no affinity change. Crystallographic and thermodynamic analysis of the inhibitor/protease complexes indicates that the entropy losses are due to a combination of conformational and solvation effects. These results provide a set of practical guidelines aimed at overcoming enthalpy/entropy compensation and improve binding potency.

  19. Limited proteolysis for assaying ligand binding affinities of nuclear receptors.

    PubMed

    Benkoussa, M; Nominé, B; Mouchon, A; Lefebvre, B; Bernardon, J M; Formstecher, P; Lefebvre, P

    1997-01-01

    The binding of natural or synthetic ligands to nuclear receptors is the triggering event leading to gene transcription activation or repression. Ligand binding to the ligand binding domain of these receptors induces conformational changes that are evidenced by an increased resistance of this domain to proteases. In vitro labeled receptors were incubated with various synthetic or natural agonists or antagonists and submitted to trypsin digestion. Proteolysis products were separated by SDS-PAGE and quantified. The amount of trypsin-resistant fragments was proportional to receptor occupancy by the ligand, and allowed the determination of dissociation constants (kDa). Using the wild-type or mutated human retinoic acid receptor alpha as a model, kDa values determined by classical competition binding assays using tritiated ligands are in agreement with those measured by the proteolytic assay. This method was successfully extended to human retinoic X receptor alpha, glucocorticoid receptor, and progesterone receptor, thus providing a basis for a new, faster assay to determine simultaneously the affinity and conformation of receptors when bound to a given ligand.

  20. Recent improvements to Binding MOAD: a resource for protein–ligand binding affinities and structures

    PubMed Central

    Ahmed, Aqeel; Smith, Richard D.; Clark, Jordan J.; Dunbar, James B.; Carlson, Heather A.

    2015-01-01

    For over 10 years, Binding MOAD (Mother of All Databases; http://www.BindingMOAD.org) has been one of the largest resources for high-quality protein–ligand complexes and associated binding affinity data. Binding MOAD has grown at the rate of 1994 complexes per year, on average. Currently, it contains 23 269 complexes and 8156 binding affinities. Our annual updates curate the data using a semi-automated literature search of the references cited within the PDB file, and we have recently upgraded our website and added new features and functionalities to better serve Binding MOAD users. In order to eliminate the legacy application server of the old platform and to accommodate new changes, the website has been completely rewritten in the LAMP (Linux, Apache, MySQL and PHP) environment. The improved user interface incorporates current third-party plugins for better visualization of protein and ligand molecules, and it provides features like sorting, filtering and filtered downloads. In addition to the field-based searching, Binding MOAD now can be searched by structural queries based on the ligand. In order to remove redundancy, Binding MOAD records are clustered in different families based on 90% sequence identity. The new Binding MOAD, with the upgraded platform, features and functionalities, is now equipped to better serve its users. PMID:25378330

  1. Osteopontin binds multiple calcium ions with high affinity and independently of phosphorylation status.

    PubMed

    Kläning, Eva; Christensen, Brian; Sørensen, Esben S; Vorup-Jensen, Thomas; Jensen, Jan K

    2014-09-01

    Osteopontin (OPN) is an acidic, intrinsically disordered extracellular matrix protein with a capacity to modulate biomineralization in vitro and in vivo. The role of posttranslational modification of osteopontin has been intensively studied. Phosphorylation of OPN has been demonstrated to play a role in inhibition of biomineral formation and growth in vitro. Here, we used isothermal titration calorimetry (ITC) to investigate the ability of OPN to bind the divalent cations Ca(2+) and Mg(2+), both essential components of inorganic minerals in vivo. We found, that bovine OPN binds ~10 Ca(2+) ions with an apparent affinity ~50-fold tighter than Mg(2+), both regardless of OPN phosphorylation, and with affinities significantly stronger than previously reported. These results were confirmed using human derived OPN. This implies that a majority of the acidic residues within OPN must be engaged in calcium interaction under physiological conditions.

  2. High Affinity Binding of Indium and Ruthenium Ions by Gastrins.

    PubMed

    Baldwin, Graham S; George, Graham N; Pushie, M Jake

    2015-01-01

    The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS) spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga3+ were fitted by a 2 site model with dissociation constants (Kd) of 3.3 x 10-7 and 1.1 x 10-6 M. Although the absorption of gastrin did not change upon the addition of In3+ ions, the changes in absorbance on Fe3+ ion binding in the presence of indium ions were fitted by a 2 site model with Kd values for In3+ of 6.5 x 10-15 and 1.7 x 10-7 M. Similar results were obtained with Ru3+ ions, although the Kd values for Ru3+ of 2.6 x 10-13 and 1.2 x 10-5 M were slightly larger than observed for In3+. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0-3.3 Å, the Ru complex clearly demonstrated a short range Ru-Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In3+ or Ru3+ ions, and that the affinity of the first site for In3+ or Ru3+ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy.

  3. High Affinity Binding of Indium and Ruthenium Ions by Gastrins

    PubMed Central

    Baldwin, Graham S.; George, Graham N.; Pushie, M. Jake

    2015-01-01

    The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS) spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga3+ were fitted by a 2 site model with dissociation constants (Kd) of 3.3 x 10−7 and 1.1 x 10−6 M. Although the absorption of gastrin did not change upon the addition of In3+ ions, the changes in absorbance on Fe3+ ion binding in the presence of indium ions were fitted by a 2 site model with Kd values for In3+ of 6.5 x 10−15 and 1.7 x 10−7 M. Similar results were obtained with Ru3+ ions, although the Kd values for Ru3+ of 2.6 x 10−13 and 1.2 x 10−5 M were slightly larger than observed for In3+. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0–3.3 Å, the Ru complex clearly demonstrated a short range Ru—Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In3+ or Ru3+ ions, and that the affinity of the first site for In3+ or Ru3+ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy. PMID:26457677

  4. Growth factors with heparin binding affinity in human synovial fluid

    SciTech Connect

    Hamerman, D.; Taylor, S.; Kirschenbaum, I.; Klagsbrun, M.; Raines, E.W.; Ross, R.; Thomas, K.A.

    1987-12-01

    Synovial effusions were obtained from the knees of 15 subjects with joint trauma, menisceal or ligamentous injury, or osteoarthritis. Heparin-Sepharose affinity chromatography of these synovial fluids revealed, in general, three major peaks of mitogenic activity as measured by incorporation of /sup 3/H-thymidine into 3T3 cells. Gradient elution patterns showed activities at 0.5M NaCl, which is characteristic of platelet derived growth factor, and at 1.1 M NaCl and 1.6M NaCl, indicative of acidic and basic fibroblast growth factors, respectively. The identities of these mitogenic fractions were confirmed by specific immunologic and receptor-binding assays. The presence of platelet derived, acidic and basic fibroblast growth factors in the synovial fluid may contribute to wound healing in the arthritic joint.

  5. Cucurbiturils: from synthesis to high-affinity binding and catalysis.

    PubMed

    Assaf, Khaleel I; Nau, Werner M

    2015-01-21

    In the wide area of supramolecular chemistry, cucurbit[n]urils (CBn) present themselves as a young family of molecular containers, able to form stable complexes with various guests, including drug molecules, amino acids and peptides, saccharides, dyes, hydrocarbons, perfluorinated hydrocarbons, and even high molecular weight guests such as proteins (e.g., human insulin). Since the discovery of the first CBn, CB6, the field has seen tremendous growth with respect to the synthesis of new homologues and derivatives, the discovery of record binding affinities of guest molecules in their hydrophobic cavity, and associated applications ranging from sensing to drug delivery. In this review, we discuss in detail the fundamental properties of CBn homologues and their cyclic derivatives with a focus on their synthesis and their applications in catalysis.

  6. Synthesis and binding affinity of an iodinated juvenile hormone

    SciTech Connect

    Prestwich, G.D.; Eng, W.S.; Robles, S.; Vogt, R.G.; Wisniewski, J.R.; Wawrzenczyk, C.

    1988-01-25

    The synthesis of the first iodinated juvenile hormone (JH) in enantiomerically enriched form is reported. This chiral compound, 12-iodo-JH I, has an iodine atom replacing a methyl group of the natural insect juvenile hormone, JH I, which is important in regulating morphogenesis and reproduction in the Lepidoptera. The unlabeled compound shows approximately 10% of the relative binding affinity for the larval hemolymph JH binding protein (JHBP) of Manduca sexta, which specifically binds natural /sup 3/H-10R,11S-JH I (labeled at 58 Ci/mmol) with a KD of 8 X 10(-8) M. It is also approximately one-tenth as biologically active as JH I in the black Manduca and epidermal commitment assays. The 12-hydroxy and 12-oxo compounds are poor competitors and are also biologically inactive. The radioiodinated (/sup 125/I)12-iodo-JH I can be prepared in low yield at greater than 2500 Ci/mmol by nucleophilic displacement using no-carrier-added /sup 125/I-labeled sodium iodide in acetone; however, synthesis using sodium iodide carrier to give the approximately 50 Ci/mmol radioiodinated ligand proceeds in higher radiochemical yield with fewer by-products and provides a radioligand which is more readily handled in binding assays. The KD of (/sup 125/I)12-iodo-JH I was determined for hemolymph JHBP of three insects: M. sexta, 795 nM; Galleria mellonella, 47 nM; Locusta migratoria, 77 nM. The selectivity of 12-iodo-JH I for the 32-kDa JHBP of M. sexta was demonstrated by direct autoradiography of a native polyacrylamide gel electrophoresis gel of larval hemolymph incubated with the radioiodinated ligand. Thus, the in vitro and in vivo activity of 12-iodo-JH I indicate that it can serve as an important new gamma-emitting probe in the search for JH receptor proteins in target tissues.

  7. Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

    PubMed Central

    2011-01-01

    Background Along with high affinity binding of epibatidine (Kd1≈10 pM) to α4β2 nicotinic acetylcholine receptor (nAChR), low affinity binding of epibatidine (Kd2≈1-10 nM) to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites. Results Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after adding a large concentration of

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

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

  9. Generating new ligand-binding RNAs by affinity maturation and disintegration of allosteric ribozymes.

    PubMed Central

    Soukup, G A; DeRose, E C; Koizumi, M; Breaker, R R

    2001-01-01

    Allosteric ribozymes are engineered RNAs that operate as molecular switches whose rates of catalytic activity are modulated by the binding of specific effector molecules. New RNA molecular switches can be created by using "allosteric selection," a molecular engineering process that combines modular rational design and in vitro evolution strategies. In this report, we describe the characterization of 3',5'-cyclic nucleotide monophosphate (cNMP)-dependent hammerhead ribozymes that were created using allosteric selection (Koizumi et al., Nat Struct Biol, 1999, 6:1062-1071). Artificial phylogeny data generated by random mutagenesis and reselection of existing cGMP-, cCMP-, and cAMP-dependent ribozymes indicate that each is comprised of distinct effector-binding and catalytic domains. In addition, patterns of nucleotide covariation and direct mutational analysis both support distinct secondary-structure organizations for the effector-binding domains. Guided by these structural models, we were able to disintegrate each allosteric ribozyme into separate ligand-binding and catalytic modules. Examinations of the independent effector-binding domains reveal that each retains its corresponding cNMP-binding function. These results validate the use of allosteric selection and modular engineering as a means of simultaneously generating new nucleic acid structures that selectively bind ligands. Furthermore, we demonstrate that the binding affinity of an allosteric ribozyme can be improved through random mutagenesis and allosteric selection under conditions that favor tighter binding. This "affinity maturation" effect is expected to be a valuable attribute of allosteric selection as future endeavors seek to apply engineered allosteric ribozymes as biosensor components and as controllable genetic switches. PMID:11345431

  10. Nucleic acid binding affinity of fd gene 5 protein in the cooperative binding mode.

    PubMed

    Bobst, A M; Ireland, J C; Bobst, E V

    1984-02-25

    A sensitive ESR method which allows a direct quantitative determination of nucleic acid binding affinities of proteins under physiologically relevant conditions has been applied to the gene 5 protein of bacteriophage fd. This was achieved with two spin-labeled nucleic acids, (ldT, dT)n and (lA,A)n, which served as macro-molecular spin probes in ESR competition experiments. With the two different macromolecular spin probes, it was possible to determine the relative apparent affinity constants, Kapp, over a large affinity domain. In 20 mM Tris X HCl (pH 8.1), 1 mM sodium EDTA, 0.1 mM dithiothreitol, 10% (w/v) glycerol, 0.05% Triton, and 125 mM NaCl, the following affinity relationship was observed: K(dT)napp = 10(3) KfdDNAapp = 2 X 10(4) K(A)napp = 6.6 X 10(4) KrRNAapp = 1.5 X 10(5) KR17RNAapp. Increasing the [NaCl] from 125 to 200 mM caused considerably less tight binding of gene 5 protein to (lA,A)n, and a typical cooperative binding isotherm was observed, whereas at the lower [NaCl] used for the competition experiments, the binding was essentially stoichiometric. A computer fit of the experimental titration data at 200 mM NaCl gave an intrinsic binding constant, Kint, of 1300 M-1 and a cooperativity factor, omega, of 60 (Kint omega = Kapp) for (lA,A)n.

  11. Phosphatidylserine Reversibly Binds Cu2+ with Extremely High Affinity

    PubMed Central

    Monson, Christopher F.; Cong, Xiao; Robison, Aaron; Pace, Hudson P.; Liu, Chunming; Poyton, Matthew F.; Cremer, Paul S.

    2012-01-01

    Phosphatidylserine (PS) embedded within supported lipid bilayers (SLBs) was found to bind Cu2+ from solution with extraordinarily high affinity. In fact, the equilibrium dissociation constant was in the femtomolar range. The resulting complex formed in a 1:2 Cu2+ to PS ratio and quenches a broad spectrum of lipid-bound fluorophores in a reversible and pH-dependent fashion. At acidic pH values, the fluorophores were almost completely unquenched, while at basic pH values significant quenching (85–90%) was observed. The pH at which the transition occurred was dependent on the PS concentration and ranged from approximately pH 5 to 8. The quenching kinetics was slow at low Cu2+ concentrations and basic values pH (up to several hours), while the unquenching reaction was orders of magnitude more rapid upon lowering the pH. This was consistent with diffusion limited complex formation at basic pH, but rapid dissociation under acidic conditions. The tight binding of Cu2+ to PS may have physiological consequences under certain circumstances. PMID:22548290

  12. Affinity ligands for glycoprotein purification based on the multi-component Ugi reaction.

    PubMed

    Chen, Chen; Khoury, Graziella El; Lowe, Christopher R

    2014-10-15

    One challenge facing the purification of therapeutic glycoproteins by affinity chromatography is creating ligands specific for the glycan moiety. Affinity chromatography of glycoproteins is currently conducted with immobilized lectins or boronates, although biomimetic ligands could present a more desirable option. This work describes the rational design and combinatorial synthesis of carbohydrate-binding ligands based on the solid phase multi-component Ugi reaction. An aldehyde-functionalized Sepharose™ solid support constitutes one component (aldehyde) in the four-component reaction, while the other three components (a primary/secondary amine, a carboxylic acid and an isocyanide) are varied in a combinatorial fashion to generate a tri-substituted Ugi scaffold which provides a degree of rigidity and is functionally suitable for interacting with the glycan moiety of glycoproteins. An Ugi library containing 48 ligands was initially screened against glucose oxidase (GOx) as the model glycoprotein to identify a candidate ligand, A13C24I8, which showed affinity to GOx through its carbohydrate moiety. Immobilized ligand A13C24I8 demonstrated a static binding capacity of 16.7mg GOx/ml resin and an apparent dissociation constant (Kd) of 1.45×10(-6)M at pH 7.4. The adsorbent can also bind 8.1mg AGP/ml resin and displays an apparent affinity constant Kd=1.44×10(-5)M. The ligand has a sugar specificity in the following sequence: sorbitol>fructose>mannitol>ribose>arabinose>xylose>galactose>mannose>glucose>fructose; however, it did not display any specificity for sialic acid or methyl α-D-glycosides. A control ligand, generated by substitution of C24 (3-carboxyphenylboronic acid) with C7 (4-hydroxyphenyl acetic acid), failed to show affinity to the carbohydrate moiety, supporting the importance of the role that boronic acid group plays in sugar binding. GOx spiked E. coli samples were loaded onto immobilized ligand A13C24I8, 3-aminophenylboronic acid (APBA) and

  13. Affinity ligands for glycoprotein purification based on the multi-component Ugi reaction.

    PubMed

    Chen, Chen; Khoury, Graziella El; Lowe, Christopher R

    2014-10-15

    One challenge facing the purification of therapeutic glycoproteins by affinity chromatography is creating ligands specific for the glycan moiety. Affinity chromatography of glycoproteins is currently conducted with immobilized lectins or boronates, although biomimetic ligands could present a more desirable option. This work describes the rational design and combinatorial synthesis of carbohydrate-binding ligands based on the solid phase multi-component Ugi reaction. An aldehyde-functionalized Sepharose™ solid support constitutes one component (aldehyde) in the four-component reaction, while the other three components (a primary/secondary amine, a carboxylic acid and an isocyanide) are varied in a combinatorial fashion to generate a tri-substituted Ugi scaffold which provides a degree of rigidity and is functionally suitable for interacting with the glycan moiety of glycoproteins. An Ugi library containing 48 ligands was initially screened against glucose oxidase (GOx) as the model glycoprotein to identify a candidate ligand, A13C24I8, which showed affinity to GOx through its carbohydrate moiety. Immobilized ligand A13C24I8 demonstrated a static binding capacity of 16.7mg GOx/ml resin and an apparent dissociation constant (Kd) of 1.45×10(-6)M at pH 7.4. The adsorbent can also bind 8.1mg AGP/ml resin and displays an apparent affinity constant Kd=1.44×10(-5)M. The ligand has a sugar specificity in the following sequence: sorbitol>fructose>mannitol>ribose>arabinose>xylose>galactose>mannose>glucose>fructose; however, it did not display any specificity for sialic acid or methyl α-D-glycosides. A control ligand, generated by substitution of C24 (3-carboxyphenylboronic acid) with C7 (4-hydroxyphenyl acetic acid), failed to show affinity to the carbohydrate moiety, supporting the importance of the role that boronic acid group plays in sugar binding. GOx spiked E. coli samples were loaded onto immobilized ligand A13C24I8, 3-aminophenylboronic acid (APBA) and

  14. Binding of the extracellular matrix component entactin to Candida albicans.

    PubMed Central

    López-Ribot, J L; Chaffin, W L

    1994-01-01

    We have investigated the interaction between Candida albicans and entactin, a recently characterized glycoprotein present in the extracellular matrix, especially in the basement membrane. Organisms of both the yeast and the hyphal morphologies of the fungus had the ability to bind recombinant entactin, as detected by an indirect immunofluorescence assay. Material present in the 2-mercaptoethanol cell wall extracts from both C. albicans growth forms was capable of binding to immobilized recombinant entactin in a dose-dependent manner. Binding to entactin was approximately twice that observed for laminin. Binding of an extract component(s) to entactin was partially inhibited by an Arg-Gly-Asp-Ser peptide. A polyclonal antientactin antiserum, as well as a pooled antiserum preparation raised against components present in different C. albicans cell wall extracts, completely or almost completely abolished binding. The existence of morphology-specific receptor-like molecules which bind to different domains of the entactin molecule was ruled out in a competition binding assay. The entactin-binding material(s) in the cell wall also displayed some ability to bind laminin and fibronectin, since preadsorption in the presence of these extracellular matrix components resulted in reduction of binding to entactin. Moieties with a molecular mass of approximately 25, 44, and 65 kDa present in the 2-mercaptoethanol cell wall extracts from both blastoconidia and germ tubes were detected in a ligand affinity blotting experiment as having the ability to bind entactin. Interactions between C. albicans and entactin could be important in mediating adhesion of the fungus to the host tissues and may play a role in the establishment of the disseminated form of the disease. Images PMID:7927722

  15. The N-terminal domain determines the affinity and specificity of H1 binding to chromatin

    SciTech Connect

    Oeberg, Christine; Belikov, Sergey

    2012-04-06

    Highlights: Black-Right-Pointing-Pointer wt Human histone H1.4 and hH1.4 devoid of N-terminal domain, {Delta}N-hH1.4, were compared. Black-Right-Pointing-Pointer Both histones bind to chromatin, however, {Delta}N-hH1.4 displays lower binding affinity. Black-Right-Pointing-Pointer Interaction of {Delta}N-hH1.4 with chromatin includes a significant unspecific component. Black-Right-Pointing-Pointer N-terminal domain is a determinant of specificity of histone H1 binding to chromatin. -- Abstract: Linker histone H1, one of the most abundant nuclear proteins in multicellular eukaryotes, is a key component of the chromatin structure mainly due to its role in the formation and maintenance of the 30 nm chromatin fiber. It has a three-domain structure; a central globular domain flanked by a short N-terminal domain and a long, highly basic C-terminal domain. Previous studies have shown that the binding abilities of H1 are at large determined by the properties of the C-terminal domain; much less attention has been paid to role of the N-terminal domain. We have previously shown that H1 can be reconstituted via cytoplasmic mRNA injection in Xenopus oocytes, cells that lack somatic H1. The heterologously expressed H1 proteins are incorporated into in vivo assembled chromatin at specific sites and the binding event is monitored as an increase in nucleosomal repeat length (NRL). Using this setup we have here compared the binding properties of wt-H1.4 and hH1.4 devoid of its N-terminal domain ({Delta}N-hH1.4). The {Delta}N-hH1.4 displays a drastically lower affinity for chromatin binding as compared to the wild type hH1.4. Our data also indicates that {Delta}N-hH1.4 is more prone to unspecific chromatin binding than the wild type. We conclude that the N-terminal domain of H1 is an important determinant of affinity and specificity of H1-chromatin interactions.

  16. Quantitative structure-activity relationships for polychlorinated hydroxybiphenyl estrogen receptor binding affinity: An assessment of conformer flexibility

    SciTech Connect

    Bradbury, S.P.; Ankley, G.T.; Mekenyan, O.G.

    1996-11-01

    A diverse group of xenobiotics has a high binding affinity to the estrogen receptor (ER), suggesting that it can accommodate large variability in ligand structure. Relationships between xenobiotic surface, binding affinity, and estrogenic response have been suggested to be dependent on the conformational structures of the ligands. To explore the influence of conformational flexibility on ER binding affinity, a quantitative structure-activity relationship (QSAR) study was undertaken with estradiol, diethylstilbestrol, and a set of polychlorinated hydroxybiphenyls (PCHBs) of environmental concern. Although the low-energy minima of the PCHB congeners suggested that interconversions among conformers were likely, the electronic parameters associated with the conformer geometries for a specific PCHB congener could vary significantly. The results of the QSAR analysis suggested that among the PCHBs studied, the most polarizable conformers (lower absolute volume polarizability values) were most closely associated with ER binding affinity. Across the set of polarizable conformers, which did not include the low-energy gas-phase conformers, the electron donating properties of the hydroxy moiety and the aromatic component of the estradiol A ring analogue in the PCHBs were found to be correlated with higher ER binding affinity.

  17. PREDICTING ER BINDING AFFINITY FOR EDC RANKING AND PRIORITIZATION: MODEL II

    EPA Science Inventory

    The training set used to derive a common reactivity pattern (COREPA) model for estrogen receptor (ER) binding affinity in Model I (see Abstract I in this series) was extended to include 47 rat estrogen receptor (rER) relative binding affinity (RBA) measurements in addition to the...

  18. High-affinity cannabinoid binding site in brain: A possible marijuana receptor

    SciTech Connect

    Nye, J.S.

    1988-01-01

    The mechanism by which delta{sup 9} tetrahydrocannabinol (delta{sup 9}THC), the major psychoactive component of marijuana or hashish, produces its potent psychological and physiological effects is unknown. To find receptor binding sites for THC, we designed a water-soluble analog for use as a radioligand. 5{prime}-Trimethylammonium-delta{sup 8}THC (TMA) is a positively charged analog of delta-{sup 8}THC modified on the 5{prime} carbon, a portion of the molecule not important for its psychoactivity. We have studied the binding of ({sup 3}H)-5{prime}-trimethylammonium-delta-{sup 8}THC (({sup 3}H)TMA) to rat neuronal membranes. ({sup 3}H)TMA binds saturably and reversibly to brain membranes with high affinity to apparently one class of sites. Highest binding site density occurs in brain, but several peripheral organs also display specific binding. Detergent solubilizes the sites without affecting their pharmacologial properties. Molecular sieve chromatography reveals a bimodal peak of ({sup 3}H)TMA binding activity of approximately 60,000 daltons apparent molecular weight.

  19. High affinity binding of an engineered, modular peptide to bone tissue.

    PubMed

    Brounts, Sabrina H; Lee, Jae Sung; Weinberg, Sean; Lan Levengood, Sheeny K; Smith, Everett L; Murphy, William L

    2013-05-01

    Bone grafting procedures have become common due in part to a global trend of population aging. Native bone graft is a popular choice when compared to various synthetic bone graft substitutes, owing to superior biological activity. Nonetheless, the insufficient ability of bone allograft to induce new bone formation and the insufficient remodeling of native bone grafts call for osteoinductive factors during bone repair, exemplified by recombinant human bone morphogenetic protein 2 (rhBMP2). We previously developed a modular bone morphogenetic peptide (mBMP) to address complications associated with the clinical use of rhBMP2 as a bone graft substitute. The mBMP is designed to strongly bind to hydroxyapatite, the main inorganic component of bone and teeth, and to provide pro-osteogenic properties analogous to rhBMP2. Our previous in vivo animal studies showed that mBMP bound to hydroxyapatite-coated orthopedic implants with high affinity and stimulated new bone formation. In this study, we demonstrate specific binding of mBMP to native bone grafts. The results show that mBMP binds with high affinity to both cortical and trabecular bones, and that the binding is dependent on the mBMP concentration and incubation time. Importantly, efficient mBMP binding is also achieved in an ex vivo bone bioreactor where bone tissue is maintained viable for several weeks. In addition, mBMP binding can be localized with spatial control on native bone tissue via simple methods, such as dip-coating, spotting, and direct writing. Taken together with the pro-osteogenic activity of mBMP established in previous bone repair models, these results suggest that mBMP may promote bone healing when coated on native bone grafts in a clinically compatible manner.

  20. Paracetamol and cytarabine binding competition in high affinity binding sites of transporting protein

    NASA Astrophysics Data System (ADS)

    Sułkowska, A.; Bojko, B.; Równicka, J.; Sułkowski, W. W.

    2006-07-01

    Paracetamol (acetaminophen, AA) the most popular analgesic drug is commonly used in the treatment of pain in patients suffering from cancer. In our studies, we evaluated the competition in binding with serum albumin between paracetamol (AA) and cytarabine, antyleukemic drug (araC). The presence of one drug can alter the binding affinity of albumin towards the second one. Such interaction can result in changing of the free fraction of the one of these drugs in blood. Two spectroscopic methods were used to determine high affinity binding sites and the competition of the drugs. Basing on the change of the serum albumin fluorescence in the presence of either of the drugs the quenching ( KQ) constants for the araC-BSA and AA-BSA systems were calculated. Analysis of UV difference spectra allowed us to describe the changes in drug-protein complexes (araC-albumin and AA-albumin) induced by the presence of the second drug (AA and araC, respectively). The mechanism of competition between araC and AA has been proposed.

  1. Purification of L-( sup 3 H) Nicotine eliminates low affinity binding

    SciTech Connect

    Romm, E.; Marks, M.J.; Collins, A.C. ); Lippiello, P.M. )

    1990-01-01

    Some studies of L-({sup 3}H) nicotine binding to rodent and human brain tissue have detected two binding sites as evidenced by nonlinear Scatchard plots. Evidence presented here indicated that the low affinity binding site is not stereospecific, is not inhibited by low concentrations of cholinergic agonists and is probably due to breakdown products of nicotine since purification of the L-({sup 3}H)nicotine eliminates the low affinity site.

  2. Anion-induced increases in the affinity of colcemid binding to tubulin.

    PubMed

    Ray, K; Bhattacharyya, B; Biswas, B B

    1984-08-01

    Colcemid binds tubulin rapidly and reversibly in contrast to colchicine which binds tubulin relatively slowly and essentially irreversibly. At 37 degrees C the association rate constant for colcemid binding is 1.88 X 10(6) M-1 h-1, about 10 times higher than that for colchicine; this is reflected in the activation energies for binding which are 51.4 kJ/mol for colcemid and 84.8 kJ/mol for colchicine. Scatchard analysis indicates two binding sites on tubulin having different affinities for colcemid. The high-affinity site (Ka = 0.7 X 10(5) M-1 at 37 degrees C) is sensitive to temperature and binds both colchicine and colcemid and hence they are mutually competitive inhibitors. The low-affinity site (Kb = 1.2 X 10(4) M-1) is rather insensitive to temperature and binds only colcemid. Like colchicine, 0.6 mol of colcemid are bound/mol of tubulin dimer (at the high-affinity site) and the reaction is entropy driven (163 J K-1 mol-1). Similar to colchicine, colcemid binding to tubulin is stimulated by certain anions (viz. sulfate and tartrate) but by a different mechanism. Colcemid binding affinity at the lower-affinity site of tubulin is increased in the presence of ammonium sulfate. Interestingly, the lower-affinity site on tubulin for colcemid, even when converted to higher affinity in presence of ammonium sulfate, is not recognized by colchicine. We conclude that tubulin possesses two binding sites, one of which specifically recognized the groups present on the B-ring of colchicine molecule and is effected by the ammonium sulfate, whereas the higher-affinity site, which could accommodate both colchicine and colcemid, possibly recognized the A and C ring of colchicine.

  3. Low affinity binding site clusters confer hox specificity and regulatory robustness.

    PubMed

    Crocker, Justin; Abe, Namiko; Rinaldi, Lucrezia; McGregor, Alistair P; Frankel, Nicolás; Wang, Shu; Alsawadi, Ahmad; Valenti, Philippe; Plaza, Serge; Payre, François; Mann, Richard S; Stern, David L

    2015-01-15

    In animals, Hox transcription factors define regional identity in distinct anatomical domains. How Hox genes encode this specificity is a paradox, because different Hox proteins bind with high affinity in vitro to similar DNA sequences. Here, we demonstrate that the Hox protein Ultrabithorax (Ubx) in complex with its cofactor Extradenticle (Exd) bound specifically to clusters of very low affinity sites in enhancers of the shavenbaby gene of Drosophila. These low affinity sites conferred specificity for Ubx binding in vivo, but multiple clustered sites were required for robust expression when embryos developed in variable environments. Although most individual Ubx binding sites are not evolutionarily conserved, the overall enhancer architecture-clusters of low affinity binding sites-is maintained and required for enhancer function. Natural selection therefore works at the level of the enhancer, requiring a particular density of low affinity Ubx sites to confer both specific and robust expression. PMID:25557079

  4. Cell-Binding Assays for Determining the Affinity of Protein-Protein Interactions: Technologies and Considerations.

    PubMed

    Hunter, S A; Cochran, J R

    2016-01-01

    Determining the equilibrium-binding affinity (Kd) of two interacting proteins is essential not only for the biochemical study of protein signaling and function but also for the engineering of improved protein and enzyme variants. One common technique for measuring protein-binding affinities uses flow cytometry to analyze ligand binding to proteins presented on the surface of a cell. However, cell-binding assays require specific considerations to accurately quantify the binding affinity of a protein-protein interaction. Here we will cover the basic assumptions in designing a cell-based binding assay, including the relevant equations and theory behind determining binding affinities. Further, two major considerations in measuring binding affinities-time to equilibrium and ligand depletion-will be discussed. As these conditions have the potential to greatly alter the Kd, methods through which to avoid or minimize them will be provided. We then outline detailed protocols for performing direct- and competitive-binding assays against proteins displayed on the surface of yeast or mammalian cells that can be used to derive accurate Kd values. Finally, a comparison of cell-based binding assays to other types of binding assays will be presented. PMID:27586327

  5. Thermodynamic mixing of molecular states of the epidermal growth factor receptor modulates macroscopic ligand binding affinity.

    PubMed Central

    Holbrook, M R; Slakey, L L; Gross, D J

    2000-01-01

    The epidermal growth factor receptor (EGFr), when expressed on the cell surface, has long been known to display two distinct affinities for epidermal growth factor (EGF) binding. In addition, the treatment of cells expressing the EGFr with phorbol esters has been shown to cause a loss of the high-affinity binding capacity of the receptor. In the present study, point mutations that alter acidic or phosphorylation sites have been made in an intracellular domain near Tyr-992 (residues 988-992) of the EGFr. Equilibrium (125)I-EGF binding studies demonstrate that the conversion of Tyr-992 into glutamate induces a 4-fold decrease in the EGFr apparent low-affinity dissociation constant, whereas the mutation of two acidic residues, Asp-988 and Glu-991, or the conversion of Tyr-992 into phenylalanine does not alter EGFr affinity. Phorbol ester treatment of EGFr-expressing Chinese hamster ovary cells results in a loss of high-affinity binding and an increase in the apparent low-affinity dissociation constant of the receptor, similar to the effect of a truncation mutant in which the C-terminal 190 residues are deleted. These results are examined in the context of a new model for regulation of the affinity of the EGFr for EGF in which a cytosolic particle stabilizes the high-affinity conformation of the EGFr and a rapid equilibrium exists between EGFr high-affinity and low-affinity conformations. This model demonstrates that the macroscopic affinities of the EGFr can differ from the affinities of individual EGFr molecules and provides a theoretical framework whereby the measured affinities of the EGFr are modulated by intracellular interactions. PMID:11062062

  6. High affinity binding of (/sup 3/H)cocaine to rat liver microsomes

    SciTech Connect

    El-Maghrabi, E.A.; Calligaro, D.O.; Eldefrawi, M.E.

    1988-01-01

    )/sup 3/H)cocaine bound reversible, with high affinity and stereospecificity to rat liver microsomes. Little binding was detected in the lysosomal, mitochondrial and nuclear fractions. The binding kinetics were slow and the kinetically calculated K/sub D/ was 2 nM. Induction of mixed function oxidases by phenobarbital did not produce significant change in (/sup 3/H)cocaine binding. On the other hand, chronic administration of cocaine reduced (/sup 3/H)cocaine binding drastically. Neither treatment affected the affinity of the liver binding protein for cocaine. Microsomes from mouse and human livers had less cocaine-binding protein and lower affinity for cocaine than those from rat liver. Binding of (/sup 3/H)cocaine to rat liver microsomes was insensitive to monovalent cations and > 10 fold less sensitive to biogenic amines than the cocaine receptor in rat striatum. However, the liver protein had higher affinity for cocaine and metabolites except for norcocaine. Amine uptake inhibitors displaced (/sup 3/H)cocaine binding to liver with a different rank order of potency than their displacement of (/sup 3/H)cocaine binding to striatum. This high affinity (/sup 3/H)cocaine binding protein in liver is not likely to be monooxygenase, but may have a role in cocaine-induced hepatotoxicity

  7. Purification of proteins specifically binding human endogenous retrovirus K long terminal repeat by affinity elution chromatography.

    PubMed

    Trubetskoy, D O; Zavalova, L L; Akopov, S B; Nikolaev, L G

    2002-11-01

    A novel affinity elution procedure for purification of DNA-binding proteins was developed and employed to purify to near homogeneity the proteins recognizing a 21 base pair sequence within the long terminal repeat of human endogenous retroviruses K. The approach involves loading the initial protein mixture on a heparin-agarose column and elution of protein(s) of interest with a solution of double-stranded oligonucleotide containing binding sites of the protein(s). The affinity elution has several advantages over conventional DNA-affinity chromatography: (i) it is easier and faster, permitting to isolate proteins in a 1 day-one stage procedure; (ii) yield of a target protein is severalfold higher than that in DNA-affinity chromatography; (iii) it is not necessary to prepare a special affinity support for each factor to be isolated. Theaffinity elution could be a useful alternative to conventional DNA-affinity chromatography.

  8. General approach for characterizing in vitro selected peptides with protein binding affinity.

    PubMed

    Larsen, Andrew C; Gillig, Annabelle; Shah, Pankti; Sau, Sujay P; Fenton, Kathryn E; Chaput, John C

    2014-08-01

    In vitro selection technologies are important tools for identifying high affinity peptides to proteins of broad medical and biological interest. However, the technological advances that have made it possible to generate long lists of candidate peptides have far outpaced our ability to characterize the binding properties of individual peptides. Here, we describe a low cost strategy to rapidly synthesize, purify, screen, and characterize peptides for high binding affinity. Peptides are assayed in a 96-well dot blot apparatus using membranes that enable partitioning of bound and unbound peptide-protein complexes. We have validated the binding affinity constants produced by this method using known peptide ligands and applied this process to discover five new peptides with nanomolar affinity to human α-thrombin. Given the need for new analytical tools that can accelerate peptide discovery and characterization, we feel that this approach would be useful to a wide range of technologies that utilize high affinity peptides.

  9. Recent advances in affinity capillary electrophoresis for binding studies.

    PubMed

    Albishri, Hassan M; El Deeb, Sami; AlGarabli, Noura; AlAstal, Raghda; Alhazmi, Hassan A; Nachbar, Markus; El-Hady, Deia Abd; Wätzig, Hermann

    2014-01-01

    The present review covers recent advances and important applications of affinity capillary electrophoresis (ACE). It provides an overview about various ACE types, including ACE-MS, the multiple injection mode, the use of microchips and field-amplified sample injection-ACE. The most common scenarios of the studied affinity interactions are protein-drug, protein-metal ion, protein-protein, protein-DNA, protein-carbohydrate, carbohydrate-drug, peptide-peptide, DNA-drug and antigen-antibody. Approaches for the improvements of ACE in term of precision, rinsing protocols and sensitivity are discussed. The combined use of computer simulation programs to support data evaluation is presented. In conclusion, the performance of ACE is compared with other techniques such as equilibrium dialysis, parallel artificial membrane permeability assay, high-performance affinity chromatography as well as surface plasmon resonance, ultraviolet, circular dichroism, nuclear magnetic resonance, Fourier transform infrared, fluorescence, MS and isothermal titration calorimetry. PMID:25534793

  10. Binding of bisbenzylisoquinoline alkaloids to phosphatidylcholine vesicles and alveolar macrophages: relationship between binding affinity and antifibrogenic potential of these drugs.

    PubMed

    Ma, J K; Mo, C G; Malanga, C J; Ma, J Y; Castranova, V

    1991-01-01

    A group of bisbenzylisoquinoline alkaloids has been shown to exhibit various degrees of effectiveness in preventing silica-induced fibrosis in animal models. The objective of the present study was to characterize the binding of several of these alkaloids to phosphatidylcholine vesicles and rat alveolar macrophages using fluorometric and equilibrium dialysis methods, respectively. The lipid binding affinity of these alkaloids was found to depend upon several structural factors including hydrophobic substitutions, chiral configurations, and double oxygen bridge-restricted confirmation of the benzylisoquinoline moieties. Tetrandrine, which is a highly effective agent in preventing fibrosis, showed strong binding to both lipid vesicles and alveolar macrophages. In contrast, certain analogues of tetrandrine such as curine and tubocurine, which have little or no effect on silicosis, exhibited only weak binding to lipid vesicles and almost no binding to cells. The moderate binding affinity of fangchinoline to vesicles and cells corresponded to a moderate effectiveness of the compound as an antifibrogenic agent. Methoxyadiantifoline, an alkaloid of unknown antifibrogenic potential, also exhibited high binding affinities for lipid and cells. In conclusion, the results of these studies indicate that alveolar macrophages exhibit large binding capacities for certain members of this class of bisbenzylisoquinoline alkaloids. A positive correlation was observed between binding affinity to alveolar macrophages and the reported antifibrotic potency of these compounds. These data also suggest that the ability of these drugs to interact with alveolar macrophages may be a key step in inhibition of the progression of silica-induced pulmonary disease. PMID:1663032

  11. The Binding of Biotin to Sepharose-Avidin Column: Demonstration of the Affinity Chromatography Technique

    ERIC Educational Resources Information Center

    Landman, A. D.; Landman, N. N.

    1976-01-01

    Describes a biochemistry experiment that illustrates the methodology of affinity chromatography by attaching avidin, a glycoprotein in egg white, to a Sepharose matrix in order to bind biotin-containing proteins. (MLH)

  12. PREDICTING ER BINDING AFFINITY FOR EDC RANKING AND PRIORITIZATION: MODEL I

    EPA Science Inventory

    A Common Reactivity Pattern (COREPA) model, based on consideration of multiple energetically reasonable conformations of flexible chemicals was developed using a training set of 232 rat estrogen receptor (rER) relative binding affinity (RBA) measurements. The training set include...

  13. Binding affinities of CRBPI and CRBPII for 9-cis-retinoids

    PubMed Central

    Kane, Maureen A.; Bright, Frank V.; Napoli, Joseph L.

    2014-01-01

    Background Cellular retinol binding-protein I (CRBPI) and cellular retinol binding-protein II (CRBPII) serve as intracellular retinoid chaperones that bind retinol and retinal with high affinity and facilitate substrate delivery to select enzymes that catalyze retinoic acid (RA) and retinyl ester biosynthesis. Recently, 9-cis-RA has been identified in vivo in the pancreas, where it contributes to regulating glucose-stimulated insulin secretion. In vitro, 9-cis-RA activates RXR (retinoid×receptors), which serve as therapeutic targets for treating cancer and metabolic diseases. Binding affinities and structure–function relationships have been well characterized for CRBPI and CRBPII with all-trans-retinoids, but not for 9-cis-retinoids. This study extended current knowledge by establishing binding affinities for CRBPI and CRBPII with 9-cis-retinoids. Methods We have determined apparent dissociation constants, Kd′, through monitoring binding of 9-cis-retinol, 9-cis-retinal, and 9-cis-RA with CRBPI and CRBPII by fluorescence spectroscopy, and analyzing the data with non-linear regression. We compared these data to the data we obtained for all-trans- and 13-cis-retinoids under identical conditions. Results CRBPI and CRBPII, respectively, bind 9-cis-retinol ( Kd′, 11 nM and 68 nM) and 9-cis-retinal ( Kd′, 8 nM and 5 nM) with high affinity. No significant 9-cis-RA binding was observed with CRBPI or CRBPII. Conclusions CRBPI and CRBPII bind 9-cis-retinol and 9-cis-retinal with high affinities, albeit with affinities somewhat lower than for all-trans-retinol and all-trans-retinal. General significance These data provide further insight into structure–binding relationships of cellular retinol binding-proteins and are consistent with a model of 9-cis-RA biosynthesis that involves chaperoned delivery of 9-cis-retinoids to enzymes that recognize retinoid binding-proteins. PMID:21382444

  14. Glycan:glycan interactions: High affinity biomolecular interactions that can mediate binding of pathogenic bacteria to host cells

    PubMed Central

    Day, Christopher J.; Tran, Elizabeth N.; Semchenko, Evgeny A.; Tram, Greg; Hartley-Tassell, Lauren E.; Ng, Preston S. K.; King, Rebecca M.; Ulanovsky, Rachel; McAtamney, Sarah; Apicella, Michael A.; Tiralongo, Joe; Morona, Renato; Korolik, Victoria; Jennings, Michael P.

    2015-01-01

    Cells from all domains of life express glycan structures attached to lipids and proteins on their surface, called glycoconjugates. Cell-to-cell contact mediated by glycan:glycan interactions have been considered to be low-affinity interactions that precede high-affinity protein–glycan or protein–protein interactions. In several pathogenic bacteria, truncation of surface glycans, lipooligosaccharide (LOS), or lipopolysaccharide (LPS) have been reported to significantly reduce bacterial adherence to host cells. Here, we show that the saccharide component of LOS/LPS have direct, high-affinity interactions with host glycans. Glycan microarrays reveal that LOS/LPS of four distinct bacterial pathogens bind to numerous host glycan structures. Surface plasmon resonance was used to determine the affinity of these interactions and revealed 66 high-affinity host–glycan:bacterial–glycan pairs with equilibrium dissociation constants (KD) ranging between 100 nM and 50 µM. These glycan:glycan affinity values are similar to those reported for lectins or antibodies with glycans. Cell assays demonstrated that glycan:glycan interaction-mediated bacterial adherence could be competitively inhibited by either host cell or bacterial glycans. This is the first report to our knowledge of high affinity glycan:glycan interactions between bacterial pathogens and the host. The discovery of large numbers of glycan:glycan interactions between a diverse range of structures suggests that these interactions may be important in all biological systems. PMID:26676578

  15. Glycan:glycan interactions: High affinity biomolecular interactions that can mediate binding of pathogenic bacteria to host cells.

    PubMed

    Day, Christopher J; Tran, Elizabeth N; Semchenko, Evgeny A; Tram, Greg; Hartley-Tassell, Lauren E; Ng, Preston S K; King, Rebecca M; Ulanovsky, Rachel; McAtamney, Sarah; Apicella, Michael A; Tiralongo, Joe; Morona, Renato; Korolik, Victoria; Jennings, Michael P

    2015-12-29

    Cells from all domains of life express glycan structures attached to lipids and proteins on their surface, called glycoconjugates. Cell-to-cell contact mediated by glycan:glycan interactions have been considered to be low-affinity interactions that precede high-affinity protein-glycan or protein-protein interactions. In several pathogenic bacteria, truncation of surface glycans, lipooligosaccharide (LOS), or lipopolysaccharide (LPS) have been reported to significantly reduce bacterial adherence to host cells. Here, we show that the saccharide component of LOS/LPS have direct, high-affinity interactions with host glycans. Glycan microarrays reveal that LOS/LPS of four distinct bacterial pathogens bind to numerous host glycan structures. Surface plasmon resonance was used to determine the affinity of these interactions and revealed 66 high-affinity host-glycan:bacterial-glycan pairs with equilibrium dissociation constants (K(D)) ranging between 100 nM and 50 µM. These glycan:glycan affinity values are similar to those reported for lectins or antibodies with glycans. Cell assays demonstrated that glycan:glycan interaction-mediated bacterial adherence could be competitively inhibited by either host cell or bacterial glycans. This is the first report to our knowledge of high affinity glycan:glycan interactions between bacterial pathogens and the host. The discovery of large numbers of glycan:glycan interactions between a diverse range of structures suggests that these interactions may be important in all biological systems. PMID:26676578

  16. Metal binding components in human amniotic fluid

    SciTech Connect

    Paterson, P.G.; Zlotkin, S.H.; Sarkar, B. )

    1990-02-26

    Amniotic fluid is a potential source of both nutritionally essential and toxic metals for the fetus. As the binding pattern of these metals in amniotic fluid may be one of the determining factors in their availability to the fetus, the objective of this study was to investigate metal binding in vitro. The binding of six trace metals, Mn(II), Ni(II), Zn(II), Cu(II), Cd(II), and Fe(III), to components of human amniotic fluid was studied by Sephadex G-100 gel filtration at physiological pH, using radioisotopes as tracers and 50 mM TRIS/HCl as the elution buffer. The amniotic fluid was collected at 16-16.5 weeks gestation by amniocentesis and pooled for analysis. Extensive amounts of Fe, Cu, Zn, and Cd and small amounts of Mn and Ni were bound to high molecular weight proteins with elution patterns similar to those seen for the binding of these metals in serum. In addition, large amounts of Fe, Mn, Ni and Cd and small amounts of Zn and Cu were associated with low molecular weight component(s). The identity of these latter components is unknown, but they play an important biological role in amniotic fluid.

  17. Generation of high-performance binding proteins for peptide motifs by affinity clamping

    PubMed Central

    Koide, Shohei; Huang, Jin

    2013-01-01

    We describe concepts and methodologies for generating “Affinity Clamps”, a new class of recombinant binding proteins that achieve high affinity and high specificity toward short peptide motifs of biological importance, which is a major challenge in protein engineering. The Affinity Clamping concept exploits the potential of nonhomologous recombination of protein domains in generating large changes in protein function and the inherent binding affinity and specificity of the so-called modular interaction domains toward short peptide motifs. Affinity Clamping creates a clamshell architecture that clamps onto a target peptide. The design processes involve (i) choosing a starting modular interaction domain appropriate for the target and applying structure-guided modifications, (ii) attaching a second domain, termed “enhancer domain” and (iii) optimizing the peptide-binding site located between the domains by directed evolution. The two connected domains work synergistically to achieve high levels of affinity and specificity that are unattainable with either domain alone. Because of the simple and modular architecture, affinity clamps are particularly well suited as building blocks for designing more complex functionalities. Affinity Clamping represents a major advance in protein design that is broadly applicable to the recognition of peptide motifs. PMID:23422435

  18. How Much Binding Affinity Can be Gained by Filling a Cavity?

    PubMed Central

    Kawasaki, Yuko; Chufan, Eduardo E.; Lafont, Virginie; Hidaka, Koushi; Kiso, Yoshiaki; Amzel, L. Mario; Freire, Ernesto

    2011-01-01

    Binding affinity optimization is critical during drug development. Here we evaluate the thermodynamic consequences of filling a binding cavity with functionalities of increasing van der Waals radii (-H, -F, -Cl and CH3) that improve the geometric fit without participating in hydrogen bonding or other specific interactions. We observe a binding affinity increase of two orders of magnitude. There appears to be three phases in the process. The first phase is associated with the formation of stable van der Waals interactions. This phase is characterized by a gain in binding enthalpy and a loss in binding entropy, attributed to a loss of conformational degrees of freedom. For the specific case presented in this paper, the enthalpy gain amounts to −1.5 kcal/mol while the entropic losses amount to +0.9 kcal/mol resulting in a net 3.5-fold affinity gain. The second phase is characterized by simultaneous enthalpic and entropic gains. This phase improves the binding affinity 25-fold. The third phase represents the collapse of the trend and is triggered by the introduction of chemical functionalities larger than the binding cavity itself (CH(CH3)2). It is characterized by large enthalpy and affinity losses. The thermodynamic signatures associated with each phase provide guidelines for lead optimization. PMID:20028396

  19. Structure-affinity relationship of the cocaine-binding aptamer with quinine derivatives.

    PubMed

    Slavkovic, Sladjana; Altunisik, Merve; Reinstein, Oren; Johnson, Philip E

    2015-05-15

    In addition to binding its target molecule, cocaine, the cocaine-binding aptamer tightly binds the alkaloid quinine. In order to understand better how the cocaine-binding aptamer interacts with quinine we have used isothermal titration calorimetry-based binding experiments to study the interaction of the cocaine-binding aptamer to a series of structural analogs of quinine. As a basis for comparison we also investigated the binding of the cocaine-binding aptamer to a set of cocaine metabolites. The bicyclic aromatic ring on quinine is essential for tight affinity by the cocaine-binding aptamer with 6-methoxyquinoline alone being sufficient for tight binding while the aliphatic portion of quinine, quinuclidine, does not show detectable binding. Compounds with three fused aromatic rings are not bound by the aptamer. Having a methoxy group at the 6-position of the bicyclic ring is important for binding as substituting it with a hydrogen, an alcohol or an amino group all result in lower binding affinity. For all ligands that bind, association is driven by a negative enthalpy compensated by unfavorable binding entropy.

  20. Structure-affinity relationship of the cocaine-binding aptamer with quinine derivatives.

    PubMed

    Slavkovic, Sladjana; Altunisik, Merve; Reinstein, Oren; Johnson, Philip E

    2015-05-15

    In addition to binding its target molecule, cocaine, the cocaine-binding aptamer tightly binds the alkaloid quinine. In order to understand better how the cocaine-binding aptamer interacts with quinine we have used isothermal titration calorimetry-based binding experiments to study the interaction of the cocaine-binding aptamer to a series of structural analogs of quinine. As a basis for comparison we also investigated the binding of the cocaine-binding aptamer to a set of cocaine metabolites. The bicyclic aromatic ring on quinine is essential for tight affinity by the cocaine-binding aptamer with 6-methoxyquinoline alone being sufficient for tight binding while the aliphatic portion of quinine, quinuclidine, does not show detectable binding. Compounds with three fused aromatic rings are not bound by the aptamer. Having a methoxy group at the 6-position of the bicyclic ring is important for binding as substituting it with a hydrogen, an alcohol or an amino group all result in lower binding affinity. For all ligands that bind, association is driven by a negative enthalpy compensated by unfavorable binding entropy. PMID:25858454

  1. A high-affinity estrogen-binding protein in rat placental trophoblast.

    PubMed

    McCormack, S A; Glasser, S R

    1976-09-01

    A high-affinity, low-capacity estradiol-binding molecule (RE) has been demonstrated in the basal zone trophoblast (BZT) of the pregnant rat. On day 11 of pregnancy (day 0 = first sperm-positive day) RE is present in BZT cytosol, where it has a ka of 1.2 X 10(6)M-1 sec-1, t1/2 = 12.7 min, at 20 C. The Kd, under similar conditions, consists of 2 components, 1.3 X 10(-4) sec-1, t1/2 = 90 min, and 5.9 X 10(-5) sec-1, t1/2 = 196 min. When one uses the faster component, the equilibrium constant, Kd, obtained from kd/ka is 1.1 X 10(-10)M, in close agreement with that obtained from Scatchard analysis of specific estradiol (E2) binding at 20 C. On day 11 there were approximately 12,000 sites/cell in BZT cytosol. Scatchard analysis of nuclear RE on day 11 indicated a Kd of 1.85 X 10(-10)M and approximately 21,000 sites/nucleus, but, in day 15 BZT, nuclear RE was undetectable. Neither cytosol nor nuclei prepared from placental labyrinthine zone (LZT) tissue (fetal placenta) showed evidence of high-affinity, low-capacity E2 binding. Sucrose density gradient analysis on 5-20% linear gradients showed the cytosol RE to be approximately 4S whether in high or low-salt conditions. When measured against binding by 3H-labeled estradiol (*E2), the cytosol BTZ RE was competed for strongly (80-90%) by estrone, estriol, diethylstilbestrol, and estradiol-17alpha at 200 times excess. Nafoxidine-HCl, also at 200X excess, competed to approximately 50%. Corticosterone, progesterone, testosterone, dehydroepiandrosterone, and pregnenolone did not compete. The hormone specificity of nuclear BZT RE was similar to that of the comparable cytosol RE with the exception that nafoxidine did not compete. This was probably due to differences in kinetics, nafoxidine requiring a longer time to reach equilibrium than the other estrogens. The size of the nuclear RE by sucrose density gradient analysis was approximately 2S by KCl extraction (which was inefficient) or 4S by trypsin extraction. We conclude that

  2. Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity.

    PubMed

    Boder, E T; Midelfort, K S; Wittrup, K D

    2000-09-26

    Single-chain antibody mutants have been evolved in vitro with antigen-binding equilibrium dissociation constant K(d) = 48 fM and slower dissociation kinetics (half-time > 5 days) than those for the streptavidin-biotin complex. These mutants possess the highest monovalent ligand-binding affinity yet reported for an engineered protein by over two orders of magnitude. Optimal kinetic screening of randomly mutagenized libraries of 10(5)-10(7) yeast surface-displayed antibodies enabled a >1,000-fold decrease in the rate of dissociation after four cycles of affinity mutagenesis and screening. The consensus mutations are generally nonconservative by comparison with naturally occurring mouse Fv sequences and with residues that do not contact the fluorescein antigen in the wild-type complex. The existence of these mutants demonstrates that the antibody Fv architecture is not intrinsically responsible for an antigen-binding affinity ceiling during in vivo affinity maturation.

  3. Integrated microfluidic approach for quantitative high-throughput measurements of transcription factor binding affinities

    PubMed Central

    Glick, Yair; Orenstein, Yaron; Chen, Dana; Avrahami, Dorit; Zor, Tsaffrir; Shamir, Ron; Gerber, Doron

    2016-01-01

    Protein binding to DNA is a fundamental process in gene regulation. Methodologies such as ChIP-Seq and mapping of DNase I hypersensitive sites provide global information on this regulation in vivo. In vitro methodologies provide valuable complementary information on protein–DNA specificities. However, current methods still do not measure absolute binding affinities. There is a real need for large-scale quantitative protein–DNA affinity measurements. We developed QPID, a microfluidic application for measuring protein–DNA affinities. A single run is equivalent to 4096 gel-shift experiments. Using QPID, we characterized the different affinities of ATF1, c-Jun, c-Fos and AP-1 to the CRE consensus motif and CRE half-site in two different genomic sequences on a single device. We discovered that binding of ATF1, but not of AP-1, to the CRE half-site is highly affected by its genomic context. This effect was highly correlated with ATF1 ChIP-seq and PBM experiments. Next, we characterized the affinities of ATF1 and ATF3 to 128 genomic CRE and CRE half-site sequences. Our affinity measurements explained that in vivo binding differences between ATF1 and ATF3 to CRE and CRE half-sites are partially mediated by differences in the minor groove width. We believe that QPID would become a central tool for quantitative characterization of biophysical aspects affecting protein–DNA binding. PMID:26635393

  4. Integrated microfluidic approach for quantitative high-throughput measurements of transcription factor binding affinities.

    PubMed

    Glick, Yair; Orenstein, Yaron; Chen, Dana; Avrahami, Dorit; Zor, Tsaffrir; Shamir, Ron; Gerber, Doron

    2016-04-01

    Protein binding to DNA is a fundamental process in gene regulation. Methodologies such as ChIP-Seq and mapping of DNase I hypersensitive sites provide global information on this regulation in vivo In vitro methodologies provide valuable complementary information on protein-DNA specificities. However, current methods still do not measure absolute binding affinities. There is a real need for large-scale quantitative protein-DNA affinity measurements. We developed QPID, a microfluidic application for measuring protein-DNA affinities. A single run is equivalent to 4096 gel-shift experiments. Using QPID, we characterized the different affinities of ATF1, c-Jun, c-Fos and AP-1 to the CRE consensus motif and CRE half-site in two different genomic sequences on a single device. We discovered that binding of ATF1, but not of AP-1, to the CRE half-site is highly affected by its genomic context. This effect was highly correlated with ATF1 ChIP-seq and PBM experiments. Next, we characterized the affinities of ATF1 and ATF3 to 128 genomic CRE and CRE half-site sequences. Our affinity measurements explained that in vivo binding differences between ATF1 and ATF3 to CRE and CRE half-sites are partially mediated by differences in the minor groove width. We believe that QPID would become a central tool for quantitative characterization of biophysical aspects affecting protein-DNA binding.

  5. The binding modes and binding affinities of epipodophyllotoxin derivatives with human topoisomerase IIα.

    PubMed

    Naik, Pradeep Kumar; Dubey, Abhishek; Soni, Komal; Kumar, Rishay; Singh, Harvinder

    2010-12-01

    Epipodophyllotoxin derivatives have important therapeutic value in the treatment of human cancers. These drugs kill cells by inhibiting the ability of topoisomerase II (TP II) to ligate nucleic acids that it cleaves during the double-stranded DNA passage reaction. The 3D structure of human TP IIα was modeled by homology modeling. A virtual library consisting of 143 epipodophyllotoxin derivatives has been developed. Their molecular interactions and binding affinities with modeled human TP IIα have been studied using the docking and Bimolecular Association with Energetics (eMBrAcE) developed by Schrödinger. Structure activity relationship models were developed between the experimental activity expressed in terms of percentage of intracellular covalent TP II-DNA complexes (log PCPDCF) of these compounds and molecular descriptors like docking score and free energy of binding. For both the cases the r2 was in the range of 0.624-0.800 indicating good data fit and r2(cv) was in the range of 0.606-774 indicating that the predictive capabilities of the models were acceptable. Low levels of root mean square error for the majority of inhibitors establish the docking and eMBrAcE based prediction model as an efficient tool for generating more potent and specific inhibitors of human TP IIα by testing rationally designed lead compounds based on epipodophyllotoxin derivatization. PMID:21075653

  6. A robust assay to measure DNA topology-dependent protein binding affinity.

    PubMed

    Litwin, Tamara R; Solà, Maria; Holt, Ian J; Neuman, Keir C

    2015-04-20

    DNA structure and topology pervasively influence aspects of DNA metabolism including replication, transcription and segregation. However, the effects of DNA topology on DNA-protein interactions have not been systematically explored due to limitations of standard affinity assays. We developed a method to measure protein binding affinity dependence on the topology (topological linking number) of supercoiled DNA. A defined range of DNA topoisomers at equilibrium with a DNA binding protein is separated into free and protein-bound DNA populations using standard nitrocellulose filter binding techniques. Electrophoretic separation and quantification of bound and free topoisomers combined with a simple normalization procedure provide the relative affinity of the protein for the DNA as a function of linking number. Employing this assay we measured topology-dependent DNA binding of a helicase, a type IB topoisomerase, a type IIA topoisomerase, a non-specific mitochondrial DNA binding protein and a type II restriction endonuclease. Most of the proteins preferentially bind negatively supercoiled DNA but the details of the topology-dependent affinity differ among proteins in ways that expose differences in their interactions with DNA. The topology-dependent binding assay provides a robust and easily implemented method to probe topological influences on DNA-protein interactions for a wide range of DNA binding proteins.

  7. Computational protocol for predicting the binding affinities of zinc containing metalloprotein-ligand complexes.

    PubMed

    Jain, Tarun; Jayaram, B

    2007-06-01

    Zinc is one of the most important metal ions found in proteins performing specific functions associated with life processes. Coordination geometry of the zinc ion in the active site of the metalloprotein-ligand complexes poses a challenge in determining ligand binding affinities accurately in structure-based drug design. We report here an all atom force field based computational protocol for estimating rapidly the binding affinities of zinc containing metalloprotein-ligand complexes, considering electrostatics, van der Waals, hydrophobicity, and loss in conformational entropy of protein side chains upon ligand binding along with a nonbonded approach to model the interactions of the zinc ion with all the other atoms of the complex. We examined the sensitivity of the binding affinity predictions to the choice of Lennard-Jones parameters, partial atomic charges, and dielectric treatments adopted for system preparation and scoring. The highest correlation obtained was R2 = 0.77 (r = 0.88) for the predicted binding affinity against the experiment on a heterogenous dataset of 90 zinc containing metalloprotein-ligand complexes consisting of five unique protein targets. Model validation and parameter analysis studies underscore the robustness and predictive ability of the scoring function. The high correlation obtained suggests the potential applicability of the methodology in designing novel ligands for zinc-metalloproteins. The scoring function has been web enabled for free access at www.scfbio-iitd.res.in/software/drugdesign/bapplz.jsp as BAPPL-Z server (Binding Affinity Prediction of Protein-Ligand complexes containing Zinc metal ions).

  8. One-step surface modification of polyurethane using affinity binding peptides for enhanced fouling resistance.

    PubMed

    Wang, Yibing; Yu, Yong; Zhang, Liting; Qin, Peng; Wang, Ping

    2015-01-01

    Affinity binding peptides were examined for surface fabrication of synthetic polymeric materials. Peptides possessing strong binding affinities toward polyurethane (PU) were discovered via biopanning of M13 phage peptide library. The apparent binding constant (K(app)) was as high as 2.68 × 10(9) M(-1) with surface peptide density exceeded 1.8 μg/cm(2). Structural analysis showed that the ideal peptide had a high content (75%) of H-donor amino acid residues, and that intensified hydrogen bond interaction was the key driving force for the highly stable binding of peptides on PU. PU treated with such affinity peptides promises applications as low-fouling materials, as peptides increased its wettability and substantially reduced protein adsorption and cell adhesion. These results demonstrated a facile but highly efficient one-step strategy for surface property modification of polymeric materials for biotechnological applications. PMID:25732121

  9. GHB receptor targets in the CNS: focus on high-affinity binding sites.

    PubMed

    Bay, Tina; Eghorn, Laura F; Klein, Anders B; Wellendorph, Petrine

    2014-01-15

    γ-Hydroxybutyric acid (GHB) is an endogenous compound in the mammalian brain with both low- and high-affinity receptor targets. GHB is used clinically in the treatment of symptoms of narcolepsy and alcoholism, but also illicitly abused as the recreational drug Fantasy. Major pharmacological effects of exogenous GHB are mediated by GABA subtype B (GABAB) receptors that bind GHB with low affinity. The existence of GHB high-affinity binding sites has been known for more than three decades, but the uncovering of their molecular identity has only recently begun. This has been prompted by the generation of molecular tools to selectively study high-affinity sites. These include both genetically modified GABAB knock-out mice and engineered selective GHB ligands. Recently, certain GABA subtype A (GABAA) receptor subtypes emerged as high-affinity GHB binding sites and potential physiological mediators of GHB effects. In this research update, a description of the various reported receptors for GHB is provided, including GABAB receptors, certain GABAA receptor subtypes and other reported GHB receptors. The main focus will thus be on the high-affinity binding targets for GHB and their potential functional roles in the mammalian brain.

  10. Relative binding affinities of bisphosphonates for human bone and relationship to antiresorptive efficacy.

    PubMed

    Leu, Chih-Tai; Luegmayr, Eva; Freedman, Leonard P; Rodan, Gideon A; Reszka, Alfred A

    2006-05-01

    Potent bisphosphonates (BPs) preferentially bind bone at sites of active osteoclastic bone resorption, where they are taken up by the osteoclast and inhibit resorption. We tested the hypothesis that BP affinity to human bone affects antiresorptive potency. [(1)(4)C]-Alendronate binding to human bone was saturable and reversible with an apparent Kd of 72 microM by Scatchard analysis. In competition binding assays, unlabeled alendronate (Ki: 61 microM) was slightly more potent than pyrophosphate (Ki = 156 microM) in blocking [(1)(4)C]-alendronate binding. Likewise, most tested BPs, including etidronate (Ki: 91 microM), ibandronate (116 microM), pamidronate (83 microM), risedronate (85 microM) and zoledronate (81 microM), showed comparable affinities. Interestingly, tiludronate (173 microM; P < 0.05 vs. all other BPs) and especially clodronate (806 microM; P > 0.0001 vs. all other BPs) displayed significantly weaker affinity for bone. The weak affinity of clodronate translated into a requirement for 10-fold higher dosing in in vitro bone resorption assays when bone was pretreated with BP and subsequently washed prior to adding osteoclasts. In stark contrast, neither alendronate nor risedronate lost any efficacy after washing the bone surface. These findings suggest that most clinically tested BPs may have similar affinities for human bone. For those with reduced affinity, this may translate into lower potency that necessitates higher dosing.

  11. Rolling adhesion of alphaL I domain mutants decorrelated from binding affinity.

    PubMed

    Pepper, Lauren R; Hammer, Daniel A; Boder, Eric T

    2006-06-30

    Activated lymphocyte function-associated antigen-1 (LFA-1, alphaLbeta2 integrin) found on leukocytes facilitates firm adhesion to endothelial cell layers by binding to intercellular adhesion molecule-1 (ICAM-1), which is up-regulated on endothelial cells at sites of inflammation. Recent work has shown that LFA-1 in a pre-activation, low-affinity state may also be involved in the initial tethering and rolling phase of the adhesion cascade. The inserted (I) domain of LFA-1 contains the ligand-binding epitope of the molecule, and a conformational change in this region during activation increases ligand affinity. We have displayed wild-type I domain on the surface of yeast and validated expression using I domain specific antibodies and flow cytometry. Surface display of I domain supports yeast rolling on ICAM-1-coated surfaces under shear flow. Expression of a locked open, high-affinity I domain mutant supports firm adhesion of yeast, while yeast displaying intermediate-affinity I domain mutants exhibit a range of rolling phenotypes. We find that rolling behavior for these mutants fails to correlate with ligand binding affinity. These results indicate that unstressed binding affinity is not the only molecular property that determines adhesive behavior under shear flow.

  12. Relationship of nonreturn rates of dairy bulls to binding affinity of heparin to sperm

    SciTech Connect

    Marks, J.L.; Ax, R.L.

    1985-08-01

    The binding of the glycosaminoglycan (3H) heparin to bull spermatozoa was compared with nonreturn rates of dairy bulls. Semen samples from five bulls above and five below an average 71% nonreturn rate were used. Samples consisted of first and second ejaculates on a single day collected 1 d/wk for up to 5 consecutive wk. Saturation binding assays using (TH) heparin were performed to quantitate the binding characteristics of each sample. Scatchard plot analyses indicated a significant difference in the binding affinity for (TH) heparin between bulls of high and low fertility. Dissociation constants were 69.0 and 119.3 pmol for bulls of high and low fertility, respectively. In contrast, the number of binding sites for (TH) heparin did not differ significantly among bulls. Differences in binding affinity of (TH) heparin to bull sperm might be used to predict relative fertility of dairy bulls.

  13. Rationally manipulating aptamer binding affinities in a stem-loop molecular beacon.

    PubMed

    Armstrong, Rachel E; Strouse, Geoffrey F

    2014-10-15

    Single-stranded DNA sequences that are highly specific for a target ligand are called aptamers. While the incorporation of aptamer sequences into stem-loop molecular beacons has become an essential tool in optical biosensors, the design principles that determine the magnitude of binding affinity and its relationship to placement of the aptamer sequence in the stem-loop architecture are not well defined. By controlled placement of the aptamer along the loop region of the molecular beacon, it is observed that the binding affinity can be tuned over 4 orders of magnitude (1.3 nM - 203 μM) for the Huizenga and Szostak ATP DNA aptamer sequence. It is observed that the Kd is enhanced for the fully exposed sequence, with reduced binding affinity when the aptamer is part of the stem region of the beacon. Analysis of the ΔG values indicate a clear correlation between the aptamer hybridized length in the stem and its observed Kd. The use of a nanometal surface energy transfer probe method for monitoring ATP binding to the aptamer sequence allows the observation of negative cooperativity between the two ATP binding events. Maintenance of the high binding affinity of this ATP aptamer and the observation of two separate Kd's for ATP binding indicate NSET as an effective, nonmanipulative, optical method for tracking biomolecular changes.

  14. Current Understanding of the Binding Sites, Capacity, Affinity, and Biological Significance of Metals in Melanin

    PubMed Central

    Hong, Lian; Simon, John D.

    2008-01-01

    Metal chelation is often invoked as one of the main biological functions of melanin. In order to understand the interaction between metals and melanin, extensive studies have been carried out to determine the nature of the metal binding sites, binding capacity and affinity. These data are central to efforts aimed at elucidating the role metal binding plays in determining the physical, structural, biological, and photochemical properties of melanin. This article examines the current state of understanding of this field. PMID:17580858

  15. A New Bisintercalating Anthracycline with Picomolar DNA Binding Affinity

    PubMed Central

    Portugal, José; Cashman, Derek J.; Trent, John O.; Ferrer-Miralles, Neus; Przewloka, Teresa; Fokt, Izabela; Priebe, Waldemar; Chaires, Jonathan B.

    2008-01-01

    A new bisintercalating anthracycline (WP762) has been designed, in which monomeric units of daunorubicin have been linked through their amino groups on the daunosamine moieties using an m-xylenyl linker. Differential scanning calorimetry and UV melting experiments were used to measure the ultratight binding of WP762 to DNA. The binding constant for the interaction of WP762 with herring sperm DNA was determined to be 7.3 (±0.2) × 1012 M−1 at 20°C. The large favorable binding free energy of −17.3 kcal mol−1 was found to result from a large negative enthalpic contribution of −33.8 kcal mol−1 and an opposing entropic term (−TΔS = +16.5 kcal mol−1). A comparative molecular modeling study rationalized the increased binding by the m-xylenyl linker of WP762 positioning in the DNA minor groove compared to the p-xylenyl linker found in WP631, the first bis-anthracycline of this type. The cytotoxicity of WP762 was compared to that of other anthracyclines in Jurkat T lymphocytes. These studies, together with an analysis of the cell-cycle traverse in the presence of WP762, suggest that in these cells the new drug is more cytotoxic than the structurally related WP631. PMID:16366602

  16. A yeast glycolipid biosurfactant, mannosylerythritol lipid, shows high binding affinity towards lectins on a self-assembled monolayer system.

    PubMed

    Konishi, Masaaki; Imura, Tomohiro; Fukuoka, Tokuma; Morita, Tomotake; Kitamoto, Dai

    2007-03-01

    Mannosylerythritol lipids (MEL), which are glycolipid biosurfactants secreted by the Pseudozyma yeasts, show not only excellent surface-active properties but also versatile biochemical actions including antitumor and cell-differentiation activities. In order to address the biochemical actions, interactions between MEL-A, the major component of MEL, and different lectins were investigated using the surface plasmon resonance spectroscopy. The monolayer of MEL-A showed high binding affinity to concanavalin A (ConA) and Maackia amurensis lectin-I (MAL-I). The observed affinity constants for ConA and MAL-I were estimated to be 9.48 +/- 1.31 x 10(6) and 3.13 +/- 0.274 x 10(6) M(-1), respectively; the value was comparable to that of Manalpha1-6(Manalpha1-3)Man, which is one of the most specific probe to ConA. Significantly, alpha-methyl-D-mannopyranoside (1 mM) exhibited no binding inhibition between MEL-A and ConA. MEL-A is thus likely to self-assemble to give a high affinity surface, where ConA binds to the hydrophilic headgroup in a different manner from that generally observed in lectin-saccharide interactions. The binding manner should be related with the biochemical actions of MEL toward mammalian cells via protein-carbohydrate interactions. PMID:17205206

  17. New horizons in mouse immunoinformatics: reliable in silico prediction of mouse class I histocompatibility major complex peptide binding affinity.

    PubMed

    Hattotuwagama, Channa K; Guan, Pingping; Doytchinova, Irini A; Flower, Darren R

    2004-11-21

    Quantitative structure-activity relationship (QSAR) analysis is a main cornerstone of modern informatic disciplines. Predictive computational models, based on QSAR technology, of peptide-major histocompatibility complex (MHC) binding affinity have now become a vital component of modern day computational immunovaccinology. Historically, such approaches have been built around semi-qualitative, classification methods, but these are now giving way to quantitative regression methods. The additive method, an established immunoinformatics technique for the quantitative prediction of peptide-protein affinity, was used here to identify the sequence dependence of peptide binding specificity for three mouse class I MHC alleles: H2-D(b), H2-K(b) and H2-K(k). As we show, in terms of reliability the resulting models represent a significant advance on existing methods. They can be used for the accurate prediction of T-cell epitopes and are freely available online ( http://www.jenner.ac.uk/MHCPred). PMID:15534705

  18. A Low Affinity Ground State Conformation for the Dynein Microtubule Binding Domain*

    PubMed Central

    McNaughton, Lynn; Tikhonenko, Irina; Banavali, Nilesh K.; LeMaster, David M.; Koonce, Michael P.

    2010-01-01

    Dynein interacts with microtubules through a dedicated binding domain that is dynamically controlled to achieve high or low affinity, depending on the state of nucleotide bound in a distant catalytic pocket. The active sites for microtubule binding and ATP hydrolysis communicate via conformational changes transduced through a ∼10-nm length antiparallel coiled-coil stalk, which connects the binding domain to the roughly 300-kDa motor core. Recently, an x-ray structure of the murine cytoplasmic dynein microtubule binding domain (MTBD) in a weak affinity conformation was published, containing a covalently constrained β+ registry for the coiled-coil stalk segment (Carter, A. P., Garbarino, J. E., Wilson-Kubalek, E. M., Shipley, W. E., Cho, C., Milligan, R. A., Vale, R. D., and Gibbons, I. R. (2008) Science 322, 1691–1695). We here present an NMR analysis of the isolated MTBD from Dictyostelium discoideum that demonstrates the coiled-coil β+ registry corresponds to the low energy conformation for this functional region of dynein. Addition of sequence encoding roughly half of the coiled-coil stalk proximal to the binding tip results in a decreased affinity of the MTBD for microtubules. In contrast, addition of the complete coiled-coil sequence drives the MTBD to the conformationally unstable, high affinity binding state. These results suggest a thermodynamic coupling between conformational free energy differences in the α and β+ registries of the coiled-coil stalk that acts as a switch between high and low affinity conformations of the MTBD. A balancing of opposing conformations in the stalk and MTBD enables potentially modest long-range interactions arising from ATP binding in the motor core to induce a relaxation of the MTBD into the stable low affinity state. PMID:20351100

  19. Evaluation of ligand-binding affinity using polynomial empirical scoring functions.

    PubMed

    de Azevedo, Walter Filgueira; Dias, Raquel

    2008-10-15

    Assessing protein-ligand interaction is of great importance for virtual screening initiatives in order to discover new drugs. The present work describes a set of empirical scoring functions to assess the binding affinity, involving terms for intermolecular hydrogen bonds and contact surface. The results show that our methodology works better to predict protein-ligand affinity when compared with XSCORE, a popular empirical scoring function.

  20. Determinants of affinity and specificity in RNA-binding proteins.

    PubMed

    Helder, Stephanie; Blythe, Amanda J; Bond, Charles S; Mackay, Joel P

    2016-06-01

    Emerging data suggest that the mechanisms by which RNA-binding proteins (RBPs) interact with RNA and the rules governing specificity might be substantially more complex than those underlying their DNA-binding counterparts. Even our knowledge of what constitutes the RNA-bound proteome is contentious; recent studies suggest that 10-30% of RBPs contain no known RNA-binding domain. Adding to this situation is a growing disconnect between the avalanche of identified interactions between proteins and long noncoding RNAs and the absence of biophysical data on these interactions. RNA-protein interactions are also at the centre of what might emerge as one of the biggest shifts in thinking about cell and molecular biology this century, following from recent reports of ribonucleoprotein complexes that drive reversible membrane-free phase separation events within the cell. Unexpectedly, low-complexity motifs are important in the formation of these structures. Here we briefly survey recent advances in our understanding of the specificity of RBPs. PMID:27315040

  1. Influence of the galloyl moiety in tea catechins on binding affinity for human serum albumin.

    PubMed

    Minoda, Kanako; Ichikawa, Tatsuya; Katsumata, Tomoharu; Onobori, Ken-ichi; Mori, Taiki; Suzuki, Yukiko; Ishii, Takeshi; Nakayama, Tsutomu

    2010-01-01

    The major catechins of green tea extract are (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg). Recent research has indicated that catechins form complexes with human serum albumin (HSA) in blood, and differences in their binding affinity toward HSA are believed to modulate their bioavailability. In this study, we kinetically investigated the interaction between the catechins and HSA immobilized on a quartz-crystal microbalance (QCM). The association constants obtained from the frequency changes of QCM revealed interactions of ECg and EGCg with HSA that are 100 times stronger than those of EC and EGC. Furthermore, comparisons of these catechins by native-gel electrophoresis/blotting with redox-cycling staining revealed that, in a phosphate buffer, ECg and EGCg have a higher binding affinity toward HSA than EC and EGC. These observations indicate that catechins with a galloyl moiety have higher binding affinities toward HSA than catechins lacking a galloyl moiety.

  2. Impedance-derived electrochemical capacitance spectroscopy for the evaluation of lectin-glycoprotein binding affinity.

    PubMed

    Santos, Adriano; Carvalho, Fernanda C; Roque-Barreira, Maria-Cristina; Bueno, Paulo R

    2014-12-15

    Characterization of lectin-carbohydrate binding using label-free methods such as impedance-derived electrochemical capacitance spectroscopy (ECS) is desirable to evaluate specific interactions, for example, ArtinM lectin and horseradish peroxidase (HRP) glycoprotein, used here as a model for protein-carbohydrate binding affinity. An electroactive molecular film comprising alkyl ferrocene as a redox probe and ArtinM as a carbohydrate receptive center to target HRP was successfully used to determine the binding affinity between ArtinM and HRP. The redox capacitance, a transducer signal associated with the alkyl ferrocene centers, was obtained by ECS and used in the Langmuir adsorption model to obtain the affinity constant (1.6±0.6)×10(8) L mol(-1). The results shown herein suggest the feasibility of ECS application for lectin glycoarray characterization. PMID:24994505

  3. Impedance-derived electrochemical capacitance spectroscopy for the evaluation of lectin-glycoprotein binding affinity.

    PubMed

    Santos, Adriano; Carvalho, Fernanda C; Roque-Barreira, Maria-Cristina; Bueno, Paulo R

    2014-12-15

    Characterization of lectin-carbohydrate binding using label-free methods such as impedance-derived electrochemical capacitance spectroscopy (ECS) is desirable to evaluate specific interactions, for example, ArtinM lectin and horseradish peroxidase (HRP) glycoprotein, used here as a model for protein-carbohydrate binding affinity. An electroactive molecular film comprising alkyl ferrocene as a redox probe and ArtinM as a carbohydrate receptive center to target HRP was successfully used to determine the binding affinity between ArtinM and HRP. The redox capacitance, a transducer signal associated with the alkyl ferrocene centers, was obtained by ECS and used in the Langmuir adsorption model to obtain the affinity constant (1.6±0.6)×10(8) L mol(-1). The results shown herein suggest the feasibility of ECS application for lectin glycoarray characterization.

  4. Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation.

    PubMed

    Zielonka, Stefan; Weber, Niklas; Becker, Stefan; Doerner, Achim; Christmann, Andreas; Christmann, Christine; Uth, Christina; Fritz, Janine; Schäfer, Elena; Steinmann, Björn; Empting, Martin; Ockelmann, Pia; Lierz, Michael; Kolmar, Harald

    2014-12-10

    A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold.

  5. Affinity regression predicts the recognition code of nucleic acid binding proteins

    PubMed Central

    Pelossof, Raphael; Singh, Irtisha; Yang, Julie L.; Weirauch, Matthew T.; Hughes, Timothy R.; Leslie, Christina S.

    2016-01-01

    Predicting the affinity profiles of nucleic acid-binding proteins directly from the protein sequence is a major unsolved problem. We present a statistical approach for learning the recognition code of a family of transcription factors (TFs) or RNA-binding proteins (RBPs) from high-throughput binding assays. Our method, called affinity regression, trains on protein binding microarray (PBM) or RNA compete experiments to learn an interaction model between proteins and nucleic acids, using only protein domain and probe sequences as inputs. By training on mouse homeodomain PBM profiles, our model correctly identifies residues that confer DNA-binding specificity and accurately predicts binding motifs for an independent set of divergent homeodomains. Similarly, learning from RNA compete profiles for diverse RBPs, our model can predict the binding affinities of held-out proteins and identify key RNA-binding residues. More broadly, we envision applying our method to model and predict biological interactions in any setting where there is a high-throughput ‘affinity’ readout. PMID:26571099

  6. High affinity binding of (/sup 3/H)neurotensin of rat uterus

    SciTech Connect

    Pettibone, D.J.; Totaro, J.A.

    1987-11-01

    (/sup 3/H)Neurotensin (NT) was found to bind specifically and with high affinity to crude membranes prepared from rat uterus. Scatchard analysis of saturation binding studies indicated that (/sup 3/H)NT apparently binds to two sites (high affinity Kd 0.5 nM; low affinity Kd 9 nM) with the density of high affinity sites (41 fmoles/mg prot.) being about one-third that of the low affinity sites (100 fmoles/mg prot.). In competition studies, NT and various fragments inhibited (/sup 3/H)NT binding with the following potencies (approximately IC50): NT 8-13 (0.4 nM), NT 1-13 (4 nM), NT 9-13 (130 nM), NT 1-11, NT 1-8 (greater than 100 microM). Quantitatively similar results were obtained using brain tissue. These findings raise the possibility of a role for NT in uterine function.

  7. On the binding affinity of macromolecular interactions: daring to ask why proteins interact

    PubMed Central

    Kastritis, Panagiotis L.; Bonvin, Alexandre M. J. J.

    2013-01-01

    Interactions between proteins are orchestrated in a precise and time-dependent manner, underlying cellular function. The binding affinity, defined as the strength of these interactions, is translated into physico-chemical terms in the dissociation constant (Kd), the latter being an experimental measure that determines whether an interaction will be formed in solution or not. Predicting binding affinity from structural models has been a matter of active research for more than 40 years because of its fundamental role in drug development. However, all available approaches are incapable of predicting the binding affinity of protein–protein complexes from coordinates alone. Here, we examine both theoretical and experimental limitations that complicate the derivation of structure–affinity relationships. Most work so far has concentrated on binary interactions. Systems of increased complexity are far from being understood. The main physico-chemical measure that relates to binding affinity is the buried surface area, but it does not hold for flexible complexes. For the latter, there must be a significant entropic contribution that will have to be approximated in the future. We foresee that any theoretical modelling of these interactions will have to follow an integrative approach considering the biology, chemistry and physics that underlie protein–protein recognition. PMID:23235262

  8. SELMAP - SELEX affinity landscape MAPping of transcription factor binding sites using integrated microfluidics

    PubMed Central

    Chen, Dana; Orenstein, Yaron; Golodnitsky, Rada; Pellach, Michal; Avrahami, Dorit; Wachtel, Chaim; Ovadia-Shochat, Avital; Shir-Shapira, Hila; Kedmi, Adi; Juven-Gershon, Tamar; Shamir, Ron; Gerber, Doron

    2016-01-01

    Transcription factors (TFs) alter gene expression in response to changes in the environment through sequence-specific interactions with the DNA. These interactions are best portrayed as a landscape of TF binding affinities. Current methods to study sequence-specific binding preferences suffer from limited dynamic range, sequence bias, lack of specificity and limited throughput. We have developed a microfluidic-based device for SELEX Affinity Landscape MAPping (SELMAP) of TF binding, which allows high-throughput measurement of 16 proteins in parallel. We used it to measure the relative affinities of Pho4, AtERF2 and Btd full-length proteins to millions of different DNA binding sites, and detected both high and low-affinity interactions in equilibrium conditions, generating a comprehensive landscape of the relative TF affinities to all possible DNA 6-mers, and even DNA10-mers with increased sequencing depth. Low quantities of both the TFs and DNA oligomers were sufficient for obtaining high-quality results, significantly reducing experimental costs. SELMAP allows in-depth screening of hundreds of TFs, and provides a means for better understanding of the regulatory processes that govern gene expression. PMID:27628341

  9. SELMAP - SELEX affinity landscape MAPping of transcription factor binding sites using integrated microfluidics.

    PubMed

    Chen, Dana; Orenstein, Yaron; Golodnitsky, Rada; Pellach, Michal; Avrahami, Dorit; Wachtel, Chaim; Ovadia-Shochat, Avital; Shir-Shapira, Hila; Kedmi, Adi; Juven-Gershon, Tamar; Shamir, Ron; Gerber, Doron

    2016-01-01

    Transcription factors (TFs) alter gene expression in response to changes in the environment through sequence-specific interactions with the DNA. These interactions are best portrayed as a landscape of TF binding affinities. Current methods to study sequence-specific binding preferences suffer from limited dynamic range, sequence bias, lack of specificity and limited throughput. We have developed a microfluidic-based device for SELEX Affinity Landscape MAPping (SELMAP) of TF binding, which allows high-throughput measurement of 16 proteins in parallel. We used it to measure the relative affinities of Pho4, AtERF2 and Btd full-length proteins to millions of different DNA binding sites, and detected both high and low-affinity interactions in equilibrium conditions, generating a comprehensive landscape of the relative TF affinities to all possible DNA 6-mers, and even DNA10-mers with increased sequencing depth. Low quantities of both the TFs and DNA oligomers were sufficient for obtaining high-quality results, significantly reducing experimental costs. SELMAP allows in-depth screening of hundreds of TFs, and provides a means for better understanding of the regulatory processes that govern gene expression. PMID:27628341

  10. Single-experiment displacement assay for quantifying high-affinity binding by isothermal titration calorimetry.

    PubMed

    Krainer, Georg; Keller, Sandro

    2015-04-01

    Isothermal titration calorimetry (ITC) is the gold standard for dissecting the thermodynamics of a biomolecular binding process within a single experiment. However, reliable determination of the dissociation constant (KD) from a single titration is typically limited to the range 100 μM>KD>1 nM. Interactions characterized by a lower KD can be assessed indirectly by so-called competition or displacement assays, provided that a suitable competitive ligand is available whose KD falls within the directly accessible window. However, this protocol is limited by the fact that it necessitates at least two titrations to characterize one high-affinity inhibitor, resulting in considerable consumption of both sample material and time. Here, we introduce a fast and efficient ITC displacement assay that allows for the simultaneous characterization of both a high-affinity ligand and a moderate-affinity ligand competing for the same binding site on a receptor within a single experiment. The protocol is based on a titration of the high-affinity ligand into a solution containing the moderate-affinity ligand bound to the receptor present in excess. The resulting biphasic binding isotherm enables accurate and precise determination of KD values and binding enthalpies (ΔH) of both ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation, explore its potential and limitations with the aid of simulations and statistical analyses, and elaborate on potential applications to protein-inhibitor interactions.

  11. Multisite phosphorylation of the NDC80 complex gradually tunes its microtubule-binding affinity

    PubMed Central

    Zaytsev, Anatoly V.; Mick, Jeanne E.; Maslennikov, Evgeny; Nikashin, Boris; DeLuca, Jennifer G.; Grishchuk, Ekaterina L.

    2015-01-01

    Microtubule (MT) attachment to kinetochores is vitally important for cell division, but how these interactions are controlled by phosphorylation is not well known. We used quantitative approaches in vitro combined with molecular dynamics simulations to examine phosphoregulation of the NDC80 complex, a core kinetochore component. We show that the outputs from multiple phosphorylation events on the unstructured tail of its Hec1 subunit are additively integrated to elicit gradual tuning of NDC80-MT binding both in vitro and in silico. Conformational plasticity of the Hec1 tail enables it to serve as a phosphorylation-controlled rheostat, providing a new paradigm for regulating the affinity of MT binders. We also show that cooperativity of NDC80 interactions is weak and is unaffected by NDC80 phosphorylation. This in vitro finding strongly supports our model that independent molecular binding events to MTs by individual NDC80 complexes, rather than their structured oligomers, regulate the dynamics and stability of kinetochore-MT attachments in dividing cells. PMID:25808492

  12. Characterization of a small acyl-CoA-binding protein (ACBP) from Helianthus annuus L. and its binding affinities.

    PubMed

    Aznar-Moreno, Jose A; Venegas-Calerón, Mónica; Du, Zhi-Yan; Garcés, Rafael; Tanner, Julian A; Chye, Mee-Len; Martínez-Force, Enrique; Salas, Joaquín J

    2016-05-01

    Acyl-CoA-binding proteins (ACBPs) bind to acyl-CoA esters and promote their interaction with other proteins, lipids and cell structures. Small class I ACBPs have been identified in different plants, such as Arabidopsis thaliana (AtACBP6), Brassica napus (BnACBP) and Oryza sativa (OsACBP1, OsACBP2, OsACBP3), and they are capable of binding to different acyl-CoA esters and phospholipids. Here we characterize HaACBP6, a class I ACBP expressed in sunflower (Helianthus annuus) tissues, studying the specificity of its corresponding recombinant HaACBP6 protein towards various acyl-CoA esters and phospholipids in vitro, particularly using isothermal titration calorimetry and protein phospholipid binding assays. This protein binds with high affinity to de novo synthetized derivatives palmitoly-CoA, stearoyl-CoA and oleoyl-CoA (Kd 0.29, 0.14 and 0.15 μM respectively). On the contrary, it showed lower affinity towards linoleoyl-CoA (Kd 5.6 μM). Moreover, rHaACBP6 binds to different phosphatidylcholine species (dipalmitoyl-PC, dioleoyl-PC and dilinoleoyl-PC), yet it displays no affinity towards other phospholipids like lyso-PC, phosphatidic acid and lysophosphatidic acid derivatives. In the light of these results, the possible involvement of this protein in sunflower oil synthesis is considered. PMID:26938582

  13. CORAL: prediction of binding affinity and efficacy of thyroid hormone receptor ligands.

    PubMed

    Toropova, A P; Toropov, A A; Benfenati, E

    2015-08-28

    Quantitative structure - activity relationships (QSARs) for binding affinity of thyroid hormone receptors based on attributes of molecular structure extracted from simplified molecular input-line entry systems (SMILES) are established using the CORAL software (http://www.insilico.eu/coral). The half maximal inhibitory concentration (IC50) is used as the measure of the binding affinity of thyroid hormone receptors. Molecular features which are statistically reliable promoters of increase and decrease for IC50 are suggested. The examples of modifications of molecular structure which lead to the increase or to the decrease of the endpoint are represented. PMID:26188619

  14. Comparison of Relative Binding Affinities for Trout and Human Estrogen Receptor Based upon Different Competitive Binding Assays

    EPA Science Inventory

    The development of a predictive model based upon a single aquatic species inevitably raises the question of whether this information is valid for other species. To partially address this question, relative binding affinities (RBA) for six alkylphenols (para-substituted, n- and b...

  15. Interactions of dopaminergic agonists and antagonists with dopaminergic D3 binding sites in rat striatum. Evidence that (/sup 3/H)dopamine can label a high affinity agonist-binding state of the D1 dopamine receptor

    SciTech Connect

    Leff, S.E.; Creese, I.

    1985-02-01

    The interactions of dopaminergic agonists and antagonists with /sup 3/H-agonist labeled D3 dopaminergic binding sites of rat striatum have been characterized by radioligand-binding techniques. When the binding of (/sup 3/H)dopamine and (/sup 3/H)apomorphine to D2 dopamine receptors is blocked by the inclusion of D2 selective concentrations of unlabeled spiroperidol or domperidone, these ligands appear to label selectively the previously termed D3 binding site. Antagonist/(/sup 3/H)dopamine competition curves are of uniformly steep slope (nH . 1.0), suggesting the presence of a single D3 binding site. The relative potencies of antagonists to inhibit D3 specific (/sup 3/H)dopamine binding are significantly correlated with their potencies to block D1 dopamine receptors as measured by the inhibition of both dopamine-stimulated adenylate cyclase and (/sup 3/H)flupentixol-binding activities. The affinities of agonists to inhibit D3 specific (/sup 3/H)dopamine binding are also correlated with estimates of these agonists affinities for the high affinity binding component of agonist/(/sup 3/H)flupentixol competition curves. Both D3 specific (/sup 3/H) dopamine binding and the high affinity agonist-binding component of dopamine/(/sup 3/H)flupentixol competition curves show a similar sensitivity to guanine nucleotides. Taken together, these data strongly suggest that the D3 binding site is related to a high affinity agonist-binding state of the D1 dopamine receptor.

  16. Binding affinities and thermodynamics of noncovalent functionalization of carbon nanotubes with surfactants.

    PubMed

    Oh, Hyunkyu; Sim, Jinsook; Ju, Sang-Yong

    2013-09-01

    Binding affinity and thermodynamic understanding between a surfactant and carbon nanotube is essential to develop various carbon nanotube applications. Flavin mononucleotide-wrapped carbon nanotubes showing a large redshift in optical signature were utilized to determine the binding affinity and related thermodynamic parameters of 12 different nanotube chiralities upon exchange with other surfactants. Determined from the midpoint of sigmoidal transition, the equilibrium constant (K), which is inversely proportional to the binding affinity of the initial surfactant-carbon nanotube, provided quantitative binding strengths of surfactants as SDBS > SC ≈ FMN > SDS, irrespective of electronic types of SWNTs. Binding affinity of metallic tubes is weaker than that of semiconducting tubes. The complex K patterns from semiconducting tubes show preference to certain SWNT chiralities and surfactant-specific cooperativity according to nanotube chirality. Controlling temperature was effective to modulate K values by 30% and enables us to probe thermodynamic parameters. Equally signed enthalpy and entropy changes produce Gibbs energy changes with a magnitude of a few kJ/mol. A greater negative Gibbs energy upon exchange of surfactant produces an enhanced nanotube photoluminescence, implying the importance of understanding thermodynamics for designing nanotube separation and supramolecular assembly of surfactant.

  17. QSAR modeling of globulin binding affinity of corticosteroids using AM1 calculations.

    PubMed

    De, Kakali; Sengupta, Chandana; Roy, Kunal

    2004-06-15

    A quantitative structure-activity analysis of binding affinity of a series of 30 steroids for corticosteroid-binding globulin was performed using Wang-Ford charges of the non-hydrogen common atoms obtained from molecular electrostatic potential surface of AM1 optimized energy-minimized geometries of the compounds. Attempts were made to include lipophilicity (logP) and molar refractivity (MR) values of the whole molecules in the multivariate relations. The final relations were subjected to 'leave-one-out' cross-validation to check their predictive potential. It was found from the study that the charges of different atoms of the steroid nucleus [atoms 3, 4, 5 (ring A), 8, 9 (fusion points of rings B and C) and 16 (ring D)] contribute significantly to the binding affinity. This suggests the importance of these atoms/sites for the globulin binding affinity, which is also supported by previous reports on structure-activity relations of corticosteroids. Further, molar refractivity shows parabolic relation with the binding affinity, which indicates the possibility of dispersion interactions. The statistical qualities of the final equations generated in the present study (predicted variance 77-82%; explained variance 83-87%) are better than those of some of the previously reported models.

  18. Enhanced antigen-antibody binding affinity mediated by an anti-idiotypic antibody

    SciTech Connect

    Sawutz, D.G.; Koury, R.; Homcy, C.J.

    1987-08-25

    The authors previously described the production of four monoclonal antibodies to the ..beta..-adrenergic receptor antagonist alprenolol. One of these antibodies, 5B7 (IgG/sub 2a/, kappa), was used to raise anti-idiotypic antisera in rabbits. In contrast to the expected results, one of the anti-idiotypic antisera (R9) promotes (/sup 125/I)iodocyanopinodolol (ICYP) binding to antibody 5B7. In the presence of R9, the dissociation constant decreases 100-fold from 20 to 0.3 nM. This increase in binding affinity of antibody 5B7 for ICYP is not observed in the presence of preimmune, rabbit anti-mouse or anti-idiotypic antisera generated to a monoclonal antibody of a different specificity. Furthermore, R9 in the absence of 5B7 does not bind ICYP. The F(ab) fragments of 5B7 and T9 behaved in a similar manner, and the soluble complex responsible for the high-affinity interaction with ICYP can be identified by gel filtration chromatography. The elution position of the complex is consistent with a 5B7 F(ab)-R9 F(ab) dimer, indicating that polyvalency is not responsible for the enhanced ligand binding. Kinetic analysis of ICYP-5B7 binding revealed that the rate of ICYP dissociation from 5B7 in the presence of R9 is approximately 100 times slower than in the absence of R9, consistent with the 100-fold change in binding affinity of 5B7 for ICYP. The available data best fit a model in which an anti-idiotypic antibody binds at or near the binding site of the idiotype participating in the formation of a hybrid ligand binding site. This would allow increased contact of the ligand with the idiotype-anti-idiotype complex and result in an enhanced affinity of the ligand interaction.

  19. Reconstitution of high-affinity opioid agonist binding in brain membranes

    SciTech Connect

    Remmers, A.E.; Medzihradsky, F. )

    1991-03-15

    In synaptosomal membranes from rat brain cortex, the {mu} selective agonist ({sup 3}H)dihydromorphine in the absence of sodium, and the nonselective antagonist ({sup 3}H)naltrexone in the presence of sodium, bound to two populations of opioid receptor sites with K{sub d} values of 0.69 and 8.7 nM for dihydromorphine, and 0.34 and 5.5 nM for naltrexone. The addition of 5 {mu}M guanosine 5{prime}-({gamma}-thio)triphosphate (GTP({gamma}S)) strongly reduced high-affinity agonist but not antagonist binding. Exposure of the membranes to high pH reduced the number of GTP({gamma}-{sup 35}S) binding sites by 90% and low K{sub m}, opioid-sensitive GTPase activity by 95%. In these membranes, high-affinity agonist binding was abolished and modulation of residual binding by GTP({gamma}S) was diminished. Alkali treatment of the glioma cell membranes prior to fusion inhibited most of the low K{sub m} GTPase activity and prevented the reconstitution of agonist binding. The results show that high-affinity opioid agonist binding reflects the ligand-occupied receptor - guanine nucleotide binding protein complex.

  20. Quantification of the Effects of Ionic Strength, Viscosity, and Hydrophobicity on Protein–Ligand Binding Affinity

    PubMed Central

    2014-01-01

    In order to quantify the interactions between molecules of biological interest, the determination of the dissociation constant (Kd) is essential. Estimation of the binding affinity in this way is routinely performed in “favorable” conditions for macromolecules. Crucial data for ligand–protein binding elucidation is mainly derived from techniques (e.g., macromolecular crystallography) that require the addition of high concentration of salts and/or other additives. In this study we have evaluated the effect of temperature, ionic strength, viscosity, and hydrophobicity on the Kd of three previously characterized protein–ligand systems, based on variation in their binding sites, in order to provide insight into how these often overlooked unconventional circumstances impact binding affinity. Our conclusions are as follows: (1) increasing solvent viscosity in general is detrimental to ligand binding, (2) moderate increases in temperature have marginal effects on the dissociation constant, and (3) the degree of hydrophobicity of the ligand and the binding site determines the extent of the influence of cosolvents and salt concentration on ligand binding affinity. PMID:25147617

  1. Oligomerization of Peptides LVEALYL and RGFFYT and Their Binding Affinity to Insulin

    PubMed Central

    Chiang, Hsin-Lin; Ngo, Son Tung; Chen, Chun-Jung; Hu, Chin-Kun; Li, Mai Suan

    2013-01-01

    Recently it has been proposed a model for fibrils of human insulin in which the fibril growth proceeds via stacking LVEALYL (fragment 11–17 from chain B of insulin) into pairs of tightly interdigitated -sheets. The experiments have also shown that LVEALYL has high propensity to self-assembly and binding to insulin. This necessitates study of oligomerization of LVEALYL and its binding affinity to full-length insulin. Using the all-atom simulations with Gromos96 43a1 force field and explicit water it is shown that LVEALYL can aggregate. Theoretical estimation of the binding free energy of LVEALYL to insulin by the molecular mechanic Poisson-Boltzmann surface area method reveals its strong binding affinity to chain B, implying that, in agreement with the experiments, LVEALYL can affect insulin aggregation via binding mechanism. We predict that, similar to LVEALYL, peptide RGFFYT (fragment B22-27) can self-assemble and bind to insulin modulating its fibril growth process. The binding affinity of RGFFYT is shown to be comparable with that of LVEALYL. PMID:23805182

  2. Protein-ligand binding affinity by nonequilibrium free energy methods.

    PubMed

    Cossins, Benjamin P; Foucher, Sebastien; Edge, Colin M; Essex, Jonathan W

    2008-11-27

    Nonequilibrium (NE) free energy methods are embarrassingly parallel and may be very conveniently run on desktop computers using distributed computing software. In recent years there has been a proliferation of NE methods, but these approaches have barely, if at all, been used in the context of calculating protein-ligand binding free energies. In a recent study by these authors, different combinations of NE methods with various test systems were compared and protocols identified which yielded results as accurate as replica exchange thermodynamic integration (RETI). The NE approaches, however, lend themselves to extensive parallelization through the use of distributed computing. Here the best performing of those NE protocols, a replica exchange method using Bennett's acceptance ratio as the free energy estimator (RENE), is applied to two sets of congeneric inhibitors bound to neuraminidase and cyclooxygenase-2. These protein-ligand systems were originally studied with RETI, giving results to which NE and RENE simulations are compared. These NE calculations were carried out on a large, highly distributed group of low-performance desktop computers which are part of a Condor pool. RENE was found to produce results of a predictive quality at least as good as RETI in less than half the wall clock time. However, non-RE NE results were found to be far less predictive. In addition, the RENE method successfully identified a localized region of rapidly changing free energy gradients without the need for prior investigation. These results suggest that the RENE protocol is appropriate for use in the context of predicting protein-ligand binding free energies and that it can offer advantages over conventional, equilibrium approaches. PMID:18973369

  3. Binding affinities of anti-acetylcholine receptor autoantibodies in myasthenia gravis

    SciTech Connect

    Bray, J.J.; Drachman, D.B.

    1982-01-01

    Antibodies directed against acetylcholine (ACh) receptors are present in the sera of nearly 90% of patients with myasthenia gravis (MG), and are involved in the pathogenesis of this autoimmune disease. However, the antibody titers measured by the standard radioimmunoassay correspond poorly with the clinical severity of the disease. To determine whether this disparity could be accounted for by differences in the binding affinities of anti-ACh receptor antibodies in different patients, we have measured the binding affinities of these autoantibodies in 15 sera from MG patients. The affinity constants (K/sub o/), as determined by Scatchard analysis, were all in the range of 10/sup 10/ M/sup -1/, comparable to the highest values reported in immunized animals. The affinity constants were truly representative of the population of autoantibodies detected by the radioimmunoassay, as shown by the remarkable linearity of the Scatchard plots (r/sup 2/>0.90) and the close correlation between the antibody titers determined by extrapolation of the Scatchard plots and by saturation analysis (r = 0.99; p < 0.001). There was only a 6-fold variation in affinity constants measured in this series of patients despite widely differing antibody titers and severity of the disease. Factors other than the titer and affinity of anti-ACh receptor antibodies may correlate better with the clinical manifestations of MG.

  4. Interplay between binding affinity and kinetics in protein-protein interactions.

    PubMed

    Cao, Huaiqing; Huang, Yongqi; Liu, Zhirong

    2016-07-01

    To clarify the interplay between the binding affinity and kinetics of protein-protein interactions, and the possible role of intrinsically disordered proteins in such interactions, molecular simulations were carried out on 20 protein complexes. With bias potential and reweighting techniques, the free energy profiles were obtained under physiological affinities, which showed that the bound-state valley is deep with a barrier height of 12 - 33 RT. From the dependence of the affinity on interface interactions, the entropic contribution to the binding affinity is approximated to be proportional to the interface area. The extracted dissociation rates based on the Arrhenius law correlate reasonably well with the experimental values (Pearson correlation coefficient R = 0.79). For each protein complex, a linear free energy relationship between binding affinity and the dissociation rate was confirmed, but the distribution of the slopes for intrinsically disordered proteins showed no essential difference with that observed for ordered proteins. A comparison with protein folding was also performed. Proteins 2016; 84:920-933. © 2016 Wiley Periodicals, Inc. PMID:27018856

  5. Interaction of nicotinic receptor affinity reagents with central nervous system. cap alpha. -bungarotoxin-binding entities

    SciTech Connect

    Lukas, R.J.; Bennett, E.L.

    1980-01-01

    Membrane-bound ..cap alpha..-bungarotoxin-binding entities derived from rat brain are found to interact specifically with the affinity reagents maleimidobenzyltrimethylammonium (MBTA) and bromoacetylcholine (BAC), originally designed to label nicotinic acetylcholine receptors from electroplax and skeletal muscle. Following treatment of membranes with dithiothreitol, all specific toxin binding sites are irreversibly blocked by reaction with MBTA or BAC. Affinity reagent labeling of dithiothreitol-reduced membranes is prevented (toxin binding sites are not blocked) by prior alkylaction with N-ethylmaleimide, by prior oxidation with dithiobis(2-nitrobenzoic acid), or by incubation with neurotoxin. Reversibly associating cholinergic agonists and antagonists retard the rate of affinity reagent interaction with toxin receptors. The apparent rates of affinity reagent alkylation of toxin receptors, and the influences of other sulfhydryl/disulfide reagents on affinity labeling are comparable to those observed for reaction with nicotinic acetylcholine receptors in the periphery. The results provide further evidence that central nervous system ..cap alpha..-bungarotoxin receptors share a remarkable number of biochemical properties with nicotinic receptors from the periphery.

  6. Interplay between binding affinity and kinetics in protein-protein interactions.

    PubMed

    Cao, Huaiqing; Huang, Yongqi; Liu, Zhirong

    2016-07-01

    To clarify the interplay between the binding affinity and kinetics of protein-protein interactions, and the possible role of intrinsically disordered proteins in such interactions, molecular simulations were carried out on 20 protein complexes. With bias potential and reweighting techniques, the free energy profiles were obtained under physiological affinities, which showed that the bound-state valley is deep with a barrier height of 12 - 33 RT. From the dependence of the affinity on interface interactions, the entropic contribution to the binding affinity is approximated to be proportional to the interface area. The extracted dissociation rates based on the Arrhenius law correlate reasonably well with the experimental values (Pearson correlation coefficient R = 0.79). For each protein complex, a linear free energy relationship between binding affinity and the dissociation rate was confirmed, but the distribution of the slopes for intrinsically disordered proteins showed no essential difference with that observed for ordered proteins. A comparison with protein folding was also performed. Proteins 2016; 84:920-933. © 2016 Wiley Periodicals, Inc.

  7. The statistical-thermodynamic basis for computation of binding affinities: a critical review.

    PubMed Central

    Gilson, M K; Given, J A; Bush, B L; McCammon, J A

    1997-01-01

    Although the statistical thermodynamics of noncovalent binding has been considered in a number of theoretical papers, few methods of computing binding affinities are derived explicitly from this underlying theory. This has contributed to uncertainty and controversy in certain areas. This article therefore reviews and extends the connections of some important computational methods with the underlying statistical thermodynamics. A derivation of the standard free energy of binding forms the basis of this review. This derivation should be useful in formulating novel computational methods for predicting binding affinities. It also permits several important points to be established. For example, it is found that the double-annihilation method of computing binding energy does not yield the standard free energy of binding, but can be modified to yield this quantity. The derivation also makes it possible to define clearly the changes in translational, rotational, configurational, and solvent entropy upon binding. It is argued that molecular mass has a negligible effect upon the standard free energy of binding for biomolecular systems, and that the cratic entropy defined by Gurney is not a useful concept. In addition, the use of continuum models of the solvent in binding calculations is reviewed, and a formalism is presented for incorporating a limited number of solvent molecules explicitly. PMID:9138555

  8. The serotonin transporter: Examination of the changes in transporter affinity induced by ligand binding

    SciTech Connect

    Humphreys, C.J.

    1989-01-01

    The plasmalemmal serotonin transporter uses transmembrane gradients of Na{sup +}, Cl{sup {minus}} and K{sup +} to accumulate serotonin within blood platelets. Transport is competitively inhibited by the antidepressant imipramine. Like serotonin transport, imipramine binding requires Na{sup +}. Unlike serotonin, however, imipramine does not appear to be transported. To gain insight into the mechanism of serotonin transport the author have analyzed the influences of Na{sup +} and Cl{sup {minus}}, the two ions cotransported with serotonin, on both serotonin transport and the interaction of imipramine and other antidepressant drugs with the plasmalemmal serotonin transporter of human platelets. Additionally, the author have synthesized, purified and characterized the binding of 2-iodoimipramine to the serotonin transporter. Finally, the author have conducted a preliminary study of the inhibition of serotonin transport and imipramine binding produced by dicyclohexylcarbodiimide. My results reveal many instances of positive heterotropic cooperativity in ligand binding to the serotonin transporter. Na{sup +} binding enhances the transporters affinity for imipramine and several other antidepressant drugs, and also increases the affinity for Cl{sup {minus}}. Cl{sup {minus}} enhances the transporters affinity for imipramine, as well as for Na{sup +}. At concentrations in the range of its K{sub M} for transport serotonin is a competitive inhibitor of imipramine binding. At much higher concentrations, however, serotonin also inhibits imipramines dissociation rate constant. This latter effect which is Na{sup +}-independent and species specific, is apparently produced by serotonin binding at a second, low affinity site on, or near, the transporter complex. Iodoimipramine competitively inhibit both ({sup 3}H)imipramine binding and ({sup 3}H)serotonin transport.

  9. Mannosylerythritol lipid, a yeast extracellular glycolipid, shows high binding affinity towards human immunoglobulin G

    PubMed Central

    Im, Jae Hong; Nakane, Takashi; Yanagishita, Hiroshi; Ikegami, Toru; Kitamoto, Dai

    2001-01-01

    Background There have been many attempts to develop new materials with stability and high affinity towards immunoglobulins. Some of glycolipids such as gangliosides exhibit a high affinity toward immunoglobulins. However, it is considerably difficult to develop these glycolipids into the practical separation ligand due to their limited amounts. We thus focused our attention on the feasible use of "mannosylerythritol lipid A", a yeast glycolipid biosurfactant, as an alternative ligand for immunoglobulins, and undertook the investigation on the binding between mannosylerythritol lipid A (MEL-A) and human immunoglobulin G (HIgG). Results In ELISA assay, MEL-A showed nearly the same binding affinity towards HIgG as that of bovine ganglioside GM1. Fab of human IgG was considered to play a more important role than Fc in the binding of HIgG by MEL-A. The bound amount of HIgG increased depending on the attached amount of MEL-A onto poly (2-hydroxyethyl methacrylate) (polyHEMA) beads, whereas the amount of human serum albumin slightly decreased. Binding-amount and -selectivity of HIgG towards MEL-A were influenced by salt species, salt concentration and pH in the buffer solution. The composite of MEL-A and polyHEMA, exhibited a significant binding constant of 1.43 × 106 (M-1) for HIgG, which is approximately 4-fold greater than that of protein A reported. Conclusions MEL-A shows high binding-affinity towards HIgG, and this is considered to be due to "multivalent effect" based on the binding molar ratio. This is the first report on the binding of a natural human antibody towards a yeast glycolipid. PMID:11604104

  10. Computational estimation of rainbow trout estrogen receptor binding affinities for environmental estrogens

    SciTech Connect

    Shyu, Conrad; Cavileer, Timothy D.; Nagler, James J.; Ytreberg, F. Marty

    2011-02-01

    Environmental estrogens have been the subject of intense research due to their documented detrimental effects on the health of fish and wildlife and their potential to negatively impact humans. A complete understanding of how these compounds affect health is complicated because environmental estrogens are a structurally heterogeneous group of compounds. In this work, computational molecular dynamics simulations were utilized to predict the binding affinity of different compounds using rainbow trout (Oncorhynchus mykiss) estrogen receptors (ERs) as a model. Specifically, this study presents a comparison of the binding affinity of the natural ligand estradiol-17{beta} to the four rainbow trout ER isoforms with that of three known environmental estrogens 17{alpha}-ethinylestradiol, bisphenol A, and raloxifene. Two additional compounds, atrazine and testosterone, that are known to be very weak or non-binders to ERs were tested. The binding affinity of these compounds to the human ER{alpha} subtype is also included for comparison. The results of this study suggest that, when compared to estradiol-17{beta}, bisphenol A binds less strongly to all four receptors, 17{alpha}-ethinylestradiol binds more strongly, and raloxifene has a high affinity for the {alpha} subtype only. The results also show that atrazine and testosterone are weak or non-binders to the ERs. All of the results are in excellent qualitative agreement with the known in vivo estrogenicity of these compounds in the rainbow trout and other fishes. Computational estimation of binding affinities could be a valuable tool for predicting the impact of environmental estrogens in fish and other animals.

  11. Routes to improve binding capacities of affinity resins demonstrated for Protein A chromatography.

    PubMed

    Müller, Egbert; Vajda, Judith

    2016-05-15

    Protein A chromatography is a well-established platform in downstream purification of monoclonal antibodies. Dynamic binding capacities are continuously increasing with almost every newly launched Protein A resin. Nevertheless, binding capacities of affinity chromatography resins cannot compete with binding capacities obtained with modern ion exchange media. Capacities of affinity resins are roughly 50% lower. High binding capacities of ion exchange media are supported by spacer technologies. In this article, we review existing spacer technologies of affinity chromatography resins. A yet known effective approach to increase the dynamic binding capacity of Protein A resins is oligomerization of the particular Protein A motifs. This resembles the tentacle technology used in ion exchange chromatography. Dynamic binding capacities of a hexameric ligand are roughly twice as high compared to capacities obtained with a tetrameric ligand. Further capacity increases up to 130mg/ml can be realized with the hexamer ligand, if the sodium phosphate buffer concentration is increased from 20 to 100mM. Equilibrium isotherms revealed a BET shape for the hexamer ligand at monoclonal antibody liquid phase concentrations higher than 9mg/ml. The apparent multilayer formation may be due to hydrophobic forces. Other quality attributes such as recovery, aggregate content, and overall purity of the captured monoclonal antibody are not affected. PMID:26830536

  12. Soybean. beta. -glucan binding sites display maximal affinity for a heptaglucoside phytoalexin-elicitor

    SciTech Connect

    Cosio, E.G.; Waldmueller, T.; Frey, T.; Ebel, J. )

    1990-05-01

    The affinity of soybean {beta}-glucan-binding sites for a synthetic heptaglucan elicitor was tested in a ligand-competition assay against a {sup 125}I-labeled 1,3-1,6-{beta}-glucan preparation (avg. DP=20). Half-maximal displacement of label (IC{sub 50}) was obtained at 9nM heptaglucan, the highest affinity of all fractions tested to date. Displacement followed a uniform sigmoidal pattern and was complete at 1{mu}M indicating access of heptaglucan to all sites available to the labeled elicitor. A mathematical model was used to predict IC{sub 50} values according to the DP of glucan fragments obtained from fungal cell walls. The lowest IC{sub 50} predicted by this model is 3nM. Binding affinity of the glucans was compared with their elicitor activity in a bioassay.

  13. Estimation of Ligand-Receptor Binding Affinity from Fluctuation of Their Interface

    NASA Astrophysics Data System (ADS)

    Iwamoto, Koji; Ode, Hirotaka; Ohta, Masami; Misu, Takashi; Hata, Masayuki; Neya, Saburo; Hoshino, Tyuji

    2005-10-01

    It is necessary for the understanding of protein interactions or in silico drug designs to accurately estimate ligand-receptor affinity. The energy calculation based on the electrostatic force, van der Waals force, and solvation effect is a direct method of computing the magnitude of the interaction between ligand and receptor. By this conventional method, however, it is difficult to estimate a slight difference in binding affinity with sufficient accuracy. We propose a novel concept for the evaluation of binding affinity between a ligand and its receptor by functionalizing the fluctuation at the ligand-receptor interface. This method enables an adequate estimation with a high accuracy compared with the conventional energetic approach. Human immunodeficiency virus type 1 (HIV-1) protease and its inhibitor are used to explain how binding affinity is extracted from the fluctuation in interfacial energy, and a combination of an antigen and its antibody is examined to demonstrate the compatibility between the estimation from the interfacial fluctuation and the experimentally measured binding energy.

  14. A strategy to enhance the binding affinity of fluorophore-aptamer pairs for RNA tagging with neomycin conjugation.

    PubMed

    Jeon, Jongho; Lee, Kyung Hyun; Rao, Jianghong

    2012-10-14

    Fluorogenic sulforhodamine-neomycin conjugates have been designed and synthesized for RNA tagging. Conjugates were fluorescently activated by binding to RNA aptamers and exhibited greater than 250-400 fold enhancement in binding affinity relative to corresponding unconjugated fluorophores.

  15. Enhanced DNA binding affinity of RecA protein from Deinococcus radiodurans.

    PubMed

    Warfel, Jaycob D; LiCata, Vince J

    2015-07-01

    Deinococcus radiodurans (Dr) has a significantly more robust DNA repair response than Escherichia coli (Ec), which helps it survive extremely high doses of ionizing radiation and prolonged periods of desiccation. DrRecA protein plays an essential part in this DNA repair capability. In this study we directly compare the binding of DrRecA and EcRecA to the same set of short, defined single (ss) and double stranded (ds) DNA oligomers. In the absence of cofactors (ATPγS or ADP), DrRecA binds to dsDNA oligomers more than 20 fold tighter than EcRecA, and binds ssDNA up to 9 fold tighter. Binding to dsDNA oligomers in the absence of cofactor presumably predominantly monitors DNA end binding, and thus suggests a significantly higher affinity of DrRecA for ds breaks. Upon addition of ATPγS, this species-specific affinity difference is nearly abolished, as ATPγS significantly decreases the affinity of DrRecA for DNA. Other findings include that: (1) both proteins exhibit a dependence of binding affinity on the length of the ssDNA oligomer, but not the dsDNA oligomer; (2) the salt dependence of binding is modest for both species of RecA, and (3) in the absence of DNA, DrRecA produces significantly shorter and/or fewer free-filaments in solution than does EcRecA. The results suggest intrinsic biothermodynamic properties of DrRecA contribute directly to the more robust DNA repair capabilities of D. radiodurans.

  16. Influence of length and flexibility of spacers on the binding affinity of divalent ligands.

    PubMed

    Liese, Susanne; Netz, Roland R

    2015-01-01

    We present a quantitative model for the binding of divalent ligand-receptor systems. We study the influence of length and flexibility of the spacers on the overall binding affinity and derive general rules for the optimal ligand design. To this end, we first compare different polymeric models and determine the probability to simultaneously bind to two neighboring receptor binding pockets. In a second step the binding affinity of divalent ligands in terms of the IC50 value is derived. We find that a divalent ligand has the potential to bind more efficiently than its monovalent counterpart only, if the monovalent dissociation constant is lower than a critical value. This critical monovalent dissociation constant depends on the ligand-spacer length and flexibility as well as on the size of the receptor. Regarding the optimal ligand-spacer length and flexibility, we find that the average spacer length should be equal or slightly smaller than the distance between the receptor binding pockets and that the end-to-end spacer length fluctuations should be in the same range as the size of a receptor binding pocket.

  17. Influence of length and flexibility of spacers on the binding affinity of divalent ligands

    PubMed Central

    Liese, Susanne

    2015-01-01

    Summary We present a quantitative model for the binding of divalent ligand–receptor systems. We study the influence of length and flexibility of the spacers on the overall binding affinity and derive general rules for the optimal ligand design. To this end, we first compare different polymeric models and determine the probability to simultaneously bind to two neighboring receptor binding pockets. In a second step the binding affinity of divalent ligands in terms of the IC50 value is derived. We find that a divalent ligand has the potential to bind more efficiently than its monovalent counterpart only, if the monovalent dissociation constant is lower than a critical value. This critical monovalent dissociation constant depends on the ligand-spacer length and flexibility as well as on the size of the receptor. Regarding the optimal ligand-spacer length and flexibility, we find that the average spacer length should be equal or slightly smaller than the distance between the receptor binding pockets and that the end-to-end spacer length fluctuations should be in the same range as the size of a receptor binding pocket. PMID:26124882

  18. Characterization of a high affinity cocaine binding site in rat brain

    SciTech Connect

    Calligaro, D.; Eldefrawi, M.

    1986-03-05

    Binding of (/sup 3/H)cocaine to synaptic membranes from whole rat brain was reversible and saturable. Nonlinear regression analysis of binding isotherms indicated two binding affinities: one with k/sub d/ = 16 nM, B/sub max/ = 0.65 pmoles/mg protein and the other with K/sub d/ = 660 nM, B/sub max/ = 5.1 pmoles/mg protein. The high-affinity binding of (/sup 3/H)cocaine was sensitive to the actions of trypsin and chymotrypsin but not carboxypeptidase, and was eliminated by exposure of the membranes to 95/sup 0/C for 5 min. Specific binding at 2 nM was higher at pH 8.8 than at pH 7.0. Binding of (/sup 3/H)cocaine (15 nM) was inhibited by increasing concentrations of Na/sup +/ ions. Several cocaine analogues, neurotransmitter uptake inhibitors and local anesthetics displaced specific (/sup 3/H)cocaine binding at 2 nM with various potencies. The cocaine analogue (-)-norcocaine was the most potent (IC/sub 50/ = 10 nM), while the local anesthetic tetracaine was the least potent in inhibiting (/sup 3/H)cocaine binding. Several biogenic amine uptake inhibitors, including tricyclic antidepressants and phencyclidine, had IC/sub 50/ values below ..mu..M concentrations.

  19. DETECTION OF HETEROGENEOUS DRUG-PROTEIN BINDING BY FRONTAL ANALYSIS AND HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Tong, Zenghan; Joseph, K.S.; Hage, David S.

    2011-01-01

    This study examined the use of frontal analysis and high-performance affinity chromatography for detecting heterogeneous binding in biomolecular interactions, using the binding of acetohexamide with human serum albumin (HSA) as a model. It was found through the use of this model system and chromatographic theory that double-reciprocal plots could be used more easily than traditional isotherms for the initial detection of binding site heterogeneity. The deviations from linearity that were seen in double-reciprocal plots as a result of heterogeneity were a function of the analyte concentration, the relative affinities of the binding sites in the system and the amount of each type of site that was present. The size of these deviations was determined and compared under various conditions. Plots were also generated to show what experimental conditions would be needed to observe these deviations for general heterogeneous systems or for cases in which some preliminary information was available on the extent of binding heterogeneity. The methods developed in this work for the detection of binding heterogeneity are not limited to drug interactions with HSA but could be applied to other types of drug-protein binding or to additional biological systems with heterogeneous binding. PMID:21612784

  20. Novel Ubiquitin-derived High Affinity Binding Proteins with Tumor Targeting Properties*

    PubMed Central

    Lorey, Susan; Fiedler, Erik; Kunert, Anja; Nerkamp, Jörg; Lange, Christian; Fiedler, Markus; Bosse-Doenecke, Eva; Meysing, Maren; Gloser, Manja; Rundfeldt, Chris; Rauchhaus, Una; Hänssgen, Ilka; Göttler, Thomas; Steuernagel, Arnd; Fiedler, Ulrike; Haupts, Ulrich

    2014-01-01

    Targeting effector molecules to tumor cells is a promising mode of action for cancer therapy and diagnostics. Binding proteins with high affinity and specificity for a tumor target that carry effector molecules such as toxins, cytokines, or radiolabels to their intended site of action are required for these applications. In order to yield high tumor accumulation while maintaining low levels in healthy tissues and blood, the half-life of such conjugates needs to be in an optimal range. Scaffold-based binding molecules are small proteins with high affinity and short systemic circulation. Due to their low molecular complexity, they are well suited for combination with effector molecules as well as half-life extension technologies yielding therapeutics with half-lives adapted to the specific therapy. We have identified ubiquitin as an ideal scaffold protein due to its outstanding biophysical and biochemical properties. Based on a dimeric ubiquitin library, high affinity and specific binding molecules, so-called Affilin® molecules, have been selected against the extradomain B of fibronectin, a target almost exclusively expressed in tumor tissues. Extradomain B-binding molecules feature high thermal and serum stability as well as strong in vitro target binding and in vivo tumor accumulation. Application of several half-life extension technologies results in molecules of largely unaffected affinity but significantly prolonged in vivo half-life and tumor retention. Our results demonstrate the utility of ubiquitin as a scaffold for the generation of high affinity binders in a modular fashion, which can be combined with effector molecules and half-life extension technologies. PMID:24474690

  1. Screening for oligonucleotide binding affinity by a convenient fluorescence competition assay.

    PubMed

    Harrison, J G; Liu, X; Balasubramanian, S

    1999-09-01

    A competitive homogeneous quenched fluorescence assay system is described for the high throughput screening of DNA conjugates that bind to single-stranded DNA. Fluorescence signal is generated by competitive binding of the sample molecule to a target strand labelled with a quencher probe, which is otherwise hybridised to a complementary strand containing a fluorescent probe. Thus fluorescence generated is related to the affinity of the sample. Competitive analysis of a number of peptide-oligonucleotide conjugates gave data that correlated well with the corresponding UV melting data. The assay will be useful for screening of large numbers of potential single-stranded binding molecules.

  2. Microcantilever-Based Label-Free Characterization of Temperature-Dependent Biomolecular Affinity Binding

    PubMed Central

    Wang, Bin; Huang, Fengliang; Nguyen, ThaiHuu; Xu, Yong; Lin, Qiao

    2014-01-01

    This paper presents label-free characterization of temperature-dependent biomolecular affinity binding on solid surfaces using a microcantilever-based device. The device consists of a Parylene cantilever one side of which is coated with a gold film and functionalized with molecules as an affinity receptor to a target analyte. The cantilever is located in a poly(dimethylsiloxane) (PDMS) microfluidic chamber that is integrated with a transparent indium tin oxide (ITO) resistive temperature sensor on the underlying substrate. The ITO sensor allows for real-time measurements of the chamber temperature, as well as unobstructed optical access for reflection-based optical detection of the cantilever deflection. To test the temperature-dependent binding between the target and receptor, the temperature of the chamber is maintained at a constant setpoint, while a solution of unlabeled analyte molecules is continuously infused through the chamber. The measured cantilever deflection is used to determine the target-receptor binding characteristics. We demonstrate label-free characterization of temperature-dependent binding kinetics of the platelet-derived growth factor (PDGF) protein with an aptamer receptor. Affinity binding properties including the association and dissociation rate constants as well as equilibrium dissociation constant are obtained, and shown to exhibit significant dependencies on temperature. PMID:24723743

  3. Optimal fusion of antibody binding domains resulted in higher affinity and wider specificity.

    PubMed

    Dong, Jinhua; Kojima, Tomoki; Ohashi, Hiroyuki; Ueda, Hiroshi

    2015-11-01

    Antibody is a very important protein in biotechnological and biomedical fields because of its high affinity and specificity to various antigens. Due to the rise of human antibody therapeutics, its cost-effective purification is an urgent issue for bio-industry. In this study, we made novel fusion proteins PAxPG with a flexible (DDAKK)n linker between the two Ig binding domains derived from Staphylococcus protein A and Streptococcus protein G. The fusion proteins bound human and mouse IgGs and their fragments with up to 58-times higher affinity and wider specificity than the parental binding domains. Interestingly, the optimal linker for human Fab fragment was n = 4, which was close to the modeled distance between the termini of domains bound to heavy chain, implying increased avidity as a possible mechanism. For binding to Fc, the longest n=6 linker gave the highest affinity, implying longer interchain distance between the two binding sites. The novel fusion protein with optimized interdomain linker length will be a useful tool for the purification and detection of various IgGs including mouse IgG1 that binds only weakly to natural protein A. PMID:25910963

  4. Ligand binding affinities of arctigenin and its demethylated metabolites to estrogen receptor alpha.

    PubMed

    Jin, Jong-Sik; Lee, Jong-Hyun; Hattori, Masao

    2013-01-01

    Phytoestrogens are defined as plant-derived compounds with estrogen-like activities according to their chemical structures and activities. Plant lignans are generally categorized as phytoestrogens. It was reported that (-)-arctigenin, the aglycone of arctiin, was demethylated to (-)-dihydroxyenterolactone (DHENL) by Eubacterium (E.) sp. ARC-2. Through stepwise demethylation, E. sp. ARC-2 produced six intermediates, three mono-desmethylarctigenins and three di-desmethylarctigenins. In the present study, ligand binding affinities of (-)-arctigenin and its seven metabolites, including DHENL, were investigated for an estrogen receptor alpha, and found that demethylated metabolites had stronger binding affinities than (-)-arctigenin using a ligand binding screen assay method. The IC(50) value of (2R,3R)-2-(4-hydroxy-3-methoxybenzyl)-3-(3,4-dihydroxybenzyl)-butyrolactone was 7.9 × 10⁻⁴ M.

  5. Ligand binding affinities of arctigenin and its demethylated metabolites to estrogen receptor alpha.

    PubMed

    Jin, Jong-Sik; Lee, Jong-Hyun; Hattori, Masao

    2013-01-01

    Phytoestrogens are defined as plant-derived compounds with estrogen-like activities according to their chemical structures and activities. Plant lignans are generally categorized as phytoestrogens. It was reported that (-)-arctigenin, the aglycone of arctiin, was demethylated to (-)-dihydroxyenterolactone (DHENL) by Eubacterium (E.) sp. ARC-2. Through stepwise demethylation, E. sp. ARC-2 produced six intermediates, three mono-desmethylarctigenins and three di-desmethylarctigenins. In the present study, ligand binding affinities of (-)-arctigenin and its seven metabolites, including DHENL, were investigated for an estrogen receptor alpha, and found that demethylated metabolites had stronger binding affinities than (-)-arctigenin using a ligand binding screen assay method. The IC(50) value of (2R,3R)-2-(4-hydroxy-3-methoxybenzyl)-3-(3,4-dihydroxybenzyl)-butyrolactone was 7.9 × 10⁻⁴ M. PMID:23325100

  6. DNA-affinity-purified chip (DAP-chip) method to determine gene targets for bacterial two component regulatory systems.

    PubMed

    Rajeev, Lara; Luning, Eric G; Mukhopadhyay, Aindrila

    2014-07-21

    In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of limited use in exploring bacterial two component regulatory systems with uncharacterized activation conditions. Such systems regulate transcription only when activated in the presence of unique signals. Since these signals are often unknown, the in vitro microarray based method described in this video article can be used to determine gene targets and binding sites for response regulators. This DNA-affinity-purified-chip method may be used for any purified regulator in any organism with a sequenced genome. The protocol involves allowing the purified tagged protein to bind to sheared genomic DNA and then affinity purifying the protein-bound DNA, followed by fluorescent labeling of the DNA and hybridization to a custom tiling array. Preceding steps that may be used to optimize the assay for specific regulators are also described. The peaks generated by the array data analysis are used to predict binding site motifs, which are then experimentally validated. The motif predictions can be further used to determine gene targets of orthologous response regulators in closely related species. We demonstrate the applicability of this method by determining the gene targets and binding site motifs and thus predicting the function for a sigma54-dependent response regulator DVU3023 in the environmental bacterium Desulfovibrio vulgaris Hildenborough.

  7. High-affinity lead binding proteins in rat kidney cytosol mediate cell-free nuclear translocation of lead

    SciTech Connect

    Mistry, P.; Lucier, G.W.; Fowler, B.A.

    1985-02-01

    The PbII binding characteristics of the previously reported PbII binding proteins of rat kidney cytosol were investigated further. Saturation and Scatchard analysis of /sup 203/Pb binding in whole cytosol and in 40% saturated ammonium sulfate precipitated fractions disclosed a class of relatively high-affinity sites with an apparent Kd of approximately 50 nM and binding capacities of approximately 41 and 9 pmol/mg of protein, respectively. Two /sup 203/Pb binding proteins with approximate molecular masses of 63K and 11.5K daltons and a high molecular weight component (greater than 200K) were isolated by Sepharose-6B column chromatography. The time course of association of /sup 203/Pb with cytosol and the 63K protein showed maximum binding at 18 hr which was stable up to 25 hr at 4 degrees C. The approximate half-time dissociation rate (T 1/2) of specifically bound /sup 203/Pb to the 63K protein was 100 min at 4 degrees C whereas the 11.5K protein showed little dissociation of specifically bound ligand at this temperature. Saturation analysis of the three isolated proteins disclosed low capacity, high-affinity sites with similar apparent Kd values to the cytosol assay. Sucrose density gradient analysis of kidney cytosol showed approximate sedimentation coefficients of 2S, 4.6S and 7S for the 11.5K, 63K and the high molecular weight proteins, respectively. Competitive binding studies with cytosol demonstrated displacement of /sup 203/Pb by PbII, CdII and ZnII ions but not CaII ions.

  8. Phosphatidylserine and Phosphatidylethanolamine Bind to Protein Z Cooperatively and with Equal Affinity.

    PubMed

    Sengupta, Tanusree; Manoj, Narayanan

    2016-01-01

    Protein Z (PZ) is an anticoagulant that binds with high affinity to Protein Z-dependent protease inhibitor (ZPI) and accelerates the rate of ZPI-mediated inhibition of factor Xa (fXa) by more than 1000-fold in the presence of Ca2+ and phospholipids. PZ promotion of the ZPI-fXa interaction results from the anchoring of the Gla domain of PZ onto phospholipid surfaces and positioning the bound ZPI in close proximity to the Gla-anchored fXa, forming a ternary complex of PZ/ZPI/fXa. Although interaction of PZ with phospholipid membrane appears to be absolutely crucial for its cofactor activity, little is known about the binding of different phospholipids to PZ. The present study was conceived to understand the interaction of different phospholipids with PZ. Experiments with both soluble lipids and model membranes revealed that PZ binds to phosphatidylserine (PS) and phosphatidylethanolamine (PE) with equal affinity (Kd~48 μM); further, PS and PE bound to PZ synergistically. Equilibrium dialysis experiments revealed two lipid-binding sites for both PS and PE. PZ binds with weaker affinity to other phospholipids, e.g., phosphatidic acid, phosphatidylglycerol, phosphatidylcholine and binding of these lipids is not synergistic with respect to PS. Both PS and PE -containing membranes supported the formation of a fXa-PZ complex. PZ protection of fXa from antithrombin inhibition were also shown to be comparable in presence of both PS: PC and PE: PC membranes. These findings are particularly important and intriguing since they suggest a special affinity of PZ, in vivo, towards activated platelets, the primary membrane involved in blood coagulation process. PMID:27584039

  9. Phosphatidylserine and Phosphatidylethanolamine Bind to Protein Z Cooperatively and with Equal Affinity

    PubMed Central

    Sengupta, Tanusree; Manoj, Narayanan

    2016-01-01

    Protein Z (PZ) is an anticoagulant that binds with high affinity to Protein Z-dependent protease inhibitor (ZPI) and accelerates the rate of ZPI-mediated inhibition of factor Xa (fXa) by more than 1000-fold in the presence of Ca2+ and phospholipids. PZ promotion of the ZPI-fXa interaction results from the anchoring of the Gla domain of PZ onto phospholipid surfaces and positioning the bound ZPI in close proximity to the Gla-anchored fXa, forming a ternary complex of PZ/ZPI/fXa. Although interaction of PZ with phospholipid membrane appears to be absolutely crucial for its cofactor activity, little is known about the binding of different phospholipids to PZ. The present study was conceived to understand the interaction of different phospholipids with PZ. Experiments with both soluble lipids and model membranes revealed that PZ binds to phosphatidylserine (PS) and phosphatidylethanolamine (PE) with equal affinity (Kd~48 μM); further, PS and PE bound to PZ synergistically. Equilibrium dialysis experiments revealed two lipid-binding sites for both PS and PE. PZ binds with weaker affinity to other phospholipids, e.g., phosphatidic acid, phosphatidylglycerol, phosphatidylcholine and binding of these lipids is not synergistic with respect to PS. Both PS and PE -containing membranes supported the formation of a fXa-PZ complex. PZ protection of fXa from antithrombin inhibition were also shown to be comparable in presence of both PS: PC and PE: PC membranes. These findings are particularly important and intriguing since they suggest a special affinity of PZ, in vivo, towards activated platelets, the primary membrane involved in blood coagulation process. PMID:27584039

  10. Comparison of biosensor platforms in the evaluation of high affinity antibody-antigen binding kinetics.

    PubMed

    Yang, Danlin; Singh, Ajit; Wu, Helen; Kroe-Barrett, Rachel

    2016-09-01

    The acquisition of reliable kinetic parameters for the characterization of biomolecular interactions is an important component of the drug discovery and development process. While several benchmark studies have explored the variability of kinetic rate constants obtained from multiple laboratories and biosensors, a direct comparison of these instruments' performance has not been undertaken, and systematic factors contributing to data variability from these systems have not been discussed. To address these questions, a panel of ten high-affinity monoclonal antibodies was simultaneously evaluated for their binding kinetics against the same antigen on four biosensor platforms: GE Healthcare's Biacore T100, Bio-Rad's ProteOn XPR36, ForteBio's Octet RED384, and Wasatch Microfluidics's IBIS MX96. We compared the strengths and weaknesses of these systems and found that despite certain inherent systematic limitations in instrumentation, the rank orders of both the association and dissociation rate constants were highly correlated between these instruments. Our results also revealed a trade-off between data reliability and sample throughput. Biacore T100, followed by ProteOn XPR36, exhibited excellent data quality and consistency, whereas Octet RED384 and IBIS MX96 demonstrated high flexibility and throughput with compromises in data accuracy and reproducibility. Our results support the need for a "fit-for-purpose" approach in instrument selection for biosensor studies. PMID:27365220

  11. Design of Cyclic Peptides That Bind Protein Surfaces with Antibody-Like Affinity

    PubMed Central

    Millward, Steven W.; Fiacco, Stephen; Austin, Ryan J.; Roberts, Richard W.

    2012-01-01

    There is a pressing need for new molecular tools to target protein surfaces with high affinity and specificity. Here, we describe cyclic messenger RNA display with a trillion-member covalent peptide macrocycle library. Using this library, we have designed a number of high-affinity, redox-insensitive, cyclic peptides that target the signaling protein Gαi1. In addition to cyclization, our library construction took advantage of an expanded genetic code, utilizing nonsense suppression to insert N-methylphenylalanine as a 21st amino acid. The designed macrocycles exhibit several intriguing features. First, the core motif seen in all of the selected variants is the same and shares an identical context with respect to the macrocyclic scaffold, consistent with the idea that selection simultaneously optimizes both the cyclization chemistry and the structural placement of the binding epitope. Second, detailed characterization of one molecule, cyclic Gαi binding peptide (cycGiBP), demonstrates substantially enhanced proteolytic stability relative to that of the parent linear molecule. Third and perhaps most important, the cycGiBP peptide binds the target with very high affinity (Ki ≈ 2.1 nM), similar to those of many of the best monoclonal antibodies and higher than that of the βγ heterodimer, an endogenous Gαi1 ligand. Overall the work provides a general route to design novel, low-molecular-weight, high-affinity ligands that target protein surfaces. PMID:17894440

  12. Design of cyclic peptides that bind protein surfaces with antibody-like affinity.

    PubMed

    Millward, Steven W; Fiacco, Stephen; Austin, Ryan J; Roberts, Richard W

    2007-09-21

    There is a pressing need for new molecular tools to target protein surfaces with high affinity and specificity. Here, we describe cyclic messenger RNA display with a trillion-member covalent peptide macrocycle library. Using this library, we have designed a number of high-affinity, redox-insensitive, cyclic peptides that target the signaling protein G alpha i1. In addition to cyclization, our library construction took advantage of an expanded genetic code, utilizing nonsense suppression to insert N-methylphenylalanine as a 21st amino acid. The designed macrocycles exhibit several intriguing features. First, the core motif seen in all of the selected variants is the same and shares an identical context with respect to the macrocyclic scaffold, consistent with the idea that selection simultaneously optimizes both the cyclization chemistry and the structural placement of the binding epitope. Second, detailed characterization of one molecule, cyclic G alpha i binding peptide (cycGiBP), demonstrates substantially enhanced proteolytic stability relative to that of the parent linear molecule. Third and perhaps most important, the cycGiBP peptide binds the target with very high affinity ( K i approximately 2.1 nM), similar to those of many of the best monoclonal antibodies and higher than that of the betagamma heterodimer, an endogenous G alpha i1 ligand. Overall the work provides a general route to design novel, low-molecular-weight, high-affinity ligands that target protein surfaces.

  13. Contacts-based prediction of binding affinity in protein–protein complexes

    PubMed Central

    Vangone, Anna; Bonvin, Alexandre MJJ

    2015-01-01

    Almost all critical functions in cells rely on specific protein–protein interactions. Understanding these is therefore crucial in the investigation of biological systems. Despite all past efforts, we still lack a thorough understanding of the energetics of association of proteins. Here, we introduce a new and simple approach to predict binding affinity based on functional and structural features of the biological system, namely the network of interfacial contacts. We assess its performance against a protein–protein binding affinity benchmark and show that both experimental methods used for affinity measurements and conformational changes have a strong impact on prediction accuracy. Using a subset of complexes with reliable experimental binding affinities and combining our contacts and contact-types-based model with recent observations on the role of the non-interacting surface in protein–protein interactions, we reach a high prediction accuracy for such a diverse dataset outperforming all other tested methods. DOI: http://dx.doi.org/10.7554/eLife.07454.001 PMID:26193119

  14. Impact of D2 Receptor Internalization on Binding Affinity of Neuroimaging Radiotracers

    PubMed Central

    Guo, Ningning; Guo, Wen; Kralikova, Michaela; Jiang, Man; Schieren, Ira; Narendran, Raj; Slifstein, Mark; Abi-Dargham, Anissa; Laruelle, Marc; Javitch, Jonathan A; Rayport, Stephen

    2010-01-01

    Synaptic dopamine (DA) levels seem to affect the in vivo binding of many D2 receptor radioligands. Thus, release of endogenous DA induced by the administration of amphetamine decreases ligand binding, whereas DA depletion increases binding. This is generally thought to be due to competition between endogenous DA and the radioligands for D2 receptors. However, the temporal discrepancy between amphetamine-induced increases in DA as measured by microdialysis, which last on the order of 2 h, and the prolonged decrease in ligand binding, which lasts up to a day, has suggested that agonist-induced D2 receptor internalization may contribute to the sustained decrease in D2 receptor-binding potential seen following a DA surge. To test this hypothesis, we developed an in vitro system showing robust agonist-induced D2 receptor internalization following treatment with the agonist quinpirole. Human embryonic kidney 293 (HEK293) cells were stably co-transfected with human D2 receptor, G-protein-coupled receptor kinase 2 and arrestin 3. Agonist-induced D2 receptor internalization was demonstrated by fluorescence microscopy, flow cytometry, and radioligand competition binding. The binding of seven D2 antagonists and four agonists to the surface and internalized receptors was measured in intact cells. All the imaging ligands bound with high affinity to both surface and internalized D2 receptors. Affinity of most of the ligands to internalized receptors was modestly lower, indicating that internalization would reduce the binding potential measured in imaging studies carried out with these ligands. However, between-ligand differences in the magnitude of the internalization-associated affinity shift only partly accounted for the data obtained in neuroimaging experiments, suggesting the involvement of mechanisms beyond competition and internalization. PMID:19956086

  15. Affinity binding of inclusion bodies on supermacroporous monolithic cryogels using labeling with specific antibodies.

    PubMed

    Ahlqvist, Josefin; Kumar, Ashok; Sundström, Heléne; Ledung, Erika; Hörnsten, E Gunnar; Enfors, Sven-Olof; Mattiasson, Bo

    2006-03-23

    A new chromatographic method based on affinity supermacroporous monolithic cryogels is developed for binding and analyzing inclusion bodies during fermentation. The work demonstrated that it is possible to bind specific IgG and IgY antibodies to the 15 and 17 amino acids at the terminus ends of a 33 kDa target protein aggregated as inclusion bodies. The antibody treated inclusion bodies from lysed fermentation broth can be specifically retained in protein A and pseudo-biospecific ligand sulfamethazine modified supermacroporous cryogels. The degree of binding of IgG and IgY treated inclusion bodies to the Protein A and sulfamethazine gels are investigated, as well as the influence of pH on the sulfamethazine ligand. Optimum binding of 78 and 72% was observed on both protein A and sulfamethazine modified cryogel columns, respectively, using IgG labeling of the inclusion bodies. The antibody treated inclusion bodies pass through unretained in the sulfamethazine supermacroporous gel at pH that does not favour the binding between the ligand on the gel and the antibodies on the surface of inclusion bodies. Also the unlabeled inclusion bodies went through the gel unretained, showing no non-specific binding or trapping within the gel. These findings may very well be the foundation for the building of a powerful analytical tool during fermentation of inclusion bodies as well as a convenient way to purify them from fermentation broth. These results also support our earlier findings [Kumar, A., Plieva, F.M., Galaev, I.Yu., Mattiasson, B., 2003. Affinity fractionation of lymphocytes using a monolithic cyogel. J. Immunol. Methods 283, 185-194] with mammalian cells that were surface labeled with specific antibodies and recognized on protein A supermacroporous gels. A general binding and separation system can be established on antibody binding cryogel affinity matrices.

  16. CHARACTERIZATION OF THE BINDING OF SULFONYLUREA DRUGS TO HSA BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY

    PubMed Central

    Joseph, K.S.; Hage, David S.

    2010-01-01

    Sulfonylurea drugs are often prescribed as a treatment for type II diabetes to help lower blood sugar levels by stimulating insulin secretion. These drugs are believed to primarily bind in blood to human serum albumin (HSA). This study used high-performance affinity chromatography (HPAC) to examine the binding of sulfonylureas to HSA. Frontal analysis with an immobilized HSA column was used to determine the association equilibrium constants (Ka) and number of binding sites on HSA for the sulfonylurea drugs acetohexamide and tolbutamide. The results from frontal analysis indicated HSA had a group of relatively high affinity binding regions and weaker binding sites for each drug, with average Ka values of 1.3 (± 0.2) × 105 M−1 and 3.5 (± 3.0) × 102 M−1 for acetohexamide and values of 8.7 (± 0.6) × 104 and 8.1 (± 1.7) × 103 M−1 for tolbutamide. Zonal elution and competition studies with site-specific probes were used to further examine the relatively high affinity interactions of these drugs by looking directly at the interactions that were occurring at Sudlow sites I and II of HSA (i.e., the major drug binding sites on this protein). It was found that acetohexamide was able to bind at both Sudlow sites I and II, with Ka values of 1.3 (± 0.1) × 105 and 4.3 (± 0.3) × 104 M−1, respectively, at 37°C. Tolbutamide also appeared to interact with both Sudlow sites I and II, with Ka values of 5.5 (± 0.2) × 104 and 5.3 (± 0.2) × 104 M−1, respectively. The results provide a more quantitative picture of how these drugs bind with HSA and illustrate how HPAC and related tools can be used to examine relatively complex drug-protein interactions. PMID:20435530

  17. Affinity purification of human granulocyte macrophage colony-stimulating factor receptor alpha-chain. Demonstration of binding by photoaffinity labeling

    SciTech Connect

    Chiba, S.; Shibuya, K.; Miyazono, K.; Tojo, A.; Oka, Y.; Miyagawa, K.; Takaku, F. )

    1990-11-15

    The human granulocyte macrophage colony-stimulating factor (GM-CSF) receptor alpha-chain, a low affinity component of the receptor, was solubilized and affinity-purified from human placenta using biotinylated GM-CSF. Scatchard analysis of {sup 125}I-GM-CSF binding to the placental membrane extract disclosed that the GM-CSF receptor had a dissociation constant (Kd) of 0.5-0.8 nM, corresponding to the Kd value of the GM-CSF receptor alpha-chain on the intact placental membrane. Affinity labeling of the solubilized protein using a photoreactive cross-linking agent, N-hydroxysuccinimidyl-4-azidobenzoate (HSAB), demonstrated a single specific band of 70-95 kDa representing a ligand-receptor complex. Approximately 2 g of the placental membrane extract was subjected to a biotinylated GM-CSF-fixed streptavidin-agarose column, resulting in a single major band at 70 kDa on a silver-stained sodium dodecyl sulfate gel. The radioiodination for the purified material disclosed that the purified protein had an approximate molecular mass of 70 kDa and a pI of 6.6. Binding activity of the purified material was demonstrated by photoaffinity labeling using HSAB-{sup 125}I-GM-CSF, producing a similar specific band at 70-95 kDa as was demonstrated for the crude protein.

  18. N-terminal engineering of amyloid-β-binding Affibody molecules yields improved chemical synthesis and higher binding affinity

    PubMed Central

    Lindgren, Joel; Wahlström, Anna; Danielsson, Jens; Markova, Natalia; Ekblad, Caroline; Gräslund, Astrid; Abrahmsén, Lars; Karlström, Amelie Eriksson; Wärmländer, Sebastian KTS

    2010-01-01

    The aggregation of amyloid-β (Aβ) peptides is believed to be a major factor in the onset and progression of Alzheimer's disease. Molecules binding with high affinity and selectivity to Aβ-peptides are important tools for investigating the aggregation process. An Aβ-binding Affibody molecule, ZAβ3, has earlier been selected by phage display and shown to bind Aβ(1–40) with nanomolar affinity and to inhibit Aβ-peptide aggregation. In this study, we create truncated functional versions of the ZAβ3 Affibody molecule better suited for chemical synthesis production. Engineered Affibody molecules of different length were produced by solid phase peptide synthesis and allowed to form covalently linked homodimers by S-S-bridges. The N-terminally truncated Affibody molecules ZAβ3(12–58), ZAβ3(15–58), and ZAβ3(18–58) were produced in considerably higher synthetic yield than the corresponding full-length molecule ZAβ3(1–58). Circular dichroism spectroscopy and surface plasmon resonance-based biosensor analysis showed that the shortest Affibody molecule, ZAβ3(18–58), exhibited complete loss of binding to the Aβ(1–40)-peptide, while the ZAβ3(12–58) and ZAβ3(15–58) Affibody molecules both displayed approximately one order of magnitude higher binding affinity to the Aβ(1–40)-peptide compared to the full-length Affibody molecule. Nuclear magnetic resonance spectroscopy showed that the structure of Aβ(1–40) in complex with the truncated Affibody dimers is very similar to the previously published solution structure of the Aβ(1–40)-peptide in complex with the full-length ZAβ3 Affibody molecule. This indicates that the N-terminally truncated Affibody molecules ZAβ3(12–58) and ZAβ3(15–58) are highly promising for further engineering and future use as binding agents to monomeric Aβ(1–40). PMID:20886513

  19. Protein unfolding as a switch from self-recognition to high-affinity client binding

    PubMed Central

    Groitl, Bastian; Horowitz, Scott; Makepeace, Karl A. T.; Petrotchenko, Evgeniy V.; Borchers, Christoph H.; Reichmann, Dana; Bardwell, James C. A.; Jakob, Ursula

    2016-01-01

    Stress-specific activation of the chaperone Hsp33 requires the unfolding of a central linker region. This activation mechanism suggests an intriguing functional relationship between the chaperone's own partial unfolding and its ability to bind other partially folded client proteins. However, identifying where Hsp33 binds its clients has remained a major gap in our understanding of Hsp33's working mechanism. By using site-specific Fluorine-19 nuclear magnetic resonance experiments guided by in vivo crosslinking studies, we now reveal that the partial unfolding of Hsp33's linker region facilitates client binding to an amphipathic docking surface on Hsp33. Furthermore, our results provide experimental evidence for the direct involvement of conditionally disordered regions in unfolded protein binding. The observed structural similarities between Hsp33's own metastable linker region and client proteins present a possible model for how Hsp33 uses protein unfolding as a switch from self-recognition to high-affinity client binding. PMID:26787517

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

    PubMed

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

    2014-05-27

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-05-27

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

  3. Identification and characterization of a novel high affinity metal-binding site in the hammerhead ribozyme.

    PubMed Central

    Hansen, M R; Simorre, J P; Hanson, P; Mokler, V; Bellon, L; Beigelman, L; Pardi, A

    1999-01-01

    A novel metal-binding site has been identified in the hammerhead ribozyme by 31P NMR. The metal-binding site is associated with the A13 phosphate in the catalytic core of the hammerhead ribozyme and is distinct from any previously identified metal-binding sites. 31P NMR spectroscopy was used to measure the metal-binding affinity for this site and leads to an apparent dissociation constant of 250-570 microM at 25 degrees C for binding of a single Mg2+ ion. The NMR data also show evidence of a structural change at this site upon metal binding and these results are compared with previous data on metal-induced structural changes in the core of the hammerhead ribozyme. These NMR data were combined with the X-ray structure of the hammerhead ribozyme (Pley HW, Flaherty KM, McKay DB. 1994. Nature 372:68-74) to model RNA ligands involved in binding the metal at this A13 site. In this model, the A13 metal-binding site is structurally similar to the previously identified A(g) metal-binding site and illustrates the symmetrical nature of the tandem G x A base pairs in domain 2 of the hammerhead ribozyme. These results demonstrate that 31P NMR represents an important method for both identification and characterization of metal-binding sites in nucleic acids. PMID:10445883

  4. The high-affinity peptidoglycan binding domain of Pseudomonas phage endolysin KZ144

    SciTech Connect

    Briers, Yves; Schmelcher, Mathias; Loessner, Martin J.; Hendrix, Jelle; Engelborghs, Yves; Volckaert, Guido; Lavigne, Rob

    2009-05-29

    The binding affinity of the N-terminal peptidoglycan binding domain of endolysin KZ144 (PBD{sub KZ}), originating from Pseudomonas aeruginosa bacteriophage {phi}KZ, has been examined using a fusion protein of PBD{sub KZ} and green fluorescent protein (PBD{sub KZ}-GFP). A fluorescence recovery after photobleaching analysis of bound PBD{sub KZ}-GFP molecules showed less than 10% fluorescence recovery in the bleached area within 15 min. Surface plasmon resonance analysis confirmed this apparent high binding affinity revealing an equilibrium affinity constant of 2.95 x 10{sup 7} M{sup -1} for the PBD{sub KZ}-peptidoglycan interaction. This unique domain, which binds to the peptidoglycan of all tested Gram-negative species, was harnessed to improve the specific activity of the peptidoglycan hydrolase domain KMV36C. The chimeric peptidoglycan hydrolase (PBD{sub KZ}-KMV36C) exhibits a threefold higher specific activity than the native catalytic domain (KMV36C). These results demonstrate that the modular assembly of functional domains is a rational approach to improve the specific activity of endolysins from phages infecting Gram-negatives.

  5. Quantitative assessment of binding affinities for nanoparticles targeted to vulnerable plaque.

    PubMed

    Tang, Tang; Tu, Chuqiao; Chow, Sarah Y; Leung, Kevin H; Du, Siyi; Louie, Angelique Y

    2015-06-17

    Recent successes in targeted immune and cell-based therapies have driven new directions for pharmaceutical research. With the rise of these new therapies there is an unfilled need for companion diagnostics to assess patients' potential for therapeutic response. Targeted nanomaterials have been widely investigated to fill this niche; however, in contrast to small molecule or peptide-based targeted agents, binding affinities are not reported for nanomaterials, and to date there has been no standard, quantitative measure for the interaction of targeted nanoparticle agents with their targets. Without a standard measure, accurate comparisons between systems and optimization of targeting behavior are challenging. Here, we demonstrate a method for quantitative assessment of the binding affinity for targeted nanoparticles to cell surface receptors in living systems and apply it to optimize the development of a novel targeted nanoprobe for imaging vulnerable atherosclerotic plaques. In this work, we developed sulfated dextran-coated iron oxide nanoparticles with specific targeting to macrophages, a cell type whose density strongly correlates with plaque vulnerability. Detailed quantitative, in vitro characterizations of (111)In(3+) radiolabeled probes show high-affinity binding to the macrophage scavenger receptor A (SR-A). Cell uptake studies illustrate that higher surface sulfation levels result in much higher uptake efficiency by macrophages. We use a modified Scatchard analysis to quantitatively describe nanoparticle binding to targeted receptors. This characterization represents a potential new standard metric for targeted nanomaterials. PMID:25970303

  6. Measurement of free glucocorticoids: quantifying corticosteroid-binding globulin binding affinity and its variation within and among mammalian species

    PubMed Central

    Delehanty, Brendan; Hossain, Sabrina; Jen, Chao Ching; Crawshaw, Graham J.; Boonstra, Rudy

    2015-01-01

    Plasma glucocorticoids (GCs) are commonly used as measures of stress in wildlife. A great deal of evidence indicates that only free GC (GC not bound by the specific binding protein, corticosteroid-binding globulin, CBG) leaves the circulation and exerts biological effects on GC-sensitive tissues. Free hormone concentrations are difficult to measure directly, so researchers estimate free GC using two measures: the binding affinity and the binding capacity in plasma. We provide an inexpensive saturation binding method for calculating the binding affinity (equilibrium dissociation constant, Kd) of CBG that can be run without specialized laboratory equipment. Given that other plasma proteins, such as albumin, also bind GCs, the method compensates for this non-specific binding. Separation of bound GC from free GC was achieved with dextran-coated charcoal. The method provides repeatable estimates (12% coefficient of variation in the red squirrel, Tamiasciurus hudsonicus), and there is little evidence of inter-individual variation in Kd (range 2.0–7.3 nM for 16 Richardson's ground squirrels, Urocitellus richardsonii). The Kd values of 28 mammalian species we assessed were mostly clustered around a median of 4 nM, but five species had values between 13 and 61 nM. This pattern may be distinct from birds, for which published values are more tightly distributed (1.5–5.1 nM). The charcoal separation method provides a reliable and robust method for measuring the Kd in a wide range of species. It uses basic laboratory equipment to provide rapid results at very low cost. Given the importance of CBG in regulating the biological activity of GCs, this method is a useful tool for physiological ecologists. PMID:27293705

  7. Measurement of free glucocorticoids: quantifying corticosteroid-binding globulin binding affinity and its variation within and among mammalian species.

    PubMed

    Delehanty, Brendan; Hossain, Sabrina; Jen, Chao Ching; Crawshaw, Graham J; Boonstra, Rudy

    2015-01-01

    Plasma glucocorticoids (GCs) are commonly used as measures of stress in wildlife. A great deal of evidence indicates that only free GC (GC not bound by the specific binding protein, corticosteroid-binding globulin, CBG) leaves the circulation and exerts biological effects on GC-sensitive tissues. Free hormone concentrations are difficult to measure directly, so researchers estimate free GC using two measures: the binding affinity and the binding capacity in plasma. We provide an inexpensive saturation binding method for calculating the binding affinity (equilibrium dissociation constant, K d) of CBG that can be run without specialized laboratory equipment. Given that other plasma proteins, such as albumin, also bind GCs, the method compensates for this non-specific binding. Separation of bound GC from free GC was achieved with dextran-coated charcoal. The method provides repeatable estimates (12% coefficient of variation in the red squirrel, Tamiasciurus hudsonicus), and there is little evidence of inter-individual variation in K d (range 2.0-7.3 nM for 16 Richardson's ground squirrels, Urocitellus richardsonii). The K d values of 28 mammalian species we assessed were mostly clustered around a median of 4 nM, but five species had values between 13 and 61 nM. This pattern may be distinct from birds, for which published values are more tightly distributed (1.5-5.1 nM). The charcoal separation method provides a reliable and robust method for measuring the K d in a wide range of species. It uses basic laboratory equipment to provide rapid results at very low cost. Given the importance of CBG in regulating the biological activity of GCs, this method is a useful tool for physiological ecologists.

  8. Probing the Determinants of Diacylglycerol Binding Affinity in C1B domain of Protein Kinase Cα

    PubMed Central

    Stewart, Mikaela D.; Morgan, Brittany; Massi, Francesca; Igumenova, Tatyana I.

    2012-01-01

    C1 domains are independently folded modules that are responsible for targeting their parent proteins to lipid membranes containing diacylglycerol (DAG), a ubiquitous second messenger. The DAG-binding affinities of C1 domains determine the threshold concentration of DAG required for the propagation of the signaling response and the selectivity of this response among the DAG receptors in the cell. The structural information currently available for C1 domains offers little insight into the molecular basis of their differential DAG-binding affinities. In this work, we characterized the C1B domain of Protein Kinase Cα (C1Bα) and its diagnostic mutant, Y123W, using solution NMR methods and molecular dynamics simulations. The mutation did not perturb the C1Bα structure or sub-nanosecond dynamics of the protein backbone, but resulted in a >100-fold increase of DAG binding affinity and substantial change in μs-timescale conformational dynamics, as quantified by NMR rotating-frame relaxation-dispersion methods. The differences in the conformational exchange behavior between the wild-type and Y123W C1Bα were localized to the hinge regions of ligand-binding loops. Molecular dynamics simulations provided insight into the identity of the exchanging conformers and revealed the significance of a particular residue, Gln128, in modulating the geometry of the ligand-binding site. Taken together with the results of binding studies, our findings suggest that the conformational dynamics and preferential partitioning of the tryptophan sidechain into the water-lipid interface are important factors that modulate the DAG-binding properties of C1 domains. PMID:21419781

  9. Cholera toxin binding affinity and specificity for gangliosides determined by surface plasmon resonance

    SciTech Connect

    Kuziemko, G.M.; Stroh, M.; Stevens, R.C. |

    1996-05-21

    The present study determines the affinity of cholera toxin for the ganglioside series GM1, GM2, GM3, GD1A, GD1B, GT1B, asialo GM1, globotriosyl ceramide, and lactosyl ceramide using real time biospecific interaction analysis (surface plasmon resonance, SPR). SPR shows that cholera toxin preferably binds to gangliosides in the following sequence: GM1 > GM2 > GD1A > GM3 > GT1B > GD1B > asialo-GM1. The measured binding affinity of cholera toxin for the ganglioside sequence ranges from 4.61 {times} 10{sup {minus}12} M for GM1 to 1.88 {times} 10{sup {minus}10} M for asialo GM1. The picomolar values obtained by surface plasmon resonance are similar to K{sub d} values determined with whole-cell binding assays. Both whole-cell assays ans SPR measurements on synthetic membranes are higher than free solution measurements by several orders of magnitude. This difference may be caused by the effects of avidity and charged lipid head-groups, which may play a major role in the binding between cholera toxin, the receptor, and the membrane surface. The primary difference between free solution binding studies and surface plasmon resonance studies is that the latter technique is performed on surfaces resembling the cell membrane. Surface plasmon resonance has the further advantage of measuring apparent kinetic association and dissociation rates in real time, providing direct information about binding events at the membrane surface. 34 refs., 8 figs., 2 tabs.

  10. Stoichiometry and Affinity of Thioflavin T Binding to Sup35p Amyloid Fibrils.

    PubMed

    Sulatskaya, Anna I; Kuznetsova, Irina M; Belousov, Mikhail V; Bondarev, Stanislav A; Zhouravleva, Galina A; Turoverov, Konstantin K

    2016-01-01

    In this work two modes of binding of the fluorescent probe thioflavin T to yeast prion protein Sup35p amyloid fibrils were revealed by absorption spectrometry of solutions prepared by equilibrium microdialysis. These binding modes exhibited significant differences in binding affinity and stoichiometry. Moreover, the absorption spectrum and the molar extinction coefficient of the dye bound in each mode were determined. The fluorescence quantum yield of the dye bound in each mode was determined via a spectrofluorimetric study of the same solutions in which the recorded fluorescence intensity was corrected for the primary inner filter effect. As previously predicted, the existence of one of the detected binding modes may be due to the incorporation of the dye into the grooves along the fiber axis perpendicular to the β-sheets of the fibrils. It was assumed that the second type of binding with higher affinity may be due to the existence of ThT binding sites that are localized to areas where amyloid fibrils are clustered.

  11. Stoichiometry and Affinity of Thioflavin T Binding to Sup35p Amyloid Fibrils

    PubMed Central

    Sulatskaya, Anna I.; Kuznetsova, Irina M.; Belousov, Mikhail V.; Bondarev, Stanislav A.; Zhouravleva, Galina A.; Turoverov, Konstantin K.

    2016-01-01

    In this work two modes of binding of the fluorescent probe thioflavin T to yeast prion protein Sup35p amyloid fibrils were revealed by absorption spectrometry of solutions prepared by equilibrium microdialysis. These binding modes exhibited significant differences in binding affinity and stoichiometry. Moreover, the absorption spectrum and the molar extinction coefficient of the dye bound in each mode were determined. The fluorescence quantum yield of the dye bound in each mode was determined via a spectrofluorimetric study of the same solutions in which the recorded fluorescence intensity was corrected for the primary inner filter effect. As previously predicted, the existence of one of the detected binding modes may be due to the incorporation of the dye into the grooves along the fiber axis perpendicular to the β-sheets of the fibrils. It was assumed that the second type of binding with higher affinity may be due to the existence of ThT binding sites that are localized to areas where amyloid fibrils are clustered. PMID:27228180

  12. Stoichiometry and Affinity of Thioflavin T Binding to Sup35p Amyloid Fibrils.

    PubMed

    Sulatskaya, Anna I; Kuznetsova, Irina M; Belousov, Mikhail V; Bondarev, Stanislav A; Zhouravleva, Galina A; Turoverov, Konstantin K

    2016-01-01

    In this work two modes of binding of the fluorescent probe thioflavin T to yeast prion protein Sup35p amyloid fibrils were revealed by absorption spectrometry of solutions prepared by equilibrium microdialysis. These binding modes exhibited significant differences in binding affinity and stoichiometry. Moreover, the absorption spectrum and the molar extinction coefficient of the dye bound in each mode were determined. The fluorescence quantum yield of the dye bound in each mode was determined via a spectrofluorimetric study of the same solutions in which the recorded fluorescence intensity was corrected for the primary inner filter effect. As previously predicted, the existence of one of the detected binding modes may be due to the incorporation of the dye into the grooves along the fiber axis perpendicular to the β-sheets of the fibrils. It was assumed that the second type of binding with higher affinity may be due to the existence of ThT binding sites that are localized to areas where amyloid fibrils are clustered. PMID:27228180

  13. A computational study of ligand binding affinities in iron(III) porphine and protoporphyrin IX complexes.

    PubMed

    Durrant, Marcus C

    2014-07-01

    The search for novel anti-malarial drugs that can disrupt biomineralization of ferriprotoporphyrin IX to haemozoin requires an understanding of the fundamental chemistry of the porphyrin's iron(iii) centre at the water-lipid interface. Towards this end, the binding affinities for a diverse set of 31 small ligands with iron(iii) porphine have been calculated using density functional theory, in the gas phase and also with implicit solvent corrections for both water and n-octanol. In addition, the binding of hydroxide, chloride, acetate, methylamine and water to ferriprotoporphyrin IX has been studied, and very similar trends are observed for the smaller and larger models. Anionic ligands generally give stronger binding than neutral ones; the strongest binding is observed for RO(-) and OH(-) ligands, whilst acetate binds relatively weakly among the anions studied. Electron-rich nitrogen donors tend to bind more strongly than electron-deficient ones, and the weakest binding is found for neutral O and S donors such as oxazole and thiophene. In all cases, ligand binding is stronger in n-octanol than in water, and the differences in binding energies for the two solvents are greater for ionic ligands than for neutrals. Finally, dimerization of ferriprotoporphyrin IX by means of iron(iii)-carboxylate bond formation has been modelled. The results are discussed in terms of haemozoin crystal growth and its disruption by known anti-malarial drugs.

  14. Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

    NASA Technical Reports Server (NTRS)

    Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

    2000-01-01

    Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

  15. Antibody Binding Selectivity: Alternative Sets of Antigen Residues Entail High-Affinity Recognition

    PubMed Central

    Nominé, Yves; Choulier, Laurence; Travé, Gilles; Vernet, Thierry; Altschuh, Danièle

    2015-01-01

    Understanding the relationship between protein sequence and molecular recognition selectivity remains a major challenge. The antibody fragment scFv1F4 recognizes with sub nM affinity a decapeptide (sequence 6TAMFQDPQER15) derived from the N-terminal end of human papilloma virus E6 oncoprotein. Using this decapeptide as antigen, we had previously shown that only the wild type amino-acid or conservative replacements were allowed at positions 9 to 12 and 15 of the peptide, indicating a strong binding selectivity. Nevertheless phenylalanine (F) was equally well tolerated as the wild type glutamine (Q) at position 13, while all other amino acids led to weaker scFv binding. The interfaces of complexes involving either Q or F are expected to diverge, due to the different physico-chemistry of these residues. This would imply that high-affinity binding can be achieved through distinct interfacial geometries. In order to investigate this point, we disrupted the scFv–peptide interface by modifying one or several peptide positions. We then analyzed the effect on binding of amino acid changes at the remaining positions, an altered susceptibility being indicative of an altered role in complex formation. The 23 starting variants analyzed contained replacements whose effects on scFv1F4 binding ranged from minor to drastic. A permutation analysis (effect of replacing each peptide position by all other amino acids except cysteine) was carried out on the 23 variants using the PEPperCHIP® Platform technology. A comparison of their permutation patterns with that of the wild type peptide indicated that starting replacements at position 11, 12 or 13 modified the tolerance to amino-acid changes at the other two positions. The interdependence between the three positions was confirmed by SPR (Biacore® technology). Our data demonstrate that binding selectivity does not preclude the existence of alternative high-affinity recognition modes. PMID:26629896

  16. Structure and Energetic Contributions of a Designed Modular Peptide-Binding Protein with Picomolar Affinity.

    PubMed

    Hansen, Simon; Tremmel, Dirk; Madhurantakam, Chaithanya; Reichen, Christian; Mittl, Peer R E; Plückthun, Andreas

    2016-03-16

    Natural armadillo repeat proteins (nArmRP) like importin-α or β-catenin bind their target peptides such that each repeat interacts with a dipeptide unit within the stretched target peptide. However, this modularity is imperfect and also restricted to short peptide stretches of usually four to six consecutive amino acids. Here we report the development and characterization of a regularized and truly modular peptide-specific binding protein, based on designed armadillo repeat proteins (dArmRP), binding to peptides of alternating lysine and arginine residues (KR)n. dArmRP were obtained from nArmRP through cycles of extensive protein engineering, which rendered them more uniform. This regularity is reflected in the consistent binding of dArmRP to (KR)-peptides, where affinities depend on the lengths of target peptides and the number of internal repeats in a very systematic manner, thus confirming the modularity of the interaction. This exponential dependency between affinity and recognition length suggests that each module adds a constant increment of binding energy to sequence-specific recognition. This relationship was confirmed by comprehensive mutagenesis studies that also reveal the importance of individual peptide side chains. The 1.83 Å resolution crystal structure of a dArmRP with five identical internal repeats in complex with the cognate (KR)5 peptide proves a modular binding mode, where each dipeptide is recognized by one internal repeat. The confirmation of this true modularity over longer peptide stretches lays the ground for the design of binders with different specificities and tailored affinities by the assembly of dipeptide-specific modules based on armadillo repeats. PMID:26878586

  17. Batroxobin binds fibrin with higher affinity and promotes clot expansion to a greater extent than thrombin.

    PubMed

    Vu, Trang T; Stafford, Alan R; Leslie, Beverly A; Kim, Paul Y; Fredenburgh, James C; Weitz, Jeffrey I

    2013-06-01

    Batroxobin is a thrombin-like serine protease from the venom of Bothrops atrox moojeni that clots fibrinogen. In contrast to thrombin, which releases fibrinopeptide A and B from the NH2-terminal domains of the Aα- and Bβ-chains of fibrinogen, respectively, batroxobin only releases fibrinopeptide A. Because the mechanism responsible for these differences is unknown, we compared the interactions of batroxobin and thrombin with the predominant γA/γA isoform of fibrin(ogen) and the γA/γ' variant with an extended γ-chain. Thrombin binds to the γ'-chain and forms a higher affinity interaction with γA/γ'-fibrin(ogen) than γA/γA-fibrin(ogen). In contrast, batroxobin binds both fibrin(ogen) isoforms with similar high affinity (Kd values of about 0.5 μM) even though it does not interact with the γ'-chain. The batroxobin-binding sites on fibrin(ogen) only partially overlap with those of thrombin because thrombin attenuates, but does not abrogate, the interaction of γA/γA-fibrinogen with batroxobin. Furthermore, although both thrombin and batroxobin bind to the central E-region of fibrinogen with a Kd value of 2-5 μM, the α(17-51) and Bβ(1-42) regions bind thrombin but not batroxobin. Once bound to fibrin, the capacity of batroxobin to promote fibrin accretion is 18-fold greater than that of thrombin, a finding that may explain the microvascular thrombosis that complicates envenomation by B. atrox moojeni. Therefore, batroxobin binds fibrin(ogen) in a manner distinct from thrombin, which may contribute to its higher affinity interaction, selective fibrinopeptide A release, and prothrombotic properties. PMID:23612970

  18. Regulation of protein-ligand binding affinity by hydrogen bond pairing.

    PubMed

    Chen, Deliang; Oezguen, Numan; Urvil, Petri; Ferguson, Colin; Dann, Sara M; Savidge, Tor C

    2016-03-01

    Hydrogen (H)-bonds potentiate diverse cellular functions by facilitating molecular interactions. The mechanism and the extent to which H-bonds regulate molecular interactions are a largely unresolved problem in biology because the H-bonding process continuously competes with bulk water. This interference may significantly alter our understanding of molecular function, for example, in the elucidation of the origin of enzymatic catalytic power. We advance this concept by showing that H-bonds regulate molecular interactions via a hitherto unappreciated donor-acceptor pairing mechanism that minimizes competition with water. On the basis of theoretical and experimental correlations between H-bond pairings and their effects on ligand binding affinity, we demonstrate that H-bonds enhance receptor-ligand interactions when both the donor and acceptor have either significantly stronger or significantly weaker H-bonding capabilities than the hydrogen and oxygen atoms in water. By contrast, mixed strong-weak H-bond pairings decrease ligand binding affinity due to interference with bulk water, offering mechanistic insight into why indiscriminate strengthening of receptor-ligand H-bonds correlates poorly with experimental binding affinity. Further support for the H-bond pairing principle is provided by the discovery and optimization of lead compounds targeting dietary melamine and Clostridium difficile toxins, which are not realized by traditional drug design methods. Synergistic H-bond pairings have therefore evolved in the natural design of high-affinity binding and provide a new conceptual framework to evaluate the H-bonding process in biological systems. Our findings may also guide wider applications of competing H-bond pairings in lead compound design and in determining the origin of enzymatic catalytic power. PMID:27051863

  19. "DAKLI": a multipurpose ligand with high affinity and selectivity for dynorphin (kappa opioid) binding sites.

    PubMed Central

    Goldstein, A; Nestor, J J; Naidu, A; Newman, S R

    1988-01-01

    We describe a synthetic ligand, "DAKLI" (Dynorphin A-analogue Kappa LIgand), related to the opioid peptide dynorphin A. A single reactive amino group at the extended carboxyl terminus permits various reporter groups to be attached, such as 125I-labeled Bolton-Hunter reagent, fluorescein isothiocyanate, or biotin. These derivatives have high affinity and selectivity for the dynorphin (kappa opioid) receptor. An incidental finding is that untreated guinea pig brain membranes have saturable avidin binding sites. PMID:2902630

  20. Regulation of protein-ligand binding affinity by hydrogen bond pairing

    PubMed Central

    Chen, Deliang; Oezguen, Numan; Urvil, Petri; Ferguson, Colin; Dann, Sara M.; Savidge, Tor C.

    2016-01-01

    Hydrogen (H)-bonds potentiate diverse cellular functions by facilitating molecular interactions. The mechanism and the extent to which H-bonds regulate molecular interactions are a largely unresolved problem in biology because the H-bonding process continuously competes with bulk water. This interference may significantly alter our understanding of molecular function, for example, in the elucidation of the origin of enzymatic catalytic power. We advance this concept by showing that H-bonds regulate molecular interactions via a hitherto unappreciated donor-acceptor pairing mechanism that minimizes competition with water. On the basis of theoretical and experimental correlations between H-bond pairings and their effects on ligand binding affinity, we demonstrate that H-bonds enhance receptor-ligand interactions when both the donor and acceptor have either significantly stronger or significantly weaker H-bonding capabilities than the hydrogen and oxygen atoms in water. By contrast, mixed strong-weak H-bond pairings decrease ligand binding affinity due to interference with bulk water, offering mechanistic insight into why indiscriminate strengthening of receptor-ligand H-bonds correlates poorly with experimental binding affinity. Further support for the H-bond pairing principle is provided by the discovery and optimization of lead compounds targeting dietary melamine and Clostridium difficile toxins, which are not realized by traditional drug design methods. Synergistic H-bond pairings have therefore evolved in the natural design of high-affinity binding and provide a new conceptual framework to evaluate the H-bonding process in biological systems. Our findings may also guide wider applications of competing H-bond pairings in lead compound design and in determining the origin of enzymatic catalytic power. PMID:27051863

  1. Learning a peptide-protein binding affinity predictor with kernel ridge regression

    PubMed Central

    2013-01-01

    Background The cellular function of a vast majority of proteins is performed through physical interactions with other biomolecules, which, most of the time, are other proteins. Peptides represent templates of choice for mimicking a secondary structure in order to modulate protein-protein interaction. They are thus an interesting class of therapeutics since they also display strong activity, high selectivity, low toxicity and few drug-drug interactions. Furthermore, predicting peptides that would bind to a specific MHC alleles would be of tremendous benefit to improve vaccine based therapy and possibly generate antibodies with greater affinity. Modern computational methods have the potential to accelerate and lower the cost of drug and vaccine discovery by selecting potential compounds for testing in silico prior to biological validation. Results We propose a specialized string kernel for small bio-molecules, peptides and pseudo-sequences of binding interfaces. The kernel incorporates physico-chemical properties of amino acids and elegantly generalizes eight kernels, comprised of the Oligo, the Weighted Degree, the Blended Spectrum, and the Radial Basis Function. We provide a low complexity dynamic programming algorithm for the exact computation of the kernel and a linear time algorithm for it’s approximation. Combined with kernel ridge regression and SupCK, a novel binding pocket kernel, the proposed kernel yields biologically relevant and good prediction accuracy on the PepX database. For the first time, a machine learning predictor is capable of predicting the binding affinity of any peptide to any protein with reasonable accuracy. The method was also applied to both single-target and pan-specific Major Histocompatibility Complex class II benchmark datasets and three Quantitative Structure Affinity Model benchmark datasets. Conclusion On all benchmarks, our method significantly (p-value ≤ 0.057) outperforms the current state-of-the-art methods at predicting

  2. Bioinspired assemblies of plant cell wall polymers unravel the affinity properties of carbohydrate-binding modules.

    PubMed

    Paës, Gabriel; von Schantz, Laura; Ohlin, Mats

    2015-09-01

    Lignocellulose-acting enzymes play a central role in the biorefinery of plant biomass to make fuels, chemicals and materials. These enzymes are often appended to carbohydrate binding modules (CBMs) that promote substrate targeting. When used in plant materials, which are complex assemblies of polymers, the binding properties of CBMs can be difficult to understand and predict, thus limiting the efficiency of enzymes. In order to gain more information on the binding properties of CBMs, some bioinspired model assemblies that contain some of the polymers and covalent interactions found in the plant cell walls have been designed. The mobility of three engineered CBMs has been investigated by FRAP in these assemblies, while varying the parameters related to the polymer concentration, the physical state of assemblies and the oligomerization state of CBMs. The features controlling the mobility of the CBMs in the assemblies have been quantified and hierarchized. We demonstrate that the parameters can have additional or opposite effects on mobility, depending on the CBM tested. We also find evidence of a relationship between the mobility of CBMs and their binding strength. Overall, bioinspired assemblies are able to reveal the unique features of affinity of CBMs. In particular, the results show that oligomerization of CBMs and the presence of ferulic acid motifs in the assemblies play an important role in the binding affinity of CBMs. Thus we propose that these features should be finely tuned when CBMs are used in plant cell walls to optimise bioprocesses. PMID:26189625

  3. DNA-binding affinity and nuclease activity of two cytotoxic copper terpyridine complexes.

    PubMed

    Shi, Pengfei; Lin, Miaoxin; Zhu, Jianhui; Zhang, Yangmiao; Jiang, Qin

    2009-01-01

    Two copper(II) terpyridine complexes, [Cu(atpy)(NO(3))(H(2)O)](NO(3)) 3H(2)O (1) and [Cu(ttpy)(NO(3))(2)] (2) (atpy = 4'-p-N9-adeninylmethyl-phenyl-2,2':6,2''-terpyridine; ttpy = 4'-p-tolyl-2,2':6,2''-terpyridine) exhibited high cytotoxicity, with average ten times more potency than cisplatin against the human cervix carcinoma cell line (HeLa), the human liver carcinoma cell line (HepG2), the human galactophore carcinoma cell line (MCF7), and the human prostate carcinoma cell line (PC-3). The cytotoxicity of the complex 1 was lower than that of the complex 2. Both complexes showed more efficient oxidative DNA cleavage activity under irradiation with UV light at 260 nm than in the presence of ascorbic acid. Especially, complex 1 exhibited evident photoinduced double-stranded DNA cleavage activity. The preliminary mechanism experiments revealed that hydrogen peroxide was involved in the oxidative DNA damage induced by both complexes. From the absorption titration data, the DNA-binding affinity of the complexes with surpersoiled plasmid pUC19 DNA, polydAdT, and polydGdC was calculated and complex 2 showed higher binding affinity than complex 1 with all these substrates. The DNA cleavage ability and DNA-binding affinity of both complexes depended on the substituent group on the terpyrdine ligands. PMID:19705364

  4. A comparison of myocardial beta-adrenoreceptor density and ligand binding affinity among selected teleost fishes.

    PubMed

    Olsson, H I; Yee, N; Shiels, H A; Brauner, C; Farrell, A P

    2000-11-01

    This study quantified the cell surface beta-adrenoreceptor density and ligand binding affinity in the ventricular tissue of seven teleost species; skipjack tuna (Katsowonus pelamis), yellowfin tuna (Thunnus albacares), Pacific mackerel (Scomber japonicus), mahimahi (dolphin fish; Coryphaena hippurus), sockeye salmon (Oncorhynchus nerka), rainbow trout (Oncorhynchus mykiss) and an Antarctic nototheniid (Trematomus bernacchii). Beta-Adrenoreceptor density varied by almost fourfold among these species, being highest for the athletic fish: sockeye salmon among the salmonids and skipjack tuna among the scombrids. Beta-Adrenoreceptor density was lowest for the Antarctic icefish. Beta-Adrenoreceptor binding affinity varied by almost threefold. We conclude that there is a significant species-specific variability in myocardial beta-adrenoreceptor density and binding affinity and these interspecific differences cannot be attributed to temperature even though intraspecifically cold temperature can stimulate an increase in myocardial beta-adrenoreceptor density. Instead, we suggest that interspecifically myocardial beta-adrenoreceptor density is highest in fish that inhabit tropical water. PMID:11128445

  5. The Quantum Nature of Drug-Receptor Interactions: Deuteration Changes Binding Affinities for Histamine Receptor Ligands

    PubMed Central

    Repič, Matej; Zakšek, Maja; Kotnik, Kristina; Fijan, Estera; Mavri, Janez

    2016-01-01

    In this article we report a combined experimental and computational study concerning the effects of deuteration on the binding of histamine and two other histaminergic agonists to 3H-tiotidine-labeled histamine H2 receptor in neonatal rat astrocytes. Binding affinities were measured by displacing radiolabeled tiotidine from H2 receptor binding sites present on cultured neonatal rat astrocytes. Quantum-chemical calculations were performed by employing the empirical quantization of nuclear motion within a cluster model of the receptor binding site extracted from the homology model of the entire H2 receptor. Structure of H2 receptor built by homology modelling is attached in the supporting information (S1 Table) Experiments clearly demonstrate that deuteration affects the binding by increasing the affinity for histamine and reducing it for 2-methylhistamine, while basically leaving it unchanged for 4-methylhistamine. Ab initio quantum-chemical calculations on the cluster system extracted from the homology H2 model along with the implicit quantization of the acidic N–H and O–H bonds demonstrate that these changes in the binding can be rationalized by the altered strength of the hydrogen bonding upon deuteration known as the Ubbelohde effect. Our computational analysis also reveals a new mechanism of histamine binding, which underlines an important role of Tyr250 residue. The present work is, to our best knowledge, the first study of nuclear quantum effects on ligand receptor binding. The ligand H/D substitution is relevant for therapy in the context of perdeuterated and thus more stable drugs that are expected to enter therapeutic practice in the near future. Moreover, presented approach may contribute towards understanding receptor activation, while a distant goal remains in silico discrimination between agonists and antagonists based on the receptor structure. PMID:27159606

  6. The Quantum Nature of Drug-Receptor Interactions: Deuteration Changes Binding Affinities for Histamine Receptor Ligands.

    PubMed

    Kržan, Mojca; Vianello, Robert; Maršavelski, Aleksandra; Repič, Matej; Zakšek, Maja; Kotnik, Kristina; Fijan, Estera; Mavri, Janez

    2016-01-01

    In this article we report a combined experimental and computational study concerning the effects of deuteration on the binding of histamine and two other histaminergic agonists to 3H-tiotidine-labeled histamine H2 receptor in neonatal rat astrocytes. Binding affinities were measured by displacing radiolabeled tiotidine from H2 receptor binding sites present on cultured neonatal rat astrocytes. Quantum-chemical calculations were performed by employing the empirical quantization of nuclear motion within a cluster model of the receptor binding site extracted from the homology model of the entire H2 receptor. Structure of H2 receptor built by homology modelling is attached in the supporting information (S1 Table) Experiments clearly demonstrate that deuteration affects the binding by increasing the affinity for histamine and reducing it for 2-methylhistamine, while basically leaving it unchanged for 4-methylhistamine. Ab initio quantum-chemical calculations on the cluster system extracted from the homology H2 model along with the implicit quantization of the acidic N-H and O-H bonds demonstrate that these changes in the binding can be rationalized by the altered strength of the hydrogen bonding upon deuteration known as the Ubbelohde effect. Our computational analysis also reveals a new mechanism of histamine binding, which underlines an important role of Tyr250 residue. The present work is, to our best knowledge, the first study of nuclear quantum effects on ligand receptor binding. The ligand H/D substitution is relevant for therapy in the context of perdeuterated and thus more stable drugs that are expected to enter therapeutic practice in the near future. Moreover, presented approach may contribute towards understanding receptor activation, while a distant goal remains in silico discrimination between agonists and antagonists based on the receptor structure. PMID:27159606

  7. Copper binding to soil fulvic and humic acids: NICA-Donnan modeling and conditional affinity spectra.

    PubMed

    Xu, Jinling; Tan, Wenfeng; Xiong, Juan; Wang, Mingxia; Fang, Linchuan; Koopal, Luuk K

    2016-07-01

    Binding of Cu(II) to soil fulvic acid (JGFA), soil humic acids (JGHA, JLHA), and lignite-based humic acid (PAHA) was investigated through NICA-Donnan modeling and conditional affinity spectrum (CAS). It is to extend the knowledge of copper binding by soil humic substances (HS) both in respect of enlarging the database of metal ion binding to HS and obtaining a good insight into Cu binding to the functional groups of FA and HA by using the NICA-Donnan model to unravel the intrinsic and conditional affinity spectra. Results showed that Cu binding to HS increased with increasing pH and decreasing ionic strength. The amount of Cu bound to the HAs was larger than the amount bound to JGFA. Milne's generic parameters did not provide satisfactory predictions for the present soil HS samples, while material-specific NICA-Donnan model parameters described and predicted Cu binding to the HS well. Both the 'low' and 'high' concentration fitting procedures indicated a substantial bidentate structure of the Cu complexes with HS. By means of CAS underlying NICA isotherm, which was scarcely used, the nature of the binding at different solution conditions for a given sample and the differences in binding mode were illustrated. It was indicated that carboxylic group played an indispensable role in Cu binding to HS in that the carboxylic CAS had stronger conditional affinity than the phenolic distribution due to its large degree of proton dissociation. The fact was especially true for JGFA and JLHA which contain much larger amount of carboxylic groups, and the occupation of phenolic sites by Cu was negligible. Comparable amounts of carboxylic and phenolic groups on PAHA and JGHA, increased the occupation of phenolic type sites by Cu. The binding strength of PAHA-Cu and JGHA-Cu was stronger than that of JGFA-Cu and JLHA-Cu. The presence of phenolic groups increased the chance of forming more stable complexes, such as the salicylate-Cu or catechol-Cu type structures. PMID:27061366

  8. Quantification of transcription factor-DNA binding affinity in a living cell.

    PubMed

    Belikov, Sergey; Berg, Otto G; Wrange, Örjan

    2016-04-20

    The apparent dissociation constant (Kd) for specific binding of glucocorticoid receptor (GR) and androgen receptor (AR) to DNA was determined in vivo in Xenopus oocytes. The total nuclear receptor concentration was quantified as specifically retained [(3)H]-hormone in manually isolated oocyte nuclei. DNA was introduced by nuclear microinjection of single stranded phagemid DNA, chromatin is then formed during second strand synthesis. The fraction of DNA sites occupied by the expressed receptor was determined by dimethylsulphate in vivo footprinting and used for calculation of the receptor-DNA binding affinity. The forkhead transcription factor FoxA1 enhanced the DNA binding by GR with an apparent Kd of ∼1 μM and dramatically stimulated DNA binding by AR with an apparent Kd of ∼0.13 μM at a composite androgen responsive DNA element containing one FoxA1 binding site and one palindromic hormone receptor binding site known to bind one receptor homodimer. FoxA1 exerted a weak constitutive- and strongly cooperative DNA binding together with AR but had a less prominent effect with GR, the difference reflecting the licensing function of FoxA1 at this androgen responsive DNA element.

  9. Synthesis and receptor binding of N-substituted tropane derivatives. High-affinity ligands for the cocaine receptor

    SciTech Connect

    Milius, R.A.; Saha, J.K.; Madras, B.K.; Neumeyer, J.L. )

    1991-05-01

    The synthesis and pharmacological characterization of a series of N-substituted 3-(4-fluorophenyl)tropane derivatives is reported. The compounds displayed binding characteristics that paralleled those of cocaine, and several had substantially higher affinity at cocaine recognition sites. Conjugate addition of 4-fluorophenyl magnesium bromide to anhydroecgonine methyl ester gave 2 beta-(carbomethoxy)-3 beta-(4-fluorophenyl)tropane (4a, designated CFT, also known as WIN 35,428) after flash chromatography. N demethylation of 4a was effected by Zn/HOAc reduction of the corresponding 2,2,2-trichloroethyl carbamate to give 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)nortropane (5), which was alkylated with allyl bromide to afford the N-allyl analogue, 6. The N-propyl analogue, 7, was prepared by catalytic reduction (Pd/C) of 6. The most potent analogue, 4a, was tritiated at a specific activity of 81.3 Ci/mmol. ({sup 3}H)4a bound rapidly and reversibly to caudate putamen membranes; the two-component binding curve typical of cocaine analogues was observed. Equilibrium was achieved within 2 h and was stable for at least 4 h. High- and low-affinity Kd values observed for ({sup 3}H)4a (4.7 and 60 nM, respectively) were more than 4 times lower than those for ({sup 3}H)cocaine, and the density of binding sites (Bmax = 50 pmol/g, high, and 290 pmol/g, low) for the two drugs were comparable. Nonspecific binding of ({sup 3}H)4a was 5-10% of total binding.

  10. Specific Internalisation of Gold Nanoparticles into Engineered Porous Protein Cages via Affinity Binding.

    PubMed

    Paramelle, David; Peng, Tao; Free, Paul; Fernig, David G; Lim, Sierin; Tomczak, Nikodem

    2016-01-01

    Porous protein cages are supramolecular protein self-assemblies presenting pores that allow the access of surrounding molecules and ions into their core in order to store and transport them in biological environments. Protein cages' pores are attractive channels for the internalisation of inorganic nanoparticles and an alternative for the preparation of hybrid bioinspired nanoparticles. However, strategies based on nanoparticle transport through the pores are largely unexplored, due to the difficulty of tailoring nanoparticles that have diameters commensurate with the pores size and simultaneously displaying specific affinity to the cages' core and low non-specific binding to the cages' outer surface. We evaluated the specific internalisation of single small gold nanoparticles, 3.9 nm in diameter, into porous protein cages via affinity binding. The E2 protein cage derived from the Geobacillus stearothermophilus presents 12 pores, 6 nm in diameter, and an empty core of 13 nm in diameter. We engineered the E2 protein by site-directed mutagenesis with oligohistidine sequences exposing them into the cage's core. Dynamic light scattering and electron microscopy analysis show that the structures of E2 protein cages mutated with bis- or penta-histidine sequences are well conserved. The surface of the gold nanoparticles was passivated with a self-assembled monolayer made of a mixture of short peptidols and thiolated alkane ethylene glycol ligands. Such monolayers are found to provide thin coatings preventing non-specific binding to proteins. Further functionalisation of the peptide coated gold nanoparticles with Ni2+ nitrilotriacetic moieties enabled the specific binding to oligohistidine tagged cages. The internalisation via affinity binding was evaluated by electron microscopy analysis. From the various mutations tested, only the penta-histidine mutated E2 protein cage showed repeatable and stable internalisation. The present work overcomes the limitations of currently

  11. Binding affinity prediction for protein-ligand complexes based on β contacts and B factor.

    PubMed

    Liu, Qian; Kwoh, Chee Keong; Li, Jinyan

    2013-11-25

    Accurate determination of protein-ligand binding affinity is a fundamental problem in biochemistry useful for many applications including drug design and protein-ligand docking. A number of scoring functions have been proposed for the prediction of protein-ligand binding affinity. However, accurate prediction is still a challenging problem because poor performance is often seen in the evaluation under the leave-one-cluster-out cross-validation (LCOCV). We introduce a new scoring function named B2BScore to improve the prediction performance. B2BScore integrates two physicochemical properties for protein-ligand binding affinity prediction. One is the property of β contacts. A β contact between two atoms requires no other atoms to interrupt the atomic contact and assumes that the two atoms should have enough direct contact area. The other is the property of B factor to capture the atomic mobility in the dynamic protein-ligand binding process. Tested on the PDBBind2009 data set, B2BScore shows superior prediction performance to existing methods on independent test data as well as under the LCOCV evaluation framework. In particular, B2BScore achieves a significant LCOCV improvement across 26 protein clusters-a big increase of the averaged Pearson's correlation coefficients from 0.418 to 0.518 and a significant decrease of standard deviation of the coefficients from 0.352 to 0.196. We also identified several important and intuitive contact descriptors of protein-ligand binding through the random forest learning in B2BScore. Some of these descriptors are closely related to contacts between carbon atoms without covalent-bond oxygen/nitrogen, preferred contacts of metal ions, interfacial backbone atoms from proteins, or π rings. Some others are negative descriptors relating to those contacts with nitrogen atoms without covalent-bond hydrogens or nonpreferred contacts of metal ions. These descriptors can be directly used to guide protein-ligand docking.

  12. Specific Internalisation of Gold Nanoparticles into Engineered Porous Protein Cages via Affinity Binding

    PubMed Central

    Peng, Tao; Free, Paul; Fernig, David G.; Lim, Sierin; Tomczak, Nikodem

    2016-01-01

    Porous protein cages are supramolecular protein self-assemblies presenting pores that allow the access of surrounding molecules and ions into their core in order to store and transport them in biological environments. Protein cages’ pores are attractive channels for the internalisation of inorganic nanoparticles and an alternative for the preparation of hybrid bioinspired nanoparticles. However, strategies based on nanoparticle transport through the pores are largely unexplored, due to the difficulty of tailoring nanoparticles that have diameters commensurate with the pores size and simultaneously displaying specific affinity to the cages’ core and low non-specific binding to the cages’ outer surface. We evaluated the specific internalisation of single small gold nanoparticles, 3.9 nm in diameter, into porous protein cages via affinity binding. The E2 protein cage derived from the Geobacillus stearothermophilus presents 12 pores, 6 nm in diameter, and an empty core of 13 nm in diameter. We engineered the E2 protein by site-directed mutagenesis with oligohistidine sequences exposing them into the cage’s core. Dynamic light scattering and electron microscopy analysis show that the structures of E2 protein cages mutated with bis- or penta-histidine sequences are well conserved. The surface of the gold nanoparticles was passivated with a self-assembled monolayer made of a mixture of short peptidols and thiolated alkane ethylene glycol ligands. Such monolayers are found to provide thin coatings preventing non-specific binding to proteins. Further functionalisation of the peptide coated gold nanoparticles with Ni2+ nitrilotriacetic moieties enabled the specific binding to oligohistidine tagged cages. The internalisation via affinity binding was evaluated by electron microscopy analysis. From the various mutations tested, only the penta-histidine mutated E2 protein cage showed repeatable and stable internalisation. The present work overcomes the limitations of

  13. Specific Internalisation of Gold Nanoparticles into Engineered Porous Protein Cages via Affinity Binding.

    PubMed

    Paramelle, David; Peng, Tao; Free, Paul; Fernig, David G; Lim, Sierin; Tomczak, Nikodem

    2016-01-01

    Porous protein cages are supramolecular protein self-assemblies presenting pores that allow the access of surrounding molecules and ions into their core in order to store and transport them in biological environments. Protein cages' pores are attractive channels for the internalisation of inorganic nanoparticles and an alternative for the preparation of hybrid bioinspired nanoparticles. However, strategies based on nanoparticle transport through the pores are largely unexplored, due to the difficulty of tailoring nanoparticles that have diameters commensurate with the pores size and simultaneously displaying specific affinity to the cages' core and low non-specific binding to the cages' outer surface. We evaluated the specific internalisation of single small gold nanoparticles, 3.9 nm in diameter, into porous protein cages via affinity binding. The E2 protein cage derived from the Geobacillus stearothermophilus presents 12 pores, 6 nm in diameter, and an empty core of 13 nm in diameter. We engineered the E2 protein by site-directed mutagenesis with oligohistidine sequences exposing them into the cage's core. Dynamic light scattering and electron microscopy analysis show that the structures of E2 protein cages mutated with bis- or penta-histidine sequences are well conserved. The surface of the gold nanoparticles was passivated with a self-assembled monolayer made of a mixture of short peptidols and thiolated alkane ethylene glycol ligands. Such monolayers are found to provide thin coatings preventing non-specific binding to proteins. Further functionalisation of the peptide coated gold nanoparticles with Ni2+ nitrilotriacetic moieties enabled the specific binding to oligohistidine tagged cages. The internalisation via affinity binding was evaluated by electron microscopy analysis. From the various mutations tested, only the penta-histidine mutated E2 protein cage showed repeatable and stable internalisation. The present work overcomes the limitations of currently

  14. Characterization of an amylase-binding component of Streptococcus gordonii G9B.

    PubMed

    Scannapieco, F A; Haraszthy, G G; Cho, M I; Levine, M J

    1992-11-01

    The goal of the present study was to begin characterizing the amylase-binding component(s) on the surface of Streptococcus gordonii G9B. Alkali extracts but not phenol-water extracts of this bacterium inhibited 125I-amylase binding to S. gordonii G9B. To identify the bacterial components involved in amylase binding, the alkali extract was subjected to affinity chromatography on amylase-Sepharose. Immunoblotting with a rabbit antiserum against S. gordonii G9B revealed that a 20-kDa streptococcal component was eluted from the amylase-Sepharose with 1% sodium dodecyl sulfate (SDS), 2 M KSCN, or 0.1 M sodium citrate buffer, pH 4.5. Subsequently, the 20-kDa component was prepared from alkali extracts by electroelution from preparative SDS electrophoresis or by gel filtration chromatography. This component was trypsin sensitive, and an antibody raised against it inhibited the binding of 125I-amylase to S. gordonii G9B. Indirect immunofluorescence microscopy and immunogold electron microscopy demonstrated that both bound amylase and the 20-kDa component were localized to the cell division septum on dividing cells or to polar zones on single cells. In addition, exponentially growing bacteria bound more 125I-amylase than stationary-phase cells did. Collectively, these results suggest that a 20-kDa amylase-binding component is present on the surface of the nascent streptococcal cell wall.

  15. Measurement of Cationic and Intracellular Modulation of Integrin Binding Affinity by AFM-Based Nanorobot

    PubMed Central

    Patterson, Kevin C.; Yang, Ruiguo; Zeng, Bixi; Song, Bo; Wang, Shouye; Xi, Ning; Basson, Marc D.

    2013-01-01

    Integrins are dynamic transmembrane cation-dependent heterodimers that both anchor cells in position and transduce signals into and out of cells. We used an atomic force microscope (AFM)-based nanorobotic system to measure integrin-binding forces in intact human intestinal epithelial Caco-2 cells. The AFM-based nanorobot enables human-directed, high-accuracy probe positioning and site-specific investigations. Functionalizing the AFM probe with an arginine-glycine-aspartate (RGD)-containing sequence (consensus binding sequence for integrins) allowed us to detect a series of peptide-cell membrane interactions with a median binding force of 115.1 ± 4.9 pN that were not detected in control interactions. Chelating divalent cations from the culture medium abolished these interactions, as did inhibiting intracellular focal adhesion kinase (FAK) using Y15. Adding 1 mM Mg2+ to the medium caused a rightward shift in the force-binding curve. Adding 1 mM Ca2+ virtually abolished the RGD-membrane specific interactions and blocked the Mg2+ effects. Cell adhesion assays demonstrated parallel effects of divalent cations and the FAK inhibitor on cell adhesion. These results demonstrate direct modulation of integrin-binding affinity by both divalent cations and intracellular signal inhibition. Additionally, three binding states (nonspecific, specific inactivated, and specific activated) were delineated from affinity measurements. Although other research has assumed that this process of integrin conformational change causes altered ligand binding, in this work we directly measured these three states in individual integrins in a physiologically based study. PMID:23823222

  16. Activated G-protein releases cGMP from high affinity binding sites on PDE from toad rod outer segments (ROS)

    SciTech Connect

    Yuen, P.S.T.; Walseth, T.F.; Panter, S.S.; Sundby, S.R.; Graeff, R.M.; Goldberg, N.D.

    1987-05-01

    cGMP binding proteins in toad ROS were identified by direct photoaffinity labeling (PAL) with /sup 32/P-cGMP and quantified by retention of complexes on nitrocellulose filters. By PAL, high affinity sites were present on the ..cap alpha.. and ..beta.. subunits of the cGMP-specific phosphodiesterase (PDE) which have MW/sub app/ of 94 and 90 kDa. A doublet was deduced from its photolabeling properties to represent PDE/sub ..gamma../ photocrosslinked with PDE/sub ..cap alpha../ or PDE/sub ..beta../, respectively. cGMP prebound to these high affinity sites was released by light-activated G-protein or its ..cap alpha.. subunit complexed with GTP..gamma..S; this inhibition of cGMP binding to PDE did not result from decreased cGMP availability due to enhanced hydrolysis. A low affinity cGMP binding component identified by PAL is tightly associated with ROS membranes. Apparent ATP/light-dependent stimulation of cGMP binding was shown to result from light activated cGMP hydrolysis in conjunction with ATP-promoted conversion of GMP to GDP/GTP and increased GDP/GTP binding. These findings coincide with a model for light-related regulation of cGMP binding and metabolism predicted from intact and cellfree kinetic measurements: in the dark state the cGMP hydrolic rate is constrained by the availability of cGMP because of its binding to high affinity sites on PDE. Light activated G-protein releases cGMP from these sites and allows for its redistribution to lower affinity sites represented by PDE catalytic site(s) and possible cGMP-dependent membrane cation channels.

  17. Relative penicillin G resistance in Neisseria meningitidis and reduced affinity of penicillin-binding protein 3.

    PubMed Central

    Mendelman, P M; Campos, J; Chaffin, D O; Serfass, D A; Smith, A L; Sáez-Nieto, J A

    1988-01-01

    We examined clinical isolates of Neisseria meningitidis relatively resistant to penicillin G (mean MIC, 0.3 micrograms/ml; range, 0.1 to 0.7 micrograms/ml), which were isolated from blood and cerebrospinal fluid for resistance mechanisms, by using susceptible isolates (mean MIC, less than or equal to 0.06 micrograms/ml) for comparison. The resistant strains did not produce detectable beta-lactamase activity, otherwise modify penicillin G, or bind less total penicillin. Penicillin-binding protein (PBP) 3 of the six resistant isolates tested uniformly bound less penicillin G in comparison to the same PBP of four susceptible isolates. Reflecting the reduced binding affinity of PBP 3 of the two resistant strains tested, the amount of 3H-labeled penicillin G required for half-maximal binding was increased in comparison with that of PBP 3 of the two susceptible isolates. We conclude that the mechanism of resistance in these meningococci relatively resistant to penicillin G was decreased affinity of PBP 3. Images PMID:3134848

  18. Dopamine transporter oligomerization: Impact of combining protomers with differential cocaine analog binding affinities

    PubMed Central

    Zhen, Juan; Antonio, Tamara; Cheng, Shu-Yuan; Ali, Solav; Jones, Kymry T.; Reith, Maarten E. A.

    2015-01-01

    Previous studies point to quaternary assembly of dopamine transporters (DATs) in oligomers. However, it is not clear whether the protomers function independently in the oligomer. Is each protomer an entirely separate unit that takes up dopamine and is inhibited by drugs known to block DAT function? In this work, human embryonic kidney 293 cells were co-transfected with DAT constructs possessing differential binding affinities for the phenyltropane cocaine analog, [3H]WIN35,428. It was assessed whether the binding properties in co-expressing cells capable of forming hetero-oligomers differ from those in preparations obtained from mixed singly transfected cells where such oligomers cannot occur. A method is described that replaces laborious “mixing” experiments with an in silico method predicting binding parameters from those observed for the singly expressed constructs. Among 5 pairs of constructs tested, statistically significant interactions were found between protomers of wild-type (WT) and D313N, WT and D345N, and WT and D436N. Compared with predicted Kd values of [3H]WIN35,428 binding to the non-interacting pairs, the observed affinity of the former pair was increased 1.7 fold while the latter two were reduced 2.2 and 4.1 fold, respectively. This is the first report of an influence of protomer composition on the properties of a DAT inhibitor, indicating cooperativity within the oligomer. PMID:25580950

  19. Identification of a high affinity nucleocapsid protein binding element from the bovine leukemia virus genome.

    PubMed

    Yildiz, F Zehra; Babalola, Kathlene; Summers, Michael F

    2013-02-01

    Retroviral genome recognition is mediated by interactions between the nucleocapsid (NC) domain of the virally encoded Gag polyprotein and cognate RNA packaging elements that, for most retroviruses, appear to reside primarily within the 5'-untranslated region (5'-UTR) of the genome. Recent studies suggest that a major packaging determinant of bovine leukemia virus (BLV), a member of the human T-cell leukemia virus (HTLV)/BLV family and a non-primate animal model for HTLV-induced leukemogenesis, resides within the gag open reading frame. We have prepared and purified the recombinant BLV NC protein and conducted electrophoretic mobility shift and isothermal titration calorimetry studies with RNA fragments corresponding to these proposed packaging elements. The gag-derived RNAs did not exhibit significant affinity for NC, suggesting an alternate role in packaging. However, an 83-nucleotide fragment of the 5'-UTR that resides just upstream of the gag start codon binds NC stoichiometrically and with high affinity (K(d)=136±21 nM). These nucleotides were predicted to form tandem hairpin structures, and studies with smaller fragments indicate that the NC binding site resides exclusively within the distal hairpin (residues G369-U399, K(d)=67±8 nM at physiological ionic strength). Unlike all other structurally characterized retroviral NC binding RNAs, this fragment is not expected to contain exposed guanosines, suggesting that RNA binding may be mediated by a previously uncharacterized mechanism.

  20. Purification of high affinity benzodiazepine receptor binding site fragments from rat brain

    SciTech Connect

    Klotz, K.L.

    1984-01-01

    In central nervous system benzodiazepine recognition sites occur on neuronal cell surfaces as one member of a multireceptor complex, including recognition sites for benzodiazepines, gamma aminobutyric acid (GABA), barbiturates and a chloride ionophore. During photoaffinity labelling, the benzodiazepine agonist, /sup 3/H-flunitrazepam, is irreversibly bound to central benzodiazepine high affinity recognition sites in the presence of ultraviolet light. In these studies a /sup 3/H-flunitrazepam radiolabel was used to track the isolation and purification of high affinity agonist binding site fragments from membrane-bound benzodiazepine receptor in rat brain. The authors present a method for limited proteolysis of /sup 3/H-flunitrazepam photoaffinity labeled rat brain membranes, generating photolabeled benzodiazepine receptor fragments containing the agonist binding site. Using trypsin chymotrypsin A/sub 4/, or a combination of these two proteases, they have demonstrated the extent and time course for partial digestion of benzodiazepine receptor, yielding photolabeled receptor binding site fragments. These photolabeled receptor fragments have been further purified on the basis of size, using ultrafiltration, gel permeation chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as on the basis of hydrophobicity, using a high performance liquid chromatography (HPLC) precolumn, several HPLC elution schemes, and two different HPLC column types. Using these procedures, they have purified three photolabeled benzodiazepine receptor fragments containing the agonist binding site which appear to have a molecular weight of less than 2000 daltons each.

  1. ANALYSIS OF DRUG-PROTEIN BINDING BY ULTRAFAST AFFINITY CHROMATOGRAPHY USING IMMOBILIZED HUMAN SERUM ALBUMIN

    PubMed Central

    Mallik, Rangan; Yoo, Michelle J.; Briscoe, Chad J.; Hage, David S.

    2010-01-01

    Human serum albumin (HSA) was explored for use as a stationary phase and ligand in affinity microcolumns for the ultrafast extraction of free drug fractions and the use of this information for the analysis of drug-protein binding. Warfarin, imipramine, and ibuprofen were used as model analytes in this study. It was found that greater than 95% extraction of all these drugs could be achieved in as little as 250 ms on HSA microcolumns. The retained drug fraction was then eluted from the same column under isocratic conditions, giving elution in less than 40 s when working at 4.5 mL/min. The chromatographic behavior of this system gave a good fit with that predicted by computer simulations based on a reversible, saturable model for the binding of an injected drug with immobilized HSA. The free fractions measured by this method were found to be comparable to those determined by ultrafiltration, and equilibrium constants estimated by this approach gave good agreement with literature values. Advantages of this method include its speed and the relatively low cost of microcolumns that contain HSA. The ability of HSA to bind many types of drugs also creates the possibility of using the same affinity microcolumn to study and measure the free fractions for a variety of pharmaceutical agents. These properties make this technique appealing for use in drug binding studies and in the high-throughput screening of new drug candidates. PMID:20227701

  2. Identification of high-affinity calmodulin-binding proteins in rat liver

    SciTech Connect

    Hanley, R.M.; Dedman, J.R.; Shenolikar, S.

    1987-03-01

    The Ca/sup 2 +/-dependent binding of (/sup 125/I) calmodulin (CaM) to hepatic proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was utilized to identify CaM binding or acceptor proteins or CAPs. Two proteins of apparent molecular weight of 60,000 (CAP-60) and 45,000 (CAP-45) comprised > 80% of the Ca/sup 2 +/-dependent CaM binding in rat liver cytosol. CAP-60 and CAP-45 were partially purified by a variety of chromatographic steps, including affinity chromatography on CaM Sepharose. CAP-60 possessed a native molecular size of 400,000, indicating it to be the CaM-binding subunit of a larger oligomeric complex. In contrast, CAP-45 was monomeric as judged by gel filtration. Neither CAP-60 nor CAP-45 possessed chromatographic properties consistent with known CaM-dependent enzymes reported in the literature. Two-dimensional peptide mapping provided convincing evidence that CAP-60 and CAP-45 were unrelated to other well-characterized CAPs, namely Ca/sup 2 +/ (CaM)-dependent protein kinase II, calcineurin, or the CaM-dependent cyclic nucleotide phosphodiesterase. The relative abundance and high affinity for CaM could suggest that these novel target proteins, CAP-60 and CAP-45, represent a dominant pathway for CaM action in the mammalian liver.

  3. High-Performance Affinity Chromatography: Applications in Drug-Protein Binding Studies and Personalized Medicine.

    PubMed

    Li, Zhao; Beeram, Sandya R; Bi, Cong; Suresh, D; Zheng, Xiwei; Hage, David S

    2016-01-01

    The binding of drugs with proteins and other agents in serum is of interest in personalized medicine because this process can affect the dosage and action of drugs. The extent of this binding may also vary with a given disease state. These interactions may involve serum proteins, such as human serum albumin or α1-acid glycoprotein, or other agents, such as lipoproteins. High-performance affinity chromatography (HPAC) is a tool that has received increasing interest as a means for studying these interactions. This review discusses the general principles of HPAC and the various approaches that have been used in this technique to examine drug-protein binding and in work related to personalized medicine. These approaches include frontal analysis and zonal elution, as well as peak decay analysis, ultrafast affinity extraction, and chromatographic immunoassays. The operation of each method is described and examples of applications for these techniques are provided. The type of information that can be obtained by these methods is also discussed, as related to the analysis of drug-protein binding and the study of clinical or pharmaceutical samples. PMID:26827600

  4. Binding of angiogenesis inhibitor kringle 5 to its specific ligands by frontal affinity chromatography.

    PubMed

    Bian, Liujiao; Li, Qian; Ji, Xu

    2015-07-01

    The interactions between angiogenesis inhibitor Kringle 5 and its five specific ligands were investigated by frontal affinity chromatography in combination with fluorescence spectra and site-directed molecular docking. The binding constants of trans-4-(aminomethyl) cyclohexane carboxylic acid (AMCHA), epsilon-aminocaproic acid (EACA), benzylamine, 7-aminoheptanoic acid (7-AHA) and L-lysine to Kringle 5 were 19.0×10(3), 7.97×10(3), 6.45×10(3), 6.07×10(3) and 4.04×10(3) L/mol, respectively. The five ligands bound to Kringle 5 on the lysine binding site in equimolar amounts, which was pushed mainly by hydrogen bond and Van der Waals force. This binding affinity was believed to be dependent on the functional group and flexible feature in ligands. This study will provide an important insight into the binding mechanism of angiogenesis inhibitor Kringle 5 to its specific ligands. PMID:25981289

  5. Determining the ice-binding planes of antifreeze proteins by fluorescence-based ice plane affinity.

    PubMed

    Basu, Koli; Garnham, Christopher P; Nishimiya, Yoshiyuki; Tsuda, Sakae; Braslavsky, Ido; Davies, Peter

    2014-01-15

    Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

  6. Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

    PubMed Central

    Basu, Koli; Garnham, Christopher P.; Nishimiya, Yoshiyuki; Tsuda, Sakae; Braslavsky, Ido; Davies, Peter

    2014-01-01

    Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms. PMID:24457629

  7. Designing a mutant CCL2-HSA chimera with high glycosaminoglycan-binding affinity and selectivity.

    PubMed

    Gerlza, Tanja; Winkler, Sophie; Atlic, Aid; Zankl, Christina; Konya, Viktoria; Kitic, Nikola; Strutzmann, Elisabeth; Knebl, Kerstin; Adage, Tiziana; Heinemann, Akos; Weis, Roland; Kungl, Andreas J

    2015-08-01

    Chemokines like CCL2 mediate leukocyte migration to inflammatory sites by binding to G-protein coupled receptors on the target cell as well as to glycosaminoglycans (GAGs) on the endothelium of the inflamed tissue. We have recently shown that the dominant-negative Met-CCL2 mutant Y13A/S21K/Q23R with improved GAG binding affinity is highly bio-active in several animal models of inflammatory diseases. For chronic indications, we have performed here a fusion to human serum albumin (HSA) in order to extend the serum half-life of the chemokine mutant. To compensate a potential drop in GAG-binding affinity due to steric hindrance by HSA, a series of novel CCL2 mutants was generated with additional basic amino acids which were genetically introduced at sites oriented towards the GAG ligand. From this set of mutants, the Met-CCL2 variant Y13A/N17K/S21K/Q23K/S34K exhibited high GAG-binding affinity and a similar selectivity as wild type (wt) CCL2. From a set of different HSA-chemokine chimeric constructs, the linked HSA(C34A)(Gly)4Ser-Met-CCL2(Y13A/N17K/S21K/Q23K/S34K) fusion protein was found to show the best overall GAG-binding characteristics. Molecular modeling demonstrated an energetically beneficial fold of this novel protein chimera. This was experimentally supported by GdmCl-induced unfolding studies, in which the fusion construct exhibited a well-defined secondary structure and a transition point significantly higher than both the wt and the unfused CCL2 mutant protein. Unlike the wt chemokine, the quaternary structure of the HSA-fusion protein is monomeric according to size-exclusion chromatography experiments. In competition experiments, the HSA-fusion construct displaced only two of seven unrelated chemokines from heparan sulfate, whereas the unfused CCL2 mutant protein displaced five other chemokines. The most effective concentration of the HSA-fusion protein in inhibiting CCL2-mediated monocyte attachment to endothelial cells, as detected in the flow chamber

  8. Protein Affinity Chromatography with Purified Yeast DNA Polymerase α Detects Proteins that Bind to DNA Polymerase

    NASA Astrophysics Data System (ADS)

    Miles, Jeff; Formosa, Tim

    1992-02-01

    We have overexpressed the POL1 gene of the yeast Saccharomyces cerevisiae and purified the resulting DNA polymerase α polypeptide in an apparently intact form. We attached the purified DNA polymerase covalently to an agarose matrix and used this matrix to chromatograph extracts prepared from yeast cells. At least six proteins bound to the yeast DNA polymerase α matrix that did not bind to a control matrix. We speculate that these proteins might be DNA polymerase α accessory proteins. Consistent with this interpretation, one of the binding proteins, which we have named POB1 (polymerase one binding), is required for normal chromosome transmission. Mutations in this gene cause increased chromosome loss and an abnormal cell morphology, phenotypes that also occur in the presence of mutations in the yeast α or δ polymerase genes. These results suggest that the interactions detected by polymerase affinity chromatography are biologically relevant and may help to illuminate the architecture of the eukaryotic DNA replication machinery.

  9. Biocompatible silicon surfaces through orthogonal click chemistries and a high affinity silicon oxide binding peptide.

    PubMed

    Hassert, Rayk; Pagel, Mareen; Ming, Zhou; Häupl, Tilmann; Abel, Bernd; Braun, Klaus; Wiessler, Manfred; Beck-Sickinger, Annette G

    2012-10-17

    Multifunctionality is gaining more and more importance in the field of improved biomaterials. Especially peptides feature a broad chemical variability and are versatile mediators between inorganic surfaces and living cells. Here, we synthesized a unique peptide that binds to SiO(2) with nM affinity. We equipped the peptide with the bioactive integrin binding c[RGDfK]-ligand and a fluorescent probe by stepwise Diels-Alder reaction with inverse electron demand and copper(I) catalyzed azide-alkyne cycloaddition. For the first time, we report the generation of a multifunctional peptide by combining these innovative coupling reactions. The resulting peptide displayed an outstanding binding to silicon oxide and induced a significant increase in cell spreading and cell viability of osteoblasts on the oxidized silicon surface.

  10. Affinity purification of proteins binding to kinase inhibitors immobilized on self-assembling monolayers.

    PubMed

    Bantscheff, Marcus; Hobson, Scott; Kuster, Bernhard

    2012-01-01

    Kinase inhibitors represent a relatively new class of drugs that offer novel therapies targeting specific -malfunctioning kinase-mediated signaling pathways in oncology and potentially inflammation. As the ATP binding sites of the ∼500 human kinases are structurally conserved and because most current drugs target the ATP binding site, there is a need to profile all the kinases that a drug may bind and/or inhibit. We have developed a chemical proteomics method that affinity purifies kinases from cell or tissue lysates using kinase inhibitors immobilized on self-assembling monolayers. The method can be applied to assess the selectivity of a given kinase inhibitor and thus to guide its preclinical or clinical development.

  11. Importin {beta}-type nuclear transport receptors have distinct binding affinities for Ran-GTP

    SciTech Connect

    Hahn, Silvia; Schlenstedt, Gabriel

    2011-03-18

    Highlights: {yields} Determination of binding properties of nuclear transport receptor/Ran-GTP complexes. {yields} Biosensor measurements provide constants for dissociation, on-rates, and off-rates. {yields} The affinity of receptors for Ran-GTP is widely divergent. {yields} Dissociation constants differ for three orders of magnitude. {yields} The cellular concentration of yeast Ran is not limiting. -- Abstract: Cargos destined to enter or leave the cell nucleus are typically transported by receptors of the importin {beta} family to pass the nuclear pore complex. The yeast Saccharomyces cerevisiae comprises 14 members of this protein family, which can be divided in importins and exportins. The Ran GTPase regulates the association and dissociation of receptors and cargos as well as the transport direction through the nuclear pore. All receptors bind to Ran exclusively in its GTP-bound state and this event is restricted to the nuclear compartment. We determined the Ran-GTP binding properties of all yeast transport receptors by biosensor measurements and observed that the affinity of importins for Ran-GTP differs significantly. The dissociation constants range from 230 pM to 270 nM, which is mostly based on a variability of the off-rate constants. The divergent affinity of importins for Ran-GTP suggests the existence of a novel mode of nucleocytoplasmic transport regulation. Furthermore, the cellular concentration of {beta}-receptors and of other Ran-binding proteins was determined. We found that the number of {beta}-receptors altogether about equals the amounts of yeast Ran, but Ran-GTP is not limiting in the nucleus. The implications of our results for nucleocytoplasmic transport mechanisms are discussed.

  12. Determination of protein binding affinities within hydrogel-based molecularly imprinted polymers (HydroMIPs).

    PubMed

    EL-Sharif, Hazim F; Hawkins, Daniel M; Stevenson, Derek; Reddy, Subrayal M

    2014-08-01

    Hydrogel-based molecularly imprinted polymers (HydroMIPs) were prepared for several proteins (haemoglobin, myoglobin and catalase) using a family of acrylamide-based monomers. Protein affinity towards the HydroMIPs was investigated under equilibrium conditions and over a range of concentrations using specific binding with Hill slope saturation profiles. We report HydroMIP binding affinities, in terms of equilibrium dissociation constants (Kd) within the micro-molar range (25 ± 4 μM, 44 ± 3 μM, 17 ± 2 μM for haemoglobin, myoglobin and catalase respectively within a polyacrylamide-based MIP). The extent of non-specific binding or cross-selectivity for non-target proteins has also been assessed. It is concluded that both selectivity and affinity for both cognate and non-cognate proteins towards the MIPs were dependent on the concentration and the complementarity of their structures and size. This is tentatively attributed to the formation of protein complexes during both the polymerisation and rebinding stages at high protein concentrations. We have used atomic force spectroscopy to characterize molecular interactions in the MIP cavities using protein-modified AFM tips. Attractive and repulsive force curves were obtained for the MIP and NIP (non-imprinted polymer) surfaces (under protein loaded or unloaded states). Our force data suggest that we have produced selective cavities for the template protein in the MIPs and we have been able to quantify the extent of non-specific protein binding on, for example, a non-imprinted polymer (NIP) control surface.

  13. Halogen bond: its role beyond drug-target binding affinity for drug discovery and development.

    PubMed

    Xu, Zhijian; Yang, Zhuo; Liu, Yingtao; Lu, Yunxiang; Chen, Kaixian; Zhu, Weiliang

    2014-01-27

    Halogen bond has attracted a great deal of attention in the past years for hit-to-lead-to-candidate optimization aiming at improving drug-target binding affinity. In general, heavy organohalogens (i.e., organochlorines, organobromines, and organoiodines) are capable of forming halogen bonds while organofluorines are not. In order to explore the possible roles that halogen bonds could play beyond improving binding affinity, we performed a detailed database survey and quantum chemistry calculation with close attention paid to (1) the change of the ratio of heavy organohalogens to organofluorines along the drug discovery and development process and (2) the halogen bonds between organohalogens and nonbiopolymers or nontarget biopolymers. Our database survey revealed that (1) an obviously increasing trend of the ratio of heavy organohalogens to organofluorines was observed along the drug discovery and development process, illustrating that more organofluorines are worn and eliminated than heavy organohalogens during the process, suggesting that heavy halogens with the capability of forming halogen bonds should have priority for lead optimization; and (2) more than 16% of the halogen bonds in PDB are formed between organohalogens and water, and nearly 20% of the halogen bonds are formed with the proteins that are involved in the ADME/T process. Our QM/MM calculations validated the contribution of the halogen bond to the binding between organohalogens and plasma transport proteins. Thus, halogen bonds could play roles not only in improving drug-target binding affinity but also in tuning ADME/T property. Therefore, we suggest that albeit halogenation is a valuable approach for improving ligand bioactivity, more attention should be paid in the future to the application of the halogen bond for ligand ADME/T property optimization.

  14. Determination of protein binding affinities within hydrogel-based molecularly imprinted polymers (HydroMIPs).

    PubMed

    EL-Sharif, Hazim F; Hawkins, Daniel M; Stevenson, Derek; Reddy, Subrayal M

    2014-08-01

    Hydrogel-based molecularly imprinted polymers (HydroMIPs) were prepared for several proteins (haemoglobin, myoglobin and catalase) using a family of acrylamide-based monomers. Protein affinity towards the HydroMIPs was investigated under equilibrium conditions and over a range of concentrations using specific binding with Hill slope saturation profiles. We report HydroMIP binding affinities, in terms of equilibrium dissociation constants (Kd) within the micro-molar range (25 ± 4 μM, 44 ± 3 μM, 17 ± 2 μM for haemoglobin, myoglobin and catalase respectively within a polyacrylamide-based MIP). The extent of non-specific binding or cross-selectivity for non-target proteins has also been assessed. It is concluded that both selectivity and affinity for both cognate and non-cognate proteins towards the MIPs were dependent on the concentration and the complementarity of their structures and size. This is tentatively attributed to the formation of protein complexes during both the polymerisation and rebinding stages at high protein concentrations. We have used atomic force spectroscopy to characterize molecular interactions in the MIP cavities using protein-modified AFM tips. Attractive and repulsive force curves were obtained for the MIP and NIP (non-imprinted polymer) surfaces (under protein loaded or unloaded states). Our force data suggest that we have produced selective cavities for the template protein in the MIPs and we have been able to quantify the extent of non-specific protein binding on, for example, a non-imprinted polymer (NIP) control surface. PMID:24950144

  15. Cyclic peptide ligand with high binding capacity for affinity purification of immunoglobulin G.

    PubMed

    Kang, Hyo Jin; Choe, Weonu; Min, Jeong-Ki; Lee, Young-Mi; Kim, B Moon; Chung, Sang J

    2016-09-30

    The rapidly increasing implementation of antibodies in therapeutic and diagnostic applications has necessitated the development of antibody production and purification technologies for both academic and industrial usage. Bacterial Protein A and Protein G are known to bind antibodies with high affinity and have facilitated the isolation and purification thereof. Recently, small peptide ligands (i.e. IgG Fc domain-binding peptides, FcBP) that specifically bind to the Fc-domain of antibodies were reported. In the present study we describe the development of a reusable high affinity column for antibody purification utilizing immobilized FcBP, comprising 13 amino acids residues, on a sepharose resin. In addition to FcBP, Cys to Ser substituted FcBP (FcBP-Ser), reduced FcBP (FcBP-Red), commercial Protein A and Protein G resins, packed into columns, were evaluated for antibody purification. All these columns except the FcBP-Ser one showed good binding capacity for a humanized IgG (trastuzumab) and a chimeric IgG (cetuximab). The column packed with FcBP-Red allowed antibody purification at a less acidic pH (pH 4.8) than was required for the other ligand affinity columns used in our experiments (i.e., pH 3.2 for Protein G and FcBP columns, and pH 3.5 for Protein A column, respectively). Utilizing the FcBP column, antibodies from swine human sera were isolated with a purity of 95%. Interestingly, the FcBP column could be easily regenerated and operated without loss of efficiency for up to 60 runs, the maximum number of runs performed in the present study.

  16. Inhibition of ornithine decarboxylase induction by retinobenzoic acids in relation to their binding affinities to cellular retinoid-binding proteins.

    PubMed

    Takagi, K; Suganuma, M; Kagechika, H; Shudo, K; Ninomiya, M; Muto, Y; Fujiki, H

    1988-01-01

    Retinobenzoic acids induce differentiation of human promyelocytic leukemia cells (HL-60). Like retinoic acid, 14 retinobenzoic acids inhibited the induction of ornithine decarboxylase (ODC) by teleocidin in mouse skin. The mechanism(s) of inhibition of ODC induction by 7 retinobenzoic acids, Am 80, Am 81, Am 580, Am 590, Am 68, Sa 80, and Ch 55 was compared with those by all-trans-retinoic acid and the arotinoid compound 19. Application of 114 nmol of Am 80, Am 81, Am 580, Am 590, Am 68, Sa 80, or Ch 55, 10 min before 11.4 nmol of teleocidin, resulted in 76.7%, 82.0%, 76.2%, 28.3%, 48.4%, 58.6%, and 85.1% inhibition of ODC induction, respectively. Since all-trans-retinoic acid and compound 19 were also inhibitory, we determined whether retinobenzoic acids bind to cellular retinoic acid-binding protein (CRABP) isolated from bovine adrenal glands. Am 80 and Am 580 inhibited the specific binding of 3H-retinoic acid to CRABP, but also showed less affinity than authentic unlabeled retinoic acid and compound 19. Am 81, Am 590, Am 68, Sa 80, and Ch 55 at up to 10 microM were not effective competitors of the binding of either 3H-retinoic acid or 3H-retinol. These results suggest that the inhibition of ODC induction can be mediated by pathways that do not involve CRABP or the cellular retinol-binding protein.

  17. The Structure of a High-Affinity Kainate Receptor: GluK4 Ligand-Binding Domain Crystallized with Kainate.

    PubMed

    Kristensen, Ole; Kristensen, Lise Baadsgaard; Møllerud, Stine; Frydenvang, Karla; Pickering, Darryl S; Kastrup, Jette Sandholm

    2016-09-01

    Ionotropic glutamate receptors play a key role in fast neurotransmission in the CNS and have been linked to several neurological diseases and disorders. One subfamily is the kainate receptors, which are grouped into low-affinity (GluK1-3) and high-affinity (GluK4-5) receptors based on their affinity for kainate. Although structures of the ligand-binding domain (LBD) of all low-affinity kainate receptors have been reported, no structures of the high-affinity receptor subunits are available. Here, we present the X-ray structure of GluK4-LBD with kainate at 2.05 Å resolution, together with thermofluor and radiolabel binding affinity data. Whereas binding-site residues in GluK4 are most similar to the AMPA receptor subfamily, the domain closure and D1-D2 interlobe contacts induced by kainate are similar to the low-affinity kainate receptor GluK1. These observations provide a likely explanation for the high binding affinity of kainate at GluK4-LBD.

  18. Copper binding affinity of rainbow trout (Oncorhynchus mykiss) and brook trout (Salvelinus fontinalis) gills: Implications for assessing bioavailable metal

    SciTech Connect

    MacRae, R.K.; Smith, D.E.; Swoboda-Colberg, N.; Meyer, J.S.; Bergman, H.L. . Dept. of Zoology and Physiology)

    1999-06-01

    In this study, the authors determined the conditional stability constant (log K[prime]) of copper for the gills of rainbow trout (Oncorhynchus mykiss; RBT) and brook trout (Salvelinus fontinalis; BT). Using toxicity-based complexation bioassays, which measure the effect of competing organic ligands on copper toxicity, the RBT gill copper log K[prime] range was 6.4 to 7.2. Using a Scatchard analysis of gill Cu accumulation, the RBT log K[prime] was 7.50 and the BT log K[prime] was 7.25. The close agreement in RBT log K[prime] values between these two methods suggests that measurement of gill copper accumulation is an acceptable alternative for determining a toxicity-based gill copper binding affinity. The results also suggest that there is either a single gill copper binding component or, more realistically, multiple components with similar binding properties that function collectively to define a single toxicologically relevant copper conditional stability constant. These results suggest analytical approaches to measuring bioavailable metal concentrations, such as geochemical modeling where biological ligands are included in speciation calculations, may adequately simulate complex biological ligands. A method to convert gill copper accumulation to a bioavailable water criterion is also discussed.

  19. Probing adenosine nucleotide-binding proteins with an affinity-labeled nucleotide probe and mass spectrometry.

    PubMed

    Qiu, Haibo; Wang, Yinsheng

    2007-08-01

    Mass spectrometry combined with chemical labeling strategies has become very important in biological analysis. Herein, we described the application of a biotin-conjugated acyl nucleotide for probing adenosine nucleotide-binding proteins. We demonstrated that the probe reacted specifically with the lysine residue at the nucleotide-binding site of two purified adenosine nucleotide-binding proteins, Escherichia coli recombinase A (RecA) and Saccharomyces cerevisiae alcohol dehydrogenase-I (YADH-I). A single conjugate peptide with a specifically labeled lysine residue was identified, by using LC-MS/MS, from the tryptic digestion mixture of the reaction products of the nucleotide analogue with RecA or YADH-I. The strategy, which involved labeling reaction, enzymatic digestion, affinity purification, and LC-MS/MS analysis, was relatively simple, fast, and straightforward. The method should be generally applicable for the identification of lysine residues at the nucleotide-binding site of other proteins. The biotin-conjugated acyl nucleotide probe also allowed for the enrichment and identification of nucleotide-binding proteins from complex protein mixtures; we showed that more than 50 adenosine nucleotide-binding proteins could be identified from the whole-cell lysates of HeLa-S3 and WM-266-4 cells.

  20. Probing adenosine nucleotide-binding proteins with an affinity labeled-nucleotide probe and mass spectrometry

    PubMed Central

    Qiu, Haibo; Wang, Yinsheng

    2008-01-01

    Mass spectrometry combined with chemical labeling strategies has become very important in biological analysis. Herein, we described the application of a biotin-conjugated acyl nucleotide for probing adenosine nucleotide-binding proteins. We demonstrated that the probe reacted specifically with the lysine residue at the nucleotide-binding site of two purified adenosine nucleotide-binding proteins, Escherichia coli RecA and Saccharomyces cerevisiae alcohol dehydrogenase-I (YADH-I). A single conjugate peptide with a specifically labeled lysine residue was identified, by using LC-MS/MS, from the tryptic digestion mixture of the reaction products of the nucleotide analog with RecA or YADH-I. The strategy, which involved labeling reaction, enzymatic digestion, affinity purification and LC-MS/MS analysis, was relatively simple, fast and straightforward. The method should be generally applicable for the identification of lysine residues at the nucleotide-binding site of other proteins. The biotin-conjugated acyl nucleotide probe also allowed for the enrichment and identification of nucleotide-binding proteins from complex protein mixtures; we showed that more than 50 adenosine nucleotide-binding proteins could be identified from the whole cell lysates of HeLa-S3 and WM-266-4 cells. PMID:17602667

  1. Enhancement of binding kinetics on affinity substrates by laser point heating induced transport.

    PubMed

    Wang, Bu; Cheng, Xuanhong

    2016-03-01

    Enhancing the time response and detection limit of affinity-binding based biosensors is an area of active research. For diffusion limited reactions, introducing active mass transport is an effective strategy to reduce the equilibration time and improve surface binding. Here, a laser is focused on the ceiling of a microchamber to generate point heating, which introduces natural advection and thermophoresis to promote mass transport to the reactive floor. We first used the COMSOL simulation to study how the kinetics of ligand binding is influenced by the optothermal effect. Afterwards, binding of biotinylated nanoparticles to NeutrAvidin-treated substrates is quantitatively measured with and without laser heating. It is discovered that laser induced point heating reduces the reaction half-life locally, and the reduction improves with the natural advection velocity. In addition, non-uniform ligand binding on the substrate is induced by the laser with predictable binding patterns. This optothermal strategy holds promise to improve the time-response and sensitivity of biosensors and microarrays. PMID:26898559

  2. Accurate electron affinity of Pb and isotope shifts of binding energies of Pb(.).

    PubMed

    Chen, Xiaolin; Ning, Chuangang

    2016-08-28

    Lead (Pb) was the last element of the group IVA whose electron affinity had a low accuracy around 10 meV before the present work. This was due to the generic threshold photodetachment measurement that cannot extent well below 0.5 eV due to the light source limitation. In the present work, the electron affinity of Pb was determined to be 2877.33(13) cm(-1) or 0.356 743(16) eV for the isotope m = 208. The accuracy was improved by a factor of 500 with respect to the previous laser photodetachment electron spectroscopy. Moreover, remarkable isotope shifts of the binding energy of Pb(-) 6p(3) (4)S3/2 - Pb 6p(2) (3)P2 were observed for m = 206, 207, and 208. PMID:27586918

  3. Accurate electron affinity of Pb and isotope shifts of binding energies of Pb-

    NASA Astrophysics Data System (ADS)

    Chen, Xiaolin; Ning, Chuangang

    2016-08-01

    Lead (Pb) was the last element of the group IVA whose electron affinity had a low accuracy around 10 meV before the present work. This was due to the generic threshold photodetachment measurement that cannot extent well below 0.5 eV due to the light source limitation. In the present work, the electron affinity of Pb was determined to be 2877.33(13) cm-1 or 0.356 743(16) eV for the isotope m = 208. The accuracy was improved by a factor of 500 with respect to the previous laser photodetachment electron spectroscopy. Moreover, remarkable isotope shifts of the binding energy of Pb- 6p3 4S3/2 - Pb 6p2 3P2 were observed for m = 206, 207, and 208.

  4. Preorganized Peptide Scaffolds as Mimics of Phosphorylated Proteins Binding Sites with a High Affinity for Uranyl.

    PubMed

    Starck, Matthieu; Sisommay, Nathalie; Laporte, Fanny A; Oros, Stéphane; Lebrun, Colette; Delangle, Pascale

    2015-12-01

    Cyclic peptides with two phosphoserines and two glutamic acids were developed to mimic high-affinity binding sites for uranyl found in proteins such as osteopontin, which is believed to be a privileged target of this ion in vivo. These peptides adopt a β-sheet structure that allows the coordination of the latter amino acid side chains in the equatorial plane of the dioxo uranyl cation. Complementary spectroscopic and analytical methods revealed that these cyclic peptides are efficient uranyl chelating peptides with a large contribution from the phosphorylated residues. The conditional affinity constants were measured by following fluorescence tryptophan quenching and are larger than 10(10) at physiological pH. These compounds are therefore promising models for understanding uranyl chelation by proteins, which is relevant to this actinide ion toxicity. PMID:26583259

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

    PubMed Central

    Keefe, Andrew J.; Jiang, Shaoyi

    2013-01-01

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

  6. pH-dependent binding engineering reveals an FcRn affinity threshold that governs IgG recycling.

    PubMed

    Borrok, M Jack; Wu, Yanli; Beyaz, Nurten; Yu, Xiang-Qing; Oganesyan, Vaheh; Dall'Acqua, William F; Tsui, Ping

    2015-02-13

    The Fc domain of IgG has been the target of multiple mutational studies aimed at altering the pH-dependent IgG/FcRn interaction to modulate IgG pharmacokinetics. These studies have yielded antibody variants with disparate pharmacokinetic characteristics, ranging from extended in vivo half-life to those exhibiting extremely rapid clearance. To better understand pH-dependent binding parameters that govern these outcomes and limit FcRn-mediated half-life extension, we generated a panel of novel Fc variants with high affinity binding at acidic pH that vary in pH 7.4 affinities and assessed pharmacokinetic outcomes. Pharmacokinetic studies in human FcRn transgenic mice and cynomolgus monkeys showed that multiple variants with increased FcRn affinities at acidic pH exhibited extended serum half-lives relative to the parental IgG. Importantly, the results reveal an underappreciated affinity threshold of neutral pH binding that determines IgG recycling efficiency. Variants with pH 7.4 FcRn affinities below this threshold recycle efficiently and can exhibit increased serum persistence. Increasing neutral pH FcRn affinity beyond this threshold reduced serum persistence by offsetting the benefits of increased pH 6.0 binding. Ultra-high affinity binding to FcRn at both acidic and neutral pH leads to rapid serum clearance.

  7. NADP+ binding to the regulatory subunit of methionine adenosyltransferase II increases intersubunit binding affinity in the hetero-trimer.

    PubMed

    González, Beatriz; Garrido, Francisco; Ortega, Rebeca; Martínez-Júlvez, Marta; Revilla-Guarinos, Ainhoa; Pérez-Pertejo, Yolanda; Velázquez-Campoy, Adrián; Sanz-Aparicio, Julia; Pajares, María A

    2012-01-01

    Mammalian methionine adenosyltransferase II (MAT II) is the only hetero-oligomer in this family of enzymes that synthesize S-adenosylmethionine using methionine and ATP as substrates. Binding of regulatory β subunits and catalytic α2 dimers is known to increase the affinity for methionine, although scarce additional information about this interaction is available. This work reports the use of recombinant α2 and β subunits to produce oligomers showing kinetic parameters comparable to MAT II purified from several tissues. According to isothermal titration calorimetry data and densitometric scanning of the stained hetero-oligomer bands on denatured gels, the composition of these oligomers is that of a hetero-trimer with α2 dimers associated to single β subunits. Additionally, the regulatory subunit is able to bind NADP(+) with a 1:1 stoichiometry, the cofactor enhancing β to α2-dimer binding affinity. Mutants lacking residues involved in NADP(+) binding and N-terminal truncations of the β subunit were able to oligomerize with α2-dimers, although the kinetic properties appeared altered. These data together suggest a role for both parts of the sequence in the regulatory role exerted by the β subunit on catalysis. Moreover, preparation of a structural model for the hetero-oligomer, using the available crystal data, allowed prediction of the regions involved in β to α2-dimer interaction. Finally, the implications that the presence of different N-terminals in the β subunit could have on MAT II behavior are discussed in light of the recent identification of several splicing forms of this subunit in hepatoma cells.

  8. NADP+ Binding to the Regulatory Subunit of Methionine Adenosyltransferase II Increases Intersubunit Binding Affinity in the Hetero-Trimer

    PubMed Central

    Ortega, Rebeca; Martínez-Júlvez, Marta; Revilla-Guarinos, Ainhoa; Pérez-Pertejo, Yolanda; Velázquez-Campoy, Adrián; Sanz-Aparicio, Julia; Pajares, María A.

    2012-01-01

    Mammalian methionine adenosyltransferase II (MAT II) is the only hetero-oligomer in this family of enzymes that synthesize S-adenosylmethionine using methionine and ATP as substrates. Binding of regulatory β subunits and catalytic α2 dimers is known to increase the affinity for methionine, although scarce additional information about this interaction is available. This work reports the use of recombinant α2 and β subunits to produce oligomers showing kinetic parameters comparable to MAT II purified from several tissues. According to isothermal titration calorimetry data and densitometric scanning of the stained hetero-oligomer bands on denatured gels, the composition of these oligomers is that of a hetero-trimer with α2 dimers associated to single β subunits. Additionally, the regulatory subunit is able to bind NADP+ with a 1∶1 stoichiometry, the cofactor enhancing β to α2-dimer binding affinity. Mutants lacking residues involved in NADP+ binding and N-terminal truncations of the β subunit were able to oligomerize with α2-dimers, although the kinetic properties appeared altered. These data together suggest a role for both parts of the sequence in the regulatory role exerted by the β subunit on catalysis. Moreover, preparation of a structural model for the hetero-oligomer, using the available crystal data, allowed prediction of the regions involved in β to α2-dimer interaction. Finally, the implications that the presence of different N-terminals in the β subunit could have on MAT II behavior are discussed in light of the recent identification of several splicing forms of this subunit in hepatoma cells. PMID:23189196

  9. Binding affinity and agonist activity of putative endogenous cannabinoids at the human neocortical CB1 receptor.

    PubMed

    Steffens, Marc; Zentner, Josef; Honegger, Jürgen; Feuerstein, Thomas J

    2005-01-01

    We investigated the affinity of putative endocannabinoids (2-arachidonylglycerol, 2-AG; noladin ether, virodhamine) for the human neocortical CB1 receptor. Functional activity of these compounds (including anandamide, AEA) was determined by examining basal and forskolin-stimulated cAMP formation. Assays were performed with synaptosomes, prepared from fresh human neocortical tissue. Receptor affinity was assessed from competition binding experiments with the CB1/2 agonist [3H]-CP55.940 in absence or presence of a protease inhibitor to assess enzymatic stability. Noladin ether and virodhamine inhibited [3H]-CP55.940 binding (Ki: 98, 1740 nM, respectively). Protease inhibition decreased the Ki value of virodhamine (Ki: 912 nM), but left that of noladin ether unchanged. 2-AG almost lacked affinity (Ki lymphoblasic )10 microM). Basal cAMP formation was unaffected by AEA and noladin ether, but strongly enhanced by 2-AG and virodhamine. Forskolin-stimulated cAMP formation was inhibited by AEA and noladin ether (IC50: 69, 427 nM, respectively) to the same extent as by CP55.940 (Imax each approximately 30%). Inhibitions by AEA or noladin ether were blocked by the CB1 receptor antagonist AM251. Virodhamine increased forskolin-stimulated cAMP formation, also in presence of AM251, by approximately 20%. 2-AG had no effect; in presence of AM251, however, 10 microM 2-AG stimulated cAMP formation by approximately 15%. Our results suggest, that AEA and noladin ether are full CB1 receptor agonists in human neocortex, whereas virodhamine may act as a CB1 receptor antagonist/inverse agonist. Particularly the (patho)physiological role of 2-AG should be further investigated, since its CB1 receptor affinity and agonist activity especially in humans might be lower than generally assumed. PMID:15588725

  10. Prediction and analysis of canonical EF hand loop and qualitative estimation of Ca²⁺ binding affinity.

    PubMed

    Mazumder, Mohit; Padhan, Narendra; Bhattacharya, Alok; Gourinath, Samudrala

    2014-01-01

    The diversity of functions carried out by EF hand-containing calcium-binding proteins is due to various interactions made by these proteins as well as the range of affinity levels for Ca²⁺ displayed by them. However, accurate methods are not available for prediction of binding affinities. Here, amino acid patterns of canonical EF hand sequences obtained from available crystal structures were used to develop a classifier that distinguishes Ca²⁺-binding loops and non Ca²⁺-binding regions with 100% accuracy. To investigate further, we performed a proteome-wide prediction for E. histolytica, and classified known EF-hand proteins. We compared our results with published methods on the E. histolytica proteome scan, and demonstrated our method to be more specific and accurate for predicting potential canonical Ca²⁺-binding loops. Furthermore, we annotated canonical EF-hand motifs and classified them based on their Ca²⁺-binding affinities using support vector machines. Using a novel method generated from position-specific scoring metrics and then tested against three different experimentally derived EF-hand-motif datasets, predictions of Ca²⁺-binding affinities were between 87 and 90% accurate. Our results show that the tool described here is capable of predicting Ca²⁺-binding affinity constants of EF-hand proteins.

  11. Picomolar-affinity binding and inhibition of adenylate cyclase activity by melatonin in Syrian hamster hypothalamus

    SciTech Connect

    Niles, L.P.; Hashemi, F. )

    1990-12-01

    1. The effect of melatonin on forskolin-stimulated adenylate cyclase activity was measured in homogenates of Syrian hamster hypothalamus. In addition, the saturation binding characteristics of the melatonin receptor ligand, ({sup 125}I)iodomelatonin, was examined using an incubation temperature (30{degree}C) similar to that used in enzyme assays. 2. At concentrations ranging from 10 pM to 1 nM, melatonin caused a significant decrease in stimulated adenylate cyclase activity with a maximum inhibition of approximately 22%. 3. Binding experiments utilizing ({sup 125}I)iodomelatonin in a range of approximately 5-80 pM indicated a single class of high-affinity sites: Kd = 55 +/- 9 pM, Bmax = 1.1 +/- 0.3 fmol/mg protein. 4. The ability of picomolar concentrations of melatonin to inhibit forskolin-stimulated adenylate cyclase activity suggests that this affect is mediated by picomolar-affinity receptor binding sites for this hormone in the hypothalamus.

  12. Cationic polymer brush-modified cellulose nanocrystals for high-affinity virus binding

    NASA Astrophysics Data System (ADS)

    Rosilo, Henna; McKee, Jason R.; Kontturi, Eero; Koho, Tiia; Hytönen, Vesa P.; Ikkala, Olli; Kostiainen, Mauri A.

    2014-09-01

    Surfaces capable of high-affinity binding of biomolecules are required in several biotechnological applications, such as purification, transfection, and sensing. Therein, the rod-shaped, colloidal cellulose nanocrystals (CNCs) are appealing due to their large surface area available for functionalization. In order to exploit electrostatic binding, their intrinsically anionic surfaces have to be cationized as biological supramolecules are predominantly anionic. Here we present a facile way to prepare cationic CNCs by surface-initiated atom-transfer radical polymerization of poly(N,N-dimethylaminoethyl methacrylate) and subsequent quaternization of the polymer pendant amino groups. The cationic polymer brush-modified CNCs maintained excellent dispersibility and colloidal stability in water and showed a ζ-potential of +38 mV. Dynamic light scattering and electron microscopy showed that the modified CNCs electrostatically bind cowpea chlorotic mottle virus and norovirus-like particles with high affinity. Addition of only a few weight percent of the modified CNCs in water dispersions sufficed to fully bind the virus capsids to form micrometer-sized assemblies. This enabled the concentration and extraction of the virus particles from solution by low-speed centrifugation. These results show the feasibility of the modified CNCs in virus binding and concentrating, and pave the way for their use as transduction enhancers for viral delivery applications.Surfaces capable of high-affinity binding of biomolecules are required in several biotechnological applications, such as purification, transfection, and sensing. Therein, the rod-shaped, colloidal cellulose nanocrystals (CNCs) are appealing due to their large surface area available for functionalization. In order to exploit electrostatic binding, their intrinsically anionic surfaces have to be cationized as biological supramolecules are predominantly anionic. Here we present a facile way to prepare cationic CNCs by surface

  13. Evaluation and optimization of the metal-binding properties of a complex ligand for immobilized metal affinity chromatography.

    PubMed

    Chen, Bin; Li, Rong; Li, Shiyu; Chen, Xiaoli; Yang, Kaidi; Chen, Guoliang; Ma, Xiaoxun

    2016-02-01

    The simultaneous determination of two binding parameters for metal ions on an immobilized metal affinity chromatography column was performed by frontal chromatography. In this study, the binding parameters of Cu(2+) to l-glutamic acid were measured, the metal ion-binding characteristics of the complex ligand were evaluated. The linear correlation coefficients were all greater than 99%, and the relative standard deviations of two binding parameters were 0.58 and 0.059%, respectively. The experiments proved that the frontal chromatography method was accurate, reproducible, and could be used to determine the metal-binding parameters of the affinity column. The effects of buffer pH, type, and concentration on binding parameters were explored by uniform design experiment. Regression, matching and residual analyses of the models were performed. Meanwhile, the optimum-binding conditions of Cu(2+) on the l-glutamic acid-silica column were obtained. Under these binding conditions, observations and regression values of two parameters were similar, and the observation values were the best. The results demonstrated that high intensity metal affinity column could be effectively prepared by measuring and evaluating binding parameters using frontal chromatography combined with a uniform design experiment. The present work provided a new mode for evaluating and preparing immobilized metal affinity column with good metal-binding behaviors. PMID:26632098

  14. Dopamine transporter oligomerization: impact of combining protomers with differential cocaine analog binding affinities.

    PubMed

    Zhen, Juan; Antonio, Tamara; Cheng, Shu-Yuan; Ali, Solav; Jones, Kymry T; Reith, Maarten E A

    2015-04-01

    Previous studies point to quaternary assembly of dopamine transporters (DATs) in oligomers. However, it is not clear whether the protomers function independently in the oligomer. Is each protomer an entirely separate unit that takes up dopamine and is inhibited by drugs known to block DAT function? In this work, human embryonic kidney 293 cells were co-transfected with DAT constructs possessing differential binding affinities for the phenyltropane cocaine analog, [³H]WIN35,428. It was assessed whether the binding properties in co-expressing cells capable of forming hetero-oligomers differ from those in preparations obtained from mixed singly transfected cells where such oligomers cannot occur. A method is described that replaces laborious 'mixing' experiments with an in silico method predicting binding parameters from those observed for the singly expressed constructs. Among five pairs of constructs tested, statistically significant interactions were found between protomers of wild-type (WT) and D313N, WT and D345N, and WT and D436N. Compared with predicted Kd values of [³H]WIN35,428 binding to the non-interacting pairs, the observed affinity of the former pair was increased 1.7 fold while the latter two were reduced 2.2 and 4.1 fold, respectively. This is the first report of an influence of protomer composition on the properties of a DAT inhibitor, indicating cooperativity within the oligomer. The dopamine transporter (DAT) can exist as an oligomer but it is unknown whether the protomers function independently. The present results indicate that protomers that are superpotent or deficient in cocaine analog binding can confer enhanced or reduced potency to the oligomer, respectively. In this respect, positive or negative cooperativity is revealed in the DAT oligomer. PMID:25580950

  15. Modeling the binding affinity of structurally diverse industrial chemicals to carbon using the artificial intelligence approaches.

    PubMed

    Gupta, Shikha; Basant, Nikita; Rai, Premanjali; Singh, Kunwar P

    2015-11-01

    Binding affinity of chemical to carbon is an important characteristic as it finds vast industrial applications. Experimental determination of the adsorption capacity of diverse chemicals onto carbon is both time and resource intensive, and development of computational approaches has widely been advocated. In this study, artificial intelligence (AI)-based ten different qualitative and quantitative structure-property relationship (QSPR) models (MLPN, RBFN, PNN/GRNN, CCN, SVM, GEP, GMDH, SDT, DTF, DTB) were established for the prediction of the adsorption capacity of structurally diverse chemicals to activated carbon following the OECD guidelines. Structural diversity of the chemicals and nonlinear dependence in the data were evaluated using the Tanimoto similarity index and Brock-Dechert-Scheinkman statistics. The generalization and prediction abilities of the constructed models were established through rigorous internal and external validation procedures performed employing a wide series of statistical checks. In complete dataset, the qualitative models rendered classification accuracies between 97.04 and 99.93%, while the quantitative models yielded correlation (R(2)) values of 0.877-0.977 between the measured and the predicted endpoint values. The quantitative prediction accuracies for the higher molecular weight (MW) compounds (class 4) were relatively better than those for the low MW compounds. Both in the qualitative and quantitative models, the Polarizability was the most influential descriptor. Structural alerts responsible for the extreme adsorption behavior of the compounds were identified. Higher number of carbon and presence of higher halogens in a molecule rendered higher binding affinity. Proposed QSPR models performed well and outperformed the previous reports. A relatively better performance of the ensemble learning models (DTF, DTB) may be attributed to the strengths of the bagging and boosting algorithms which enhance the predictive accuracies. The

  16. Pyrrolic tripodal receptors effectively recognizing monosaccharides. Affinity assessment through a generalized binding descriptor.

    PubMed

    Nativi, Cristina; Cacciarini, Martina; Francesconi, Oscar; Vacca, Alberto; Moneti, Gloriano; Ienco, Andrea; Roelens, Stefano

    2007-04-11

    Pyrrolic and imino (3) or amino (4) H-bonding ligands were incorporated into a benzene-based tripodal scaffold to develop a new generation of receptors for molecular recognition of carbohydrates. Receptors 3 and 4 effectively bound a set of octylglycosides of biologically relevant monosaccharides, including glucose (Glc), galactose (Gal), mannose (Man), and N-acetyl-glucosamine (GlcNAc), showing micromolar affinities in CDCl3 and millimolar affinities in CD3CN by NMR titrations. Both receptors selectively recognized Glc among the investigated monosaccharides, with 3 generally less effective than 4 but showing selectivities for the all-equatorial beta-glycosides of Glc and GlcNAc among the largest reported for H-bonding synthetic receptors. Selectivities in CDCl3 spanned a range of nearly 250-fold for 3 and over 30-fold for 4. Affinities and selectivities were univocally assessed through the BC50 descriptor, for which a generalized treatment is described that extends the scope of the descriptor to include any two-reagent host-guest system featuring any number of binding constants. ITC titrations of betaGlc in acetonitrile evidenced, for both receptors, a strong enthalpic contribution to the binding interaction, suggesting multiple H bonding. Selectivity trends toward alphaGlc and betaGlc analogous to those obtained in solution were also observed in the gas phase for 3 and 4 by collision-induced dissociation experiments. From comparison with appropriate reference compounds, a substantial contribution to carbohydrate binding emerged for both the imino/amino and the pyrrolic H-bonding groups but not for the amidic group. This previously undocumented behavior, supported by crystallographic evidence, has been discussed in terms of geometric, functional, and coordinative complementarity between H-bonding groups and glycosidic hydroxyls and opens the way to a new designer strategy of H-bonding receptors for carbohydrates.

  17. Hydroxamate-based iron chelators: combinatorial syntheses of desferrioxamine B analogues and evaluation of binding affinities.

    PubMed

    Poreddy, Amruta R; Schall, Otto F; Osiek, Todd A; Wheatley, James R; Beusen, Denise D; Marshall, Garland R; Slomczynska, Urszula

    2004-01-01

    This article describes the solid-phase combinatorial methods developed for the synthesis of polyhydroxamate-based siderophores. This strategy was applied to generate several libraries of structural DFO (1a) analogues that include DFO variants, non-amide analogues, C-terminal modified analogues, reverse-amide analogues, and hybrid analogues. To assess the relative iron-binding affinities of these compounds, a high-throughput spectrophotometric screening method based on competition with 8-hydroxyquinoline-5-sulfonic acid was developed. Some of the promising candidates containing various terminal functional groups were identified and prepared on large scale to enable future studies in animal models for iron-overload diseases.

  18. Copper(II) ions and the Alzheimer's amyloid-β peptide: Affinity and stoichiometry of binding

    NASA Astrophysics Data System (ADS)

    Tõugu, Vello; Friedemann, Merlin; Tiiman, Ann; Palumaa, Peep

    2014-10-01

    Deposition of amyloid beta (Aβ) peptides into amyloid plaques is the hallmark of Alzheimer's disease. According to the amyloid cascade hypothesis this deposition is an early event and primary cause of the disease, however, the mechanisms that cause this deposition remain elusive. An increasing amount of evidence shows that the interactions of biometals can contribute to the fibrillization and amyloid formation by amyloidogenic peptides. From different anions the copper ions deserve the most attention since it can contribute not only toamyloid formation but also to its toxicity due to the generation of ROS. In this thesis we focus on the affinity and stoichiometry of copper(II) binding to the Aβ molecule.

  19. Active site - a site of binding of affinity inhibitors in baker's yeast inorganic pyrophosphatase

    SciTech Connect

    Svyato, I.E.; Sklyankina, V.A.; Avaeva, S.M.

    1986-03-20

    The interaction of the enzyme-substrate complex with methyl phosphate, O-phosphoethanolamine, O-phosphopropanolamine, N-acetylphosphoserine, and phosphoglyolic acid, as well as pyrophosphatase, modified by monoesters of phosphoric acid, with pyrophosphate and tripolyphosphate, was investigated. It was shown that the enzyme containing the substrate in the active site does not react with monophosphates, but modified pyrophosphatase entirely retains the ability to bind polyanions to the regulatory site. It is concluded that the inactivation of baker's yeast inorganic pyrophosphatase by monoesters of phosphoric acid, which are affinity inhibitors of it, is the result of modification of the active site of the enzyme.

  20. Asparagine deamidation reduces DNA-binding affinity of the Drosophila melanogaster Scr homeodomain.

    PubMed

    O'Connell, Nichole E; Lelli, Katherine; Mann, Richard S; Palmer, Arthur G

    2015-10-24

    Spontaneous deamidation of asparagine is a non-enzymatic post-translational modification of proteins. Residue Asn 321 is the main site of deamidation of the Drosophila melanogaster Hox transcription factor Sex Combs Reduced (Scr). Formation of iso-aspartate, the major deamidation product, is detected by HNCACB triple-resonance NMR spectroscopy. The rate of deamidation is quantified by fitting the decay of Asn NH2 side-chain signals in a time-series of (15)N-(1)H HSQC NMR spectra. The deamidated form of Scr binds to specific DNA target sequences with reduced affinity as determined by an electrophoretic mobility shift assay.

  1. A protein engineered to bind uranyl selectively and with femtomolar affinity

    NASA Astrophysics Data System (ADS)

    Zhou, Lu; Bosscher, Mike; Zhang, Changsheng; Özçubukçu, Salih; Zhang, Liang; Zhang, Wen; Li, Charles J.; Liu, Jianzhao; Jensen, Mark P.; Lai, Luhua; He, Chuan

    2014-03-01

    Uranyl (UO22+), the predominant aerobic form of uranium, is present in the ocean at a concentration of ~3.2 parts per 109 (13.7 nM) however, the successful enrichment of uranyl from this vast resource has been limited by the high concentrations of metal ions of similar size and charge, which makes it difficult to design a binding motif that is selective for uranyl. Here we report the design and rational development of a uranyl-binding protein using a computational screening process in the initial search for potential uranyl-binding sites. The engineered protein is thermally stable and offers very high affinity and selectivity for uranyl with a Kd of 7.4 femtomolar (fM) and >10,000-fold selectivity over other metal ions. We also demonstrated that the uranyl-binding protein can repeatedly sequester 30-60% of the uranyl in synthetic sea water. The chemical strategy employed here may be applied to engineer other selective metal-binding proteins for biotechnology and remediation applications.

  2. Soluble low affinity adenosine A/sub 2/ binding site from human placenta: reconstitution and characteristics

    SciTech Connect

    Hutchison, K.; Prasad, M.; Fox, I.H.

    1987-05-01

    The authors have developed a vesicle reconstitution technique that allows for rapid vacuum filtration assay, and have characterized the soluble A/sub 2/ site from placental membranes. The overall yield of reconstituted binding is 60%. Competition analysis of membranes and reconstituted vesicles yields identical agonist potency orders and affinities: N-ethylcarboxamidoadenosine (NECA) (Kd-330 nM)>2-chloroadenosine (Kd=1.7 ..mu..M) > L-phenylisopropyladenosine (Kd > 1 mM). Equilibrium binding to membranes and reconstituted vesicles of (/sup 3/H)-NECA, an adenosine agonist, was not reduced by guanine nulceotides. HPLC gel permeation chromatography of extracts from membranes preincubated with 5 mM MgCl/sub 2/ and 100 ..mu..M NECA revealed a peak of binding with kD of 0.07. Extracts prepared with either an antagonist or NECA and 100 ..mu..M guanylyl 5'-imidodiphosphate revealed a peak of binding with a kD of 0.09. These data suggest that the adenosine A/sub 2/ receptor retains its binding properties upon reconstitution and may couple to a guanine nucleotide regulatory protein.

  3. Structure and Ligand Binding Properties of the Epoxidase Component of Styrene Monooxygenase

    SciTech Connect

    Ukaegbu, Uchechi E.; Kantz, Auric; Beaton, Michelle; Gassner, George T.; Rosenzweig, Amy C.

    2010-07-23

    Styrene monooxygenase (SMO) is a two-component flavoprotein monooxygenase that transforms styrene to styrene oxide in the first step of the styrene catabolic and detoxification pathway of Pseudomonas putida S12. The crystal structure of the N-terminally histidine-tagged epoxidase component of this system, NSMOA, determined to 2.3 {angstrom} resolution, indicates the enzyme exists as a homodimer in which each monomer forms two distinct domains. The overall architecture is most similar to that of p-hydroxybenzoate hydroxylase (PHBH), although there are some significant differences in secondary structure. Structural comparisons suggest that a large cavity open to the surface forms the FAD binding site. At the base of this pocket is another cavity that likely represents the styrene binding site. Flavin binding and redox equilibria are tightly coupled such that reduced FAD binds apo NSMOA {approx}8000 times more tightly than the oxidized coenzyme. Equilibrium fluorescence and isothermal titration calorimetry data using benzene as a substrate analogue indicate that the oxidized flavin and substrate analogue binding equilibria of NSMOA are linked such that the binding affinity of each is increased by 60-fold when the enzyme is saturated with the other. A much weaker {approx}2-fold positive cooperative interaction is observed for the linked binding equilibria of benzene and reduced FAD. The low affinity of the substrate analogue for the reduced FAD complex of NSMOA is consistent with a preferred reaction order in which flavin reduction and reaction with oxygen precede the binding of styrene, identifying the apoenzyme structure as the key catalytic resting state of NSMOA poised to bind reduced FAD and initiate the oxygen reaction.

  4. Positron-attachment to small molecules: Vibrational enhancement of positron affinities with configuration interaction level of multi-component molecular orbital approach

    SciTech Connect

    Tachikawa, Masanori

    2015-12-31

    To theoretically demonstrate the binding of a positron to small polarized molecules, we have calculated the vibrational averaged positron affinity (PA) values along the local vibrational contribution with the configuration interaction level of multi-component molecular orbital method. This method can take the electron-positron correlation contribution into account through single electronic - single positronic excitation configurations. The PA values are enhanced by including the local vibrational contribution from vertical PA values due to the anharmonicity of the potential.

  5. Biological effect of varying peptide binding affinity to the BoLA-DRB3*2703 allele

    PubMed Central

    2003-01-01

    MHC class I and II molecules are immunoregulatory cell surface glycoproteins, which selectively bind to and present antigenic peptides to T-lymphocytes. Murine and human studies show that variable peptide binding affinity to MHC II molecules influences Th1/Th2 responses by inducing distinctive cytokine expression. To examine the biological effects of peptide binding affinity to bovine MHC (BoLA), various self peptides (BoLA-DQ and fibrinogen fragments) and non-self peptides from ovalbumin (OVA), as well as VP2 and VP4 peptides from foot and mouth disease virus (FMD-V) were used to (1) determine binding affinities to the BoLA-DRB3*2703 allele, previously associated with mastitis susceptibility and (2) determine whether peptide binding affinity influences T-lymphocyte function. Peptide binding affinity was determined by a competitive assay using high affinity biotinylated self-peptide incubated with purified BoLA-DRB3*2703 in the presence of various concentrations of competing peptides. The concentrations of non-self peptide required to inhibit self-peptide binding by 50% (IC50) were variable, ranging from 26.92 to > 320 μM. Peptide-specific T-lymphocyte function was determined by measuring DNA synthesis, cell division, and IFN-γ production in cultures of mononuclear cells from a BoLA-DRB3*2703 homozygous cow. When compared to non-stimulated control cultures, differences in lymphocyte function were observed for all of the assessed parameters; however, peptide-binding affinity did not always account for the observed differences in lymphocyte function. PMID:12927080

  6. Neutrophil recruitment limited by high-affinity bent β2 integrin binding ligand in cis

    PubMed Central

    Fan, Zhichao; McArdle, Sara; Marki, Alex; Mikulski, Zbigniew; Gutierrez, Edgar; Engelhardt, Britta; Deutsch, Urban; Ginsberg, Mark; Groisman, Alex; Ley, Klaus

    2016-01-01

    Neutrophils are essential for innate immunity and inflammation and many neutrophil functions are β2 integrin-dependent. Integrins can extend (E+) and acquire a high-affinity conformation with an ‘open' headpiece (H+). The canonical switchblade model of integrin activation proposes that the E+ conformation precedes H+, and the two are believed to be structurally linked. Here we show, using high-resolution quantitative dynamic footprinting (qDF) microscopy combined with a homogenous conformation-reporter binding assay in a microfluidic device, that a substantial fraction of β2 integrins on human neutrophils acquire an unexpected E−H+ conformation. E−H+ β2 integrins bind intercellular adhesion molecules (ICAMs) in cis, which inhibits leukocyte adhesion in vitro and in vivo. This endogenous anti-inflammatory mechanism inhibits neutrophil aggregation, accumulation and inflammation. PMID:27578049

  7. Two Affinity Sites of the Cannabinoid Subtype 2 Receptor Identified by a Novel Homogeneous Binding Assay.

    PubMed

    Martínez-Pinilla, Eva; Rabal, Obdulia; Reyes-Resina, Irene; Zamarbide, Marta; Navarro, Gemma; Sánchez-Arias, Juan A; de Miguel, Irene; Lanciego, José L; Oyarzabal, Julen; Franco, Rafael

    2016-09-01

    Endocannabinoids act on G protein-coupled receptors that are considered potential targets for a variety of diseases. There are two different cannabinoid receptor types: ligands for cannabinoid type 2 receptors (CB2Rs) show more promise than those for cannabinoid type 1 receptors (CB1Rs) because they lack psychotropic actions. However, the complex pharmacology of these receptors, coupled with the lipophilic nature of ligands, is delaying the translational success of medications targeting the endocannabinoid system. We here report the discovery and synthesis of a fluorophore-conjugated CB2R-selective compound, CM-157 (3-[[4-[2-tert-butyl-1-(tetrahydropyran-4-ylmethyl)benzimidazol-5-yl]sulfonyl-2-pyridyl]oxy]propan-1-amine), which was useful for pharmacological characterization of CB2R by using a time-resolved fluorescence resonance energy transfer assay. This methodology does not require radiolabeled compounds and may be undertaken in homogeneous conditions and in living cells (i.e., without the need to isolate receptor-containing membranes). The affinity of the labeled compound was similar to that of the unlabeled molecule. Time-resolved fluorescence resonance energy transfer assays disclosed a previously unreported second affinity site and showed conformational changes in CB2R forming receptor heteromers with G protein-coupled receptor GPR55, a receptor for l-α-lysophosphatidylinositol. The populations displaying subnanomolar and nanomolar affinities were undisclosed in competitive assays using a well known cannabinoid receptor ligand, AM630 (1-[2-(morpholin-4-yl)ethyl]-2-methyl-3-(4-methoxybenzoyl)-6-iodoindole), and TH-chrysenediol, not previously tested on binding to cannabinoid receptors. Variations in binding parameters upon formation of dimers with GPR55 may reflect decreases in binding sites or alterations of the quaternary structure of the macromolecular G protein-coupled receptor complexes. In summary, the homogeneous binding assay described here may

  8. Monoclonal IgM antibody exhibiting high-affinity binding and cryoglobulin properties.

    PubMed Central

    Ballard, D W; Kranz, D M; Voss, E W

    1983-01-01

    A monoclonal IgM antibody (18-2-3) derived from cell fusion of (NZB X NZW) F1 splenocytes following secondary immunization with fluorescein-conjugated keyhole limpet hemocyanin was shown to exhibit high intrinsic binding affinity and cryoinsolubility. Affinity-purified preparations were determined to be IgM by immunochemical, electrophoretic, and chromatographic analyses. An intrinsic association constant (Ka) of 2.9 X 10(10) M-1 (at 2 degrees C) was measured by first-order dissociation-rate analysis. Antibody solubility at low concentration (approximately equal to 50 micrograms/ml) was shown, by absorption spectroscopy, to be temperature dependent between 4 degrees C and 32 degrees C. Insolubility at low temperature (4 degrees C) was reversible in the presence of homologous fluorescyl hapten, indicative of active site involvement in the mechanism of cryoglobulin-18-2-3 complex formation. Characteristics of clone 18-2-3 are discussed in terms of (i) its potential use as a model for examining the mechanism of cryoprecipitation and (ii) the proposed relationship between affinity maturation and the IgM to IgG class switch. Images PMID:6348779

  9. A cleavable silica-binding affinity tag for rapid and inexpensive protein purification.

    PubMed

    Coyle, Brandon L; Baneyx, François

    2014-10-01

    We describe a new affinity purification tag called Car9 that confers proteins to which it is fused micromolar affinity for unmodified silica. When appended to the C-terminus of GFPmut2 through a flexible linker, Car9 promotes efficient adsorption to silica gel and the fusion protein can be released from the particles by incubation with L-lysine. Using a silica gel column and the lysine elution approach in fast protein liquid chromatography (FPLC) mode, Car9-tagged versions of GFPmut2, mCherry and maltose binding protein (MBP) can be recovered from clarified lysates with a purity of 80-90%. Capitalizing on silica's ability to handle large pressure drops, we further show that it is possible to go from cell lysates to purified protein in less than 15 min using a fully disposable device. Finally, we demonstrate that the linker-Car9 region is susceptible to proteolysis by E. coli OmpT and take advantage of this observation to excise the C-terminal extension of GFPmut2-Car9 by incubating purified fusion protein with cells that overproduce the outer membrane protease OmpT. The set of strategies described herein, should reduce the cost of affinity purification by at least 10-fold, cut down purification times to minutes, and allow for the production of proteins with native (or nearly native) termini from their C-terminally-tagged versions.

  10. Measurements of relative binding of cohesin and dockerin mutants using an advanced ELISA technique for high-affinity interactions.

    PubMed

    Slutzki, Michal; Barak, Yoav; Reshef, Dan; Schueler-Furman, Ora; Lamed, Raphael; Bayer, Edward A

    2012-01-01

    The cellulosome is a large bacterial extracellular multienzyme complex able to degrade crystalline cellulosic substrates. The complex contains catalytic and noncatalytic subunits, interconnected by high-affinity cohesin-dockerin interactions. In this chapter, we introduce an optimized method for comparative binding among different cohesins or cohesin mutants to the dockerin partner. This assay offers advantages over other methods (such as ELISA, cELIA, SPR, and ITC) for particularly high-affinity binding interactions. In this approach, the high-affinity interaction of interest occurs in the liquid phase during the equilibrated binding step, whereas the interaction with the immobilized phase is used only for detection of the unbound dockerins that remain in the solution phase. Once equilibrium conditions are reached, the change in free energy of binding (ΔΔG(binding)), as well as the affinity constant of mutants, can be estimated against the known affinity constant of the wild-type interaction. In light of the above, we propose this method as a preferred alternative for the relative quantification of high-affinity protein interactions. PMID:22608739

  11. Water channel in the binding site of a high affinity anti-methotrexate antibody.

    PubMed

    Gayda, Susan; Longenecker, Kenton L; Manoj, Sharmila; Judge, Russell A; Saldana, Sylvia C; Ruan, Qiaoqiao; Swift, Kerry M; Tetin, Sergey Y

    2014-06-17

    In the present study, we report the structure of the free and drug-bound Fab fragment of a high affinity anti-methotrexate antibody and perform a thermodynamic analysis of the binding process. The anti-methotrexate Fab fragment features a remarkably rigid tunnel-like binding site that extends into a water channel serving as a specialized route to move solvent out and into the site upon ligand binding and dissociation. This new finding in antibody structure-function relationships directly relates to the fast association (1 × 10⁷ M⁻¹ s⁻¹) and slow dissociation (4 × 10⁻⁵ s⁻¹) rates determined for mAb ADD056, resulting in a very strong binding with a K(D) ~ 3.6 pM at 20 °C. As follows from the X-ray data analysis, the methotrexate-antibody complex is stabilized by an extended network of hydrogen bonds and stacking interactions. The analysis also shows structural involvement of the CDR H3 in formation of the water channel revealing another important role of this hypervariable region. This suggests a new direction in natural affinity maturation and opens a new possibility in antibody engineering. Methotrexate is a widely used therapeutic agent for many malignant diseases and inflammatory disorders. Unfortunately, it may also interfere with central aspects of metabolism and thereby cause inevitable side effects. Therefore, methotrexate therapy requires careful monitoring of drug blood levels, which is traditionally done by immunoassays. An understanding of the structure-function properties of antibodies selected for drug monitoring substantiates the performance and robustness of such tests.

  12. Identification of an Orthogonal Peptide Binding Motif for Biarsenical Multiuse Affinity Probes

    SciTech Connect

    Chen, Baowei; Cao, Haishi; Yan, Ping; Mayer, M. Uljana; Squier, Thomas C.

    2007-07-01

    Biarsenical multiuse affinity probes (MAPs) complexed with ethanedithiol (EDT) permit the selective cellular labeling of proteins engineered with tetracysteine motifs, but are limited by the availability of a single binding motif (i.e., CCPGCC or PG tag) that prevents the differential labeling of co-expressed proteins. To overcome this problem, we have used a high-throughput peptide screen to identify an alternate binding motif (i.e., CCKACC or KA tag), which has a similar brightness to the classical sequence upon MAP binding, but displays altered rates and affinities of association that permit the differential labeling of these peptide sequences by the red probe 4,5-bis(1,3,2-dithiarsolan-2-yl)-resorufin (ReAsH-EDT2) or its green cognate 4’,5’-bis(1,3,2-dithoarsolan-2-yl)fluorescein-(1,2-ethanedithiol)2 (FLAsH-EDT2). The utility of this labeling strategy was demonstrated following the expression of PG- and KA-tagged subunits of RNA polymerase expressed in E. coli. Specific labeling of two subunits of RNA polymerase in cellular lysates was achieved, whereby ReAsH-EDT2 is shown to selectively label the PG-tag on RNA polymerase alpha subunit prior to the labeling of the KA-tag sequence of the beta subunit of RNA polymerase with FlAsH-EDT2. These results demonstrate the ability to selectively label multiple individual proteins with orthogonal sequence tags in complex cellular lystates with spectroscopically distinct MAPs, and indicate the absolute specificity of ReAsH to target expressed proteins with essentially no nonspecific binding interactions.

  13. Water channel in the binding site of a high affinity anti-methotrexate antibody.

    PubMed

    Gayda, Susan; Longenecker, Kenton L; Manoj, Sharmila; Judge, Russell A; Saldana, Sylvia C; Ruan, Qiaoqiao; Swift, Kerry M; Tetin, Sergey Y

    2014-06-17

    In the present study, we report the structure of the free and drug-bound Fab fragment of a high affinity anti-methotrexate antibody and perform a thermodynamic analysis of the binding process. The anti-methotrexate Fab fragment features a remarkably rigid tunnel-like binding site that extends into a water channel serving as a specialized route to move solvent out and into the site upon ligand binding and dissociation. This new finding in antibody structure-function relationships directly relates to the fast association (1 × 10⁷ M⁻¹ s⁻¹) and slow dissociation (4 × 10⁻⁵ s⁻¹) rates determined for mAb ADD056, resulting in a very strong binding with a K(D) ~ 3.6 pM at 20 °C. As follows from the X-ray data analysis, the methotrexate-antibody complex is stabilized by an extended network of hydrogen bonds and stacking interactions. The analysis also shows structural involvement of the CDR H3 in formation of the water channel revealing another important role of this hypervariable region. This suggests a new direction in natural affinity maturation and opens a new possibility in antibody engineering. Methotrexate is a widely used therapeutic agent for many malignant diseases and inflammatory disorders. Unfortunately, it may also interfere with central aspects of metabolism and thereby cause inevitable side effects. Therefore, methotrexate therapy requires careful monitoring of drug blood levels, which is traditionally done by immunoassays. An understanding of the structure-function properties of antibodies selected for drug monitoring substantiates the performance and robustness of such tests. PMID:24832237

  14. A structure-based benchmark for protein-protein binding affinity.

    PubMed

    Kastritis, Panagiotis L; Moal, Iain H; Hwang, Howook; Weng, Zhiping; Bates, Paul A; Bonvin, Alexandre M J J; Janin, Joël

    2011-03-01

    We have assembled a nonredundant set of 144 protein-protein complexes that have high-resolution structures available for both the complexes and their unbound components, and for which dissociation constants have been measured by biophysical methods. The set is diverse in terms of the biological functions it represents, with complexes that involve G-proteins and receptor extracellular domains, as well as antigen/antibody, enzyme/inhibitor, and enzyme/substrate complexes. It is also diverse in terms of the partners' affinity for each other, with K(d) ranging between 10(-5) and 10(-14) M. Nine pairs of entries represent closely related complexes that have a similar structure, but a very different affinity, each pair comprising a cognate and a noncognate assembly. The unbound structures of the component proteins being available, conformation changes can be assessed. They are significant in most of the complexes, and large movements or disorder-to-order transitions are frequently observed. The set may be used to benchmark biophysical models aiming to relate affinity to structure in protein-protein interactions, taking into account the reactants and the conformation changes that accompany the association reaction, instead of just the final product.

  15. Design and synthesis of novel hydrophilic spacers for the reduction of nonspecific binding proteins on affinity resins.

    PubMed

    Shiyama, Takaaki; Furuya, Minoru; Yamazaki, Akira; Terada, Tomohiro; Tanaka, Akito

    2004-06-01

    Tubulin and actin often bind nonspecifically to affinity chromatography resins, complicating research toward identifying the cellular targets. Reduction of nonspecific binding proteins is important for success in finding such targets. We herein disclose the design, synthesis, and effectiveness in reduction of nonspecific binding proteins, of novel hydrophilic spacers (2-5), which were introduced between matrices and a ligand. Among them, tartaric acid derivative (5) exhibited the most effective reduction of nonspecific binding proteins, whilst maintaining binding of the target protein. Introduction of 5 on TOYOPEARL reduced tubulin and actin by almost 65% and 90% compared to that without the hydrophilic spacer, respectively, with effective binding to the target protein, FKBP12.

  16. Isomer-Specific Binding Affinity of Perfluorooctanesulfonate (PFOS) and Perfluorooctanoate (PFOA) to Serum Proteins.

    PubMed

    Beesoon, Sanjay; Martin, Jonathan W

    2015-05-01

    Perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) are among the most prominent contaminants in human serum, and these were historically manufactured as technical mixtures of linear and branched isomers. The isomers display unique pharmacokinetics in humans and in animal models, but molecular mechanisms underlying isomer-specific PFOS and PFOA disposition have not previously been studied. Here, ultrafiltration devices were used to examine (i) the dissociation constants (Kd) of individual PFOS and PFOA isomers with human serum albumin (HSA) and (ii) relative binding affinity of isomers in technical mixtures spiked to whole calf serum and human serum. Measurement of HSA Kd's demonstrated that linear PFOS (Kd=8(±4)×10(-8) M) was much more tightly bound than branched PFOS isomers (Kd range from 8(±1)×10(-5) M to 4(±2)×10(-4) M). Similarly, linear PFOA (Kd=1(±0.9)×10(-4) M) was more strongly bound to HSA compared to branched PFOA isomers (Kd range from 4(±2)×10(-4) M to 3(±2)×10(-4) M). The higher binding affinities of linear PFOS and PFOA to total serum protein were confirmed when both calf serum and human serum were spiked with technical mixtures. Overall, these data provide a mechanistic explanation for the longer biological half-life of PFOS in humans, compared to PFOA, and for the higher transplacental transfer efficiencies and renal clearance of branched PFOS and PFOA isomers, compared to the respective linear isomer.

  17. Bicarbonate increases binding affinity of Vibrio cholerae ToxT to virulence gene promoters.

    PubMed

    Thomson, Joshua J; Withey, Jeffrey H

    2014-11-01

    The major Vibrio cholerae virulence gene transcription activator, ToxT, is responsible for the production of the diarrhea-inducing cholera toxin (CT) and the major colonization factor, toxin coregulated pilus (TCP). In addition to the two primary virulence factors mentioned, ToxT is responsible for the activation of accessory virulence genes, such as aldA, tagA, acfA, acfD, tcpI, and tarAB. ToxT activity is negatively modulated by bile and unsaturated fatty acids found in the upper small intestine. Conversely, previous work identified another intestinal signal, bicarbonate, which enhances the ability of ToxT to activate production of CT and TCP. The work presented here further elucidates the mechanism for the enhancement of ToxT activity by bicarbonate. Bicarbonate was found to increase the activation of ToxT-dependent accessory virulence promoters in addition to those that produce CT and TCP. Bicarbonate is taken up into the V. cholerae cell, where it positively affects ToxT activity by increasing DNA binding affinity for the virulence gene promoters that ToxT activates regardless of toxbox configuration. The increase in ToxT binding affinity in the presence of bicarbonate explains the elevated level of virulence gene transcription.

  18. Enhancing ligand-protein binding in affinity thermoprecipitation: elucidation of spacer effects

    PubMed

    Vaidya; Lele; Kulkarni; Mashelkar

    1999-08-20

    Copolymers of N-isopropylacrylamide and N-acryloyl amino acid spacers of varying chain length were synthesized. p-Aminobenzamidine (PABA) was chemically linked to the pendant carboxyl groups of these polymers to obtain thermoprecipitating affinity polymers. The inhibition constant (Ki) of these polymers for trypsin decreased, i. e., the efficiency of PABA-trypsin binding increased with increase in the spacer chain length. The polymer to which PABA was linked through a spacer of five methylene groups exhibited eleven times lower Ki than that of the polymer containing PABA without a spacer. Investigations on model inhibitors N-acyl-p-aminobenzamidines showed that this enhancement in trypsin binding by the polymers was due to the spacer as well as to microenvironmental effects. Recovery and specific activity of the trypsin recovered increased with the spacer chain length. Separation of trypsin from a mixture of trypsin and chymotrypsin was also enhanced with the spacer chain length. The inhibition constants of these affinity polymers were not adversely affected by the crowding effect. Copyright 1999 John Wiley & Sons, Inc. PMID:10397880

  19. Advances and applications of binding affinity prediction methods in drug discovery.

    PubMed

    Parenti, Marco Daniele; Rastelli, Giulio

    2012-01-01

    Nowadays, the improvement of R&D productivity is the primary commitment in pharmaceutical research, both in big pharma and smaller biotech companies. To reduce costs, to speed up the discovery process and to increase the chance of success, advanced methods of rational drug design are very helpful, as demonstrated by several successful applications. Among these, computational methods able to predict the binding affinity of small molecules to specific biological targets are of special interest because they can accelerate the discovery of new hit compounds. Here we provide an overview of the most widely used methods in the field of binding affinity prediction, as well as of our own work in developing BEAR, an innovative methodology specifically devised to overtake some limitations in existing approaches. The BEAR method was successfully validated against different biological targets, and proved its efficacy in retrieving active compounds from virtual screening campaigns. The results obtained so far indicate that BEAR may become a leading tool in the drug discovery pipeline. We primarily discuss advantages and drawbacks of each technique and show relevant examples and applications in drug discovery.

  20. (/sup 3/H)dihydroergotamine as a high-affinity, slowly dissociating radioligand for 5-HT1B binding sites in rat brain membranes: evidence for guanine nucleotide regulation of agonist affinity states

    SciTech Connect

    Hamblin, M.W.; Ariani, K.; Adriaenssens, P.I.; Ciaranello, R.D.

    1987-12-01

    (/sup 3/H)Dihydroergotamine (DE) labels a population of binding sites in rat brain membranes with an affinity of approximately 70 pM in both hippocampus (maximal binding at saturation (Bmax) = 340 fmol/mg of protein) and cerebral cortex (Bmax = 250 fmol/mg of protein). Specific binding typically comprises about 97% of total binding at the Kd of the radioligand when nonspecific binding is determined in the presence of 100 nM unlabeled DE. Association kinetics at 37 degrees C are consistent with a uniform association rate constant for all sites labeled. Specific binding is completely reversible with addition of excess unlabeled DE, but dissociation does not proceed with simple first-order kinetics, suggesting the presence of more than one discrete binding site. Competition studies with selective drugs reveal alpha adrenergic, 5-HT1A and 5-HT1B components of (/sup 3/H)DE specific binding. When phentolamine (500 nM) is included to block alpha receptors and DPAT (100 nM) or spiroxatrine (500 nM) is included to block 5-HT1A receptors, specific binding is exclusively to sites with drug affinities characteristic of 5-HT1B receptors. Under these 5-HT1B-selective conditions, (/sup 3/H)DE binding is about 90% specific, with a Kd of about 50 to 60 pM and a Bmax of 96 fmol/mg of protein in hippocampus and 77 fmol/mg of protein in cortex. (/sup 3/H)DE binding to 5-HT1B sites is very slowly dissociable, with a T1/2 of greater than 2 h at 37 degrees C. 5-HT1B antagonists and DE itself yield competition curves at (/sup 3/H)DE-labeled 5-HT1B sites that are adequately fit assuming a single site in nonlinear regression analysis. Addition of 100 microM guanylyl 5'-imidodiphosphate appears to convert nearly all 5-HT1B sites to those having low affinity for agonists while having a much smaller effect on the binding of (/sup 3/H)DE.

  1. Deciphering the specific high-affinity binding of cucurbit[7]uril to amino acids in water.

    PubMed

    Lee, Jong Wha; Lee, Hyun Hee L; Ko, Young Ho; Kim, Kimoon; Kim, Hugh I

    2015-04-01

    This work presents a systematic study on the host-guest interactions between the macrocyclic host molecule cucurbit[7]uril (CB[7]) and amino acids (AAs) including three basic AAs (Lys, Arg, and His) and three aromatic AAs (Phe, Tyr, and Trp) to elucidate the origin of the high selectivity of CB[7] toward AA residues in proteins. Complex formation between CB[7] and each AA was examined in solution (by isothermal titration calorimetry and NMR) as well as in the gas phase (by ion mobility mass spectrometry and collision-induced dissociation), and the results were further combined with computational investigations. Generally, the aromatic AAs show higher binding affinities than the basic AAs in buffer solutions with various pH values. On the contrary, the gas-phase stabilities of the basic AA complex ions are higher than those of the aromatic AA complex ions, suggesting that the direct ion-dipole interactions between the charged side chains of the basic AAs and the polar carbonyl groups of CB[7] predominate in the absence of water. The ion-dipole interactions are less significant in water, since the original interactions of the guests with water are lost upon complex formation. In contrast, the transfer of the hydrophobic groups from the bulk into the hydrophobic CB[7] cavity suffers less from the desolvation penalty, resulting in higher binding affinities in water. Therefore, initial guest solvation is another key factor which should be considered when designing high-affinity host-guest systems, in addition to the contribution from the release of high-energy water molecules from the CB[7] cavity (J. Am. Chem. Soc. 2012, 134, 15318-15323).

  2. Deciphering the specific high-affinity binding of cucurbit[7]uril to amino acids in water.

    PubMed

    Lee, Jong Wha; Lee, Hyun Hee L; Ko, Young Ho; Kim, Kimoon; Kim, Hugh I

    2015-04-01

    This work presents a systematic study on the host-guest interactions between the macrocyclic host molecule cucurbit[7]uril (CB[7]) and amino acids (AAs) including three basic AAs (Lys, Arg, and His) and three aromatic AAs (Phe, Tyr, and Trp) to elucidate the origin of the high selectivity of CB[7] toward AA residues in proteins. Complex formation between CB[7] and each AA was examined in solution (by isothermal titration calorimetry and NMR) as well as in the gas phase (by ion mobility mass spectrometry and collision-induced dissociation), and the results were further combined with computational investigations. Generally, the aromatic AAs show higher binding affinities than the basic AAs in buffer solutions with various pH values. On the contrary, the gas-phase stabilities of the basic AA complex ions are higher than those of the aromatic AA complex ions, suggesting that the direct ion-dipole interactions between the charged side chains of the basic AAs and the polar carbonyl groups of CB[7] predominate in the absence of water. The ion-dipole interactions are less significant in water, since the original interactions of the guests with water are lost upon complex formation. In contrast, the transfer of the hydrophobic groups from the bulk into the hydrophobic CB[7] cavity suffers less from the desolvation penalty, resulting in higher binding affinities in water. Therefore, initial guest solvation is another key factor which should be considered when designing high-affinity host-guest systems, in addition to the contribution from the release of high-energy water molecules from the CB[7] cavity (J. Am. Chem. Soc. 2012, 134, 15318-15323). PMID:25757499

  3. Thermodynamics of Calcium binding to the Calmodulin N-terminal domain to evaluate site-specific affinity constants and cooperativity.

    PubMed

    Beccia, Maria Rosa; Sauge-Merle, Sandrine; Lemaire, David; Brémond, Nicolas; Pardoux, Romain; Blangy, Stéphanie; Guilbaud, Philippe; Berthomieu, Catherine

    2015-07-01

    Calmodulin (CaM) is an essential Ca(II)-dependent regulator of cell physiology. To understand its interaction with Ca(II) at a molecular level, it is essential to examine Ca(II) binding at each site of the protein, even if it is challenging to estimate the site-specific binding properties of the interdependent CaM-binding sites. In this study, we evaluated the site-specific Ca(II)-binding affinity of sites I and II of the N-terminal domain by combining site-directed mutagenesis and spectrofluorimetry. The mutations had very low impact on the protein structure and stability. We used these binding constants to evaluate the inter-site cooperativity energy and compared it with its lower limit value usually reported in the literature. We found that site I affinity for Ca(II) was 1.5 times that of site II and that cooperativity induced an approximately tenfold higher affinity for the second Ca(II)-binding event, as compared to the first one. We further showed that insertion of a tryptophan at position 7 of site II binding loop significantly increased site II affinity for Ca(II) and the intra-domain cooperativity. ΔH and ΔS parameters were studied by isothermal titration calorimetry for Ca(II) binding to site I, site II and to the entire N-terminal domain. They showed that calcium binding is mainly entropy driven for the first and second binding events. These findings provide molecular information on the structure-affinity relationship of the individual sites of the CaM N-terminal domain and new perspectives for the optimization of metal ion binding by mutating the EF-hand loops sequences.

  4. Measuring Binding Affinity of Protein-Ligand Interaction Using Spectrophotometry: Binding of Neutral Red to Riboflavin-Binding Protein

    ERIC Educational Resources Information Center

    Chenprakhon, Pirom; Sucharitakul, Jeerus; Panijpan, Bhinyo; Chaiyen, Pimchai

    2010-01-01

    The dissociation constant, K[subscript d], of the binding of riboflavin-binding protein (RP) with neutral red (NR) can be determined by titrating RP to a fixed concentration of NR. Upon adding RP to the NR solution, the maximum absorption peak of NR shifts to 545 nm from 450 nm for the free NR. The change of the absorption can be used to determine…

  5. Response Element Composition Governs Correlations between Binding Site Affinity and Transcription in Glucocorticoid Receptor Feed-forward Loops.

    PubMed

    Sasse, Sarah K; Zuo, Zheng; Kadiyala, Vineela; Zhang, Liyang; Pufall, Miles A; Jain, Mukesh K; Phang, Tzu L; Stormo, Gary D; Gerber, Anthony N

    2015-08-01

    Combinatorial gene regulation through feed-forward loops (FFLs) can bestow specificity and temporal control to client gene expression; however, characteristics of binding sites that mediate these effects are not established. We previously showed that the glucocorticoid receptor (GR) and KLF15 form coherent FFLs that cooperatively induce targets such as the amino acid-metabolizing enzymes AASS and PRODH and incoherent FFLs exemplified by repression of MT2A by KLF15. Here, we demonstrate that GR and KLF15 physically interact and identify low affinity GR binding sites within glucocorticoid response elements (GREs) for PRODH and AASS that contribute to combinatorial regulation with KLF15. We used deep sequencing and electrophoretic mobility shift assays to derive in vitro GR binding affinities across sequence space. We applied these data to show that AASS GRE activity correlated (r(2) = 0.73) with predicted GR binding affinities across a 50-fold affinity range in transfection assays; however, the slope of the linear relationship more than doubled when KLF15 was expressed. Whereas activity of the MT2A GRE was even more strongly (r(2) = 0.89) correlated with GR binding site affinity, the slope of the linear relationship was sharply reduced by KLF15, consistent with incoherent FFL logic. Thus, GRE architecture and co-regulator expression together determine the functional parameters that relate GR binding site affinity to hormone-induced transcriptional responses. Utilization of specific affinity response functions and GR binding sites by FFLs may contribute to the diversity of gene expression patterns within GR-regulated transcriptomes. PMID:26088140

  6. Specific high-affinity binding of thiazole orange to triplex and G-quadruplex DNA.

    PubMed

    Lubitz, Irit; Zikich, Dragoslav; Kotlyar, Alexander

    2010-05-01

    Interaction of Thiazole Orange (TO) with double-, triple-, and quadruple-stranded forms of DNA was studied. We have demonstrated by UV-vis absorption, circular dichroism (CD), and fluorescence spectroscopy that TO binds with much higher affinity to triplex and G-quadruplex DNA structures compared to double-stranded (ds) DNA. Complexes of the dye with DNA triplexes and G-quadruplexes are very stable and do not dissociate during chromatography and gel electrophoresis. TO binding to either triple- or quadruple-stranded DNA structures results in a >1000-fold increase in dye fluorescence. The fluorescence titration data showed that TO to triad and tetrad ratios, in tight complexes with the triplex and the G-quadruplex, are equal to 0.5 and 1, respectively. Preferential binding of TO to triplexes and G-quadruplexes enables selective detection of only these DNA forms in gels in the absence of free TO in electrophoresis running buffer. We have also demonstrated that incubation of U2OS cells with submicromolar concentrations of TO results in preferential staining of certain areas in the nucleus in contrast to DAPI which binds to dsDNA and efficiently stains regions that are unstained with TO. We suggest that TO staining may be useful for the detection of noncanonical structural motifs in genomic DNA.

  7. New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-affinity γ-Hydroxybutyrate (GHB) Binding Sites

    PubMed Central

    Vogensen, Stine B.; Marek, Aleš; Bay, Tina; Wellendorph, Petrine; Kehler, Jan; Bundgaard, Christoffer; Frølund, Bente; Pedersen, Martin H.F.; Clausen, Rasmus P.

    2013-01-01

    3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screening of 1 against different CNS targets establishes a high selectivity and we demonstrate in vivo brain penetration. In vitro characterization of [3H]-1 binding shows high specificity to the high-affinity GHB binding sites. PMID:24053696

  8. Exploring binding affinity of oxaliplatin and carboplatin, to nucleoprotein structure of chromatin: spectroscopic study and histone proteins as a target.

    PubMed

    Soori, Hosna; Rabbani-Chadegani, Azra; Davoodi, Jamshid

    2015-01-01

    Platinum drugs are potent chemotherapeutic agents widely used in cancer therapy. They exert their biological activity by binding to DNA, producing DNA adducts; however, in the cell nucleus, DNA is complexed with histone proteins into a nucleoprotein structure known as chromatin. The aim of this study was to explore the binding affinity of oxaliplatin and carboplatin to chromatin using spectroscopic as well as thermal denaturation and equilibrium dialysis techniques. The results showed that the drugs quenched with chromophores of chromatin and the quenching effect for oxaliplatin (Ksv = 3.156) was higher than carboplatin (Ksv = 0.28). The binding of the drugs exhibited hypochromicity both in thermal denaturation profiles and UV absorbance at 210 nm. The binding was positive cooperation with spontaneous reaction and oxaliplatin (Ka = 5.3 × 10(3) M(-1), n = 1.7) exhibited higher binding constant and number of binding sites than carboplatin (Ka = 0.33 × 10(3) M(-1), n = 1.0) upon binding to chromatin. Also secondary structure of chromatin proteins was altered upon drugs binding. It is concluded that oxaliplatin represents higher binding affinity to chromatin compared to carboplatin. In chromatin where DNA is compacted into nucleosomes structure with histones, the affinity of the platinated drugs is reduced and histone proteins may play a fundamental role in this binding process.

  9. Identification of BZR1-interacting Proteins as Potential Components of the Brassinosteroid Signaling Pathway in Arabidopsis Through Tandem Affinity Purification*

    PubMed Central

    Wang, Chunming; Shang, Jian-Xiu; Chen, Qi-Xiu; Oses-Prieto, Juan A.; Bai, Ming-Yi; Yang, Yihong; Yuan, Min; Zhang, Yu-Lan; Mu, Cong-Cong; Deng, Zhiping; Wei, Chuang-Qi; Burlingame, Alma L.; Wang, Zhi-Yong; Sun, Ying

    2013-01-01

    Brassinosteroids (BRs) are essential phytohormones for plant growth and development. BRs are perceived by the cell surface receptor kinase BRI1, and downstream signal transduction through multiple components leads to activation of the transcription factors BZR1 and BZR2/BES1. BZR1 activity is highly controlled by BR through reversible phosphorylation, protein degradation, and nucleocytoplasmic shuttling. To further understand the molecular function of BZR1, we performed tandem affinity purification of the BZR1 complex and identified BZR1-associated proteins using mass spectrometry. These BZR1-associated proteins included several known BR signaling components, such as BIN2, BSK1, 14–3-3λ, and PP2A, as well as a large number of proteins with previously unknown functions in BR signal transduction, including the kinases MKK5 and MAPK4, histone deacetylase 19, cysteine proteinase inhibitor 6, a DEAD-box RNA helicase, cysteine endopeptidases RD21A and RD21B, calmodulin-binding transcription activator 5, ubiquitin protease 12, cyclophilin 59, and phospholipid-binding protein synaptotagmin A. Their interactions with BZR1 were confirmed by in vivo and in vitro assays. Furthermore, MKK5 was found to phosphorylate BZR1 in vitro. This study demonstrates an effective method for purifying proteins associated with low-abundance transcription factors, and identifies new BZR1-interacting proteins with potentially important roles in BR response. PMID:24019147

  10. Identification of BZR1-interacting proteins as potential components of the brassinosteroid signaling pathway in Arabidopsis through tandem affinity purification.

    PubMed

    Wang, Chunming; Shang, Jian-Xiu; Chen, Qi-Xiu; Oses-Prieto, Juan A; Bai, Ming-Yi; Yang, Yihong; Yuan, Min; Zhang, Yu-Lan; Mu, Cong-Cong; Deng, Zhiping; Wei, Chuang-Qi; Burlingame, Alma L; Wang, Zhi-Yong; Sun, Ying

    2013-12-01

    Brassinosteroids (BRs) are essential phytohormones for plant growth and development. BRs are perceived by the cell surface receptor kinase BRI1, and downstream signal transduction through multiple components leads to activation of the transcription factors BZR1 and BZR2/BES1. BZR1 activity is highly controlled by BR through reversible phosphorylation, protein degradation, and nucleocytoplasmic shuttling. To further understand the molecular function of BZR1, we performed tandem affinity purification of the BZR1 complex and identified BZR1-associated proteins using mass spectrometry. These BZR1-associated proteins included several known BR signaling components, such as BIN2, BSK1, 14-3-3λ, and PP2A, as well as a large number of proteins with previously unknown functions in BR signal transduction, including the kinases MKK5 and MAPK4, histone deacetylase 19, cysteine proteinase inhibitor 6, a DEAD-box RNA helicase, cysteine endopeptidases RD21A and RD21B, calmodulin-binding transcription activator 5, ubiquitin protease 12, cyclophilin 59, and phospholipid-binding protein synaptotagmin A. Their interactions with BZR1 were confirmed by in vivo and in vitro assays. Furthermore, MKK5 was found to phosphorylate BZR1 in vitro. This study demonstrates an effective method for purifying proteins associated with low-abundance transcription factors, and identifies new BZR1-interacting proteins with potentially important roles in BR response. PMID:24019147

  11. The gall bladder cholecystokinin receptor exists in two guanine nucleotide-binding protein-regulated affinity states

    SciTech Connect

    Molero, X.; Miller, L.J. )

    1991-02-01

    To study proximal events in cholecystokinin (CCK) action on bovine gall bladder smooth muscle, we used the hormone analogue D-Tyr-Gly-((N1e28,31)CCK-26-32)-phenethyl ester (OPE), which has unique biological properties. This fully efficacious agonist differs from native CCK by not expressing supramaximal inhibition of cell shortening, yet it clearly interacts with the same receptor molecule. This was demonstrated in binding and affinity labeling studies, where both peptides label the same Mr 70,000-85,000 protein and both fully compete for binding of the other ligand. Further, its relatively high affinity for the low affinity CCK receptor permits the clear demonstration of two affinity states of a CCK receptor on a membrane preparation and makes possible evaluation of the molecular basis of these affinity states and their regulation. Analysis of homologous and heterologous binding curves performed with both CCK and OPE peptides and radioligands demonstrated the presence of two affinity states, with CCK being able to distinguish them (Kd1 = 0.48 +/- 0.04 nM and Kd2 = 56.5 +/- 7.4 nM) and OPE recognizing them equally (Kd1 = 0.94 +/- 0.31 nM and Kd2 = 0.96 +/- 0.23 nM). In the presence of nonhydrolyzable GTP analogues, there was a shift in distribution of receptors toward the low affinity state, with the total number of receptors and their absolute affinities for each peptide remaining constant. Thus, the gall bladder CCK receptor is a single molecule capable of assuming two interconvertible affinity states, regulated by a guanine nucleotide-binding protein. Two full agonists are capable of interacting with this molecule to yield different biological responses via different molecular events.

  12. Analysis of a nucleotide-binding site of 5-lipoxygenase by affinity labelling: binding characteristics and amino acid sequences.

    PubMed Central

    Zhang, Y Y; Hammarberg, T; Radmark, O; Samuelsson, B; Ng, C F; Funk, C D; Loscalzo, J

    2000-01-01

    5-Lipoxygenase (5LO) catalyses the first two steps in the biosynthesis of leukotrienes, which are inflammatory mediators derived from arachidonic acid. 5LO activity is stimulated by ATP; however, a consensus ATP-binding site or nucleotide-binding site has not been found in its protein sequence. In the present study, affinity and photoaffinity labelling of 5LO with 5'-p-fluorosulphonylbenzoyladenosine (FSBA) and 2-azido-ATP showed that 5LO bound to the ATP analogues quantitatively and specifically and that the incorporation of either analogue inhibited ATP stimulation of 5LO activity. The stoichiometry of the labelling was 1.4 mol of FSBA/mol of 5LO (of which ATP competed with 1 mol/mol) or 0.94 mol of 2-azido-ATP/mol of 5LO (of which ATP competed with 0.77 mol/mol). Labelling with FSBA prevented further labelling with 2-azido-ATP, indicating that the same binding site was occupied by both analogues. Other nucleotides (ADP, AMP, GTP, CTP and UTP) also competed with 2-azido-ATP labelling, suggesting that the site was a general nucleotide-binding site rather than a strict ATP-binding site. Ca(2+), which also stimulates 5LO activity, had no effect on the labelling of the nucleotide-binding site. Digestion with trypsin and peptide sequencing showed that two fragments of 5LO were labelled by 2-azido-ATP. These fragments correspond to residues 73-83 (KYWLNDDWYLK, in single-letter amino acid code) and 193-209 (FMHMFQSSWNDFADFEK) in the 5LO sequence. Trp-75 and Trp-201 in these peptides were modified by the labelling, suggesting that they were immediately adjacent to the C-2 position of the adenine ring of ATP. Given the stoichiometry of the labelling, the two peptide sequences of 5LO were probably near each other in the enzyme's tertiary structure, composing or surrounding the ATP-binding site of 5LO. PMID:11042125

  13. DNA-binding affinity and sequence permutation preference of the telomere protein from Euplotes crassus

    PubMed Central

    Suzuki, Takahito; McKenzie, Margaret; Ott, Elizabeth; Ilkun, Olesya; Horvath, Martin P.

    2008-01-01

    Telomere end binding proteins from diverse organisms use various forms of an ancient protein structure to recognize and bind with single strand DNA found at the ends of telomeres. To further understand the biochemistry and evolution of these proteins we have characterized the DNA-binding properties of the telomere end binding protein from Euplotes crassus (EcTEBP). EcTEBP and its predicted amino-terminal DNA-binding domain, EcTEBP-N, were expressed in E. coli and purified. Each protein formed stoichiometric (1:1) complexes with single strand DNA oligos derived from the precisely defined d(TTTTGGGGTTTTGG) sequence found at DNA termini in Euplotes. Dissociation constants for DNA•EcTEBP and DNA•EcTEBP-N were comparable, with KD-DNA = 38 ± 2 nM for the full-length protein and KD-DNA = 60 ± 4 nM for the N-terminal domain, indicating that the N-terminal domain retains high affinity for DNA even in the absence of potentially stabilizing moieties located in the C-terminal domain. Rate constants for DNA association and DNA dissociation corroborated a slightly improved DNA binding performance for the full-length protein (ka = 45 ± 4 μM-1 s-1, kd = 0.10 ± 0.02 s-1) relative to the N-terminal domain (ka = 18 ± 1 μM-1 s-1, kd = 0.15 ± 0.01 s-1). Equilibrium dissociation constants measured for sequence permutations of the telomere repeat spanned a 55 – 1400 nM range, with EcTEBP and EcTEBP-N binding most tightly to d(TTGGGGTTTTGG) — the sequence corresponding with that of mature DNA termini. Additionally, competition experiments showed that EcTEBP recognizes and binds the telomere-derived 14-nucleotide DNA in preference to shorter 5′ -truncation variants. Compared with multi-subunit complexes assembled with telomere single strand DNA from Oxytricha nova, our results highlight the relative simplicity of the Euplotes crassus system where a telomere end binding protein has biochemical properties indicating one protein subunit caps the single strand DNA. PMID

  14. Phosphorylation of CREB affects its binding to high and low affinity sites: implications for cAMP induced gene transcription.

    PubMed Central

    Nichols, M; Weih, F; Schmid, W; DeVack, C; Kowenz-Leutz, E; Luckow, B; Boshart, M; Schütz, G

    1992-01-01

    Cyclic AMP treatment of hepatoma cells leads to increased protein binding at the cyclic AMP response element (CRE) of the tyrosine aminotransferase (TAT) gene in vivo, as revealed by genomic footprinting, whereas no increase is observed at the CRE of the phosphoenolpyruvate carboxykinase (PEPCK) gene. Several criteria establish that the 43 kDa CREB protein is interacting with both of these sites. Two classes of CRE with different affinity for CREB are described. One class, including the TATCRE, is characterized by asymmetric and weak binding sites (CGTCA), whereas the second class containing symmetrical TGACGTCA sites shows a much higher binding affinity for CREB. Both classes show an increase in binding after phosphorylation of CREB by protein kinase A (PKA). An in vivo phosphorylation-dependent change in binding of CREB increases the occupancy of weak binding sites used for transactivation, such as the TATCRE, while high affinity sites may have constitutive binding of transcriptionally active and inactive CREB dimers, as demonstrated by in vivo footprinting at the PEPCK CRE. Thus, lower basal level and higher relative stimulation of transcription by cyclic AMP through low affinity CREs should result, allowing finely tuned control of gene activation. Images PMID:1354612

  15. Tuning the affinity of anion binding sites in porin channels with negatively charged residues: molecular details for OprP.

    PubMed

    Modi, Niraj; Bárcena-Uribarri, Iván; Bains, Manjeet; Benz, Roland; Hancock, Robert E W; Kleinekathöfer, Ulrich

    2015-02-20

    The cell envelope of the Gram negative opportunistic pathogen Pseudomonas aeruginosa is poorly permeable to many classes of hydrophilic molecules including antibiotics due to the presence of the narrow and selective porins. Here we focused on one of the narrow-channel porins, that is, OprP, which is responsible for the high-affinity uptake of phosphate ions. Its two central binding sites for phosphate contain a number of positively charged amino acids together with a single negatively charged residue (D94). The presence of this negatively charged residue in a binding site for negatively charged phosphate ions is highly surprising due to the potentially reduced binding affinity. The goal of this study was to better understand the role of D94 in phosphate binding, selectivity, and transport using a combination of mutagenesis, electrophysiology, and free-energy calculations. The presence of a negatively charged residue in the binding site is critical for this specific porin OprP as emphasized by the evolutionary conservation of such negatively charged residue in the binding site of several anion-selective porins. Mutations of D94 in OprP to any positively charged or neutral residue increased the binding affinity of phosphate for OprP. Detailed analysis indicated that this anionic residue in the phosphate binding site of OprP, despite its negative charge, maintained energetically favorable phosphate binding sites in the central region of the channel and at the same time decreased residence time thus preventing excessively strong binding of phosphate that would oppose phosphate flux through the channel. Intriguingly mutations of D94 to positively charged residues, lysine and arginine, resulted in very different binding affinities and free energy profiles, indicating the importance of side chain conformations of these positively charged residues in phosphate binding to OprP.

  16. A carbohydrate-binding affinity ligand for the specific enrichment of glycoproteins.

    PubMed

    Chen, Chen; El Khoury, Graziella; Zhang, Peiqing; Rudd, Pauline M; Lowe, Christopher R

    2016-04-29

    One challenge facing the production of glycoprotein therapeutics is the lack of stable and selective affinity ligands for their enrichment. Synthetic affinity ligands based on the solid phase multi-component Ugi reaction represent a desirable option, particularly those incorporating benzoboroxole and its derivatives, which have been shown to enrich glycoproteins under physiological conditions. Thus, an Ugi ligand, A21C11I8, comprising 5-amino-2-hydroxymethylphenylboronic acid was synthesised on aldehyde-functionalised Sepharose™. Immobilised A21C11I8 displayed affinity for the glycosylated protein, glucose oxidase (GOx), which bound primarily through its glycan moiety. The ligand had a preference for sugar alcohols and the furanose form of the monosaccharides tested. Compared with immobilised 3-aminophenylboronic acid and Concanavalin A, the Ugi ligand was able to purify GOx from spiked Escherichia coli supernatants with retention of its maximum enzymatic activity and protein recovery. Glycan profiles of human immunoglobulin G tested on A21C11I8 columns suggested that the adsorbent possesses the potential to resolve sialylated and neutral glycoforms. The benzoboroxole-functionalised Ugi ligand may find application in selective glycoform separation.

  17. High-affinity binding of short peptides to major histocompatibility complex class II molecules by anchor combinations.

    PubMed Central

    Hammer, J; Belunis, C; Bolin, D; Papadopoulos, J; Walsky, R; Higelin, J; Danho, W; Sinigaglia, F; Nagy, Z A

    1994-01-01

    We have previously identified four anchor positions in HLA-DRB1*0101-binding peptides, and three anchors involved in peptide binding to DRB1*0401 and DRB1*1101 molecules, by screening of an M13 peptide display library (approximately 20 million independent nonapeptides) for DR-binding activity. In this study, high stringency screening of the M13 library for DRB1*0401 binding has resulted in identification of three further anchor positions. Taken together, a peptide-binding motif has been obtained, in which six of seven positions show enrichment of certain residues. We have demonstrated an additive effect of anchors in two different ways: (i) the addition of more anchors is shown to compensate for progressive truncation of designer peptides; (ii) the incorporation of an increasing number of anchors into 6- or 7-residue-long designer peptides is shown to result in a gradual increase of binding affinity to the level of 13-residue-long high-affinity epitopes. The anchor at relative position 1 seems to be obligatory, in that its substitution abrogates binding completely, whereas the elimination of other anchors results only in partial loss of binding affinity. The spacing between anchors is critical, since their effect is lost by shifting them one position toward the N or C terminus. The information born out of this study has been successfully used to identify DR-binding sequences from natural proteins. PMID:8183931

  18. Ultrasensitive Analysis of Binding Affinity of HIV Receptor and Neutralizing Antibody Using Solution-Phase Electrochemiluminescence Assay

    PubMed Central

    Xu, Xiao-Hong Nancy; Wen, Zhaoyang; Brownlow, William J.

    2012-01-01

    Binding of a few ligand molecules with its receptors on cell surface can initiate cellular signaling transduction pathways, and trigger viral infection of host cells. HIV-1 infects host T-cells by binding its viral envelope protein (gp120) with its receptor (a glycoprotein, CD4) on T cells. Primary strategies to prevent and treat HIV infection is to develop therapies (e.g., neutralizing antibodies) that can block specific binding of CD4 with gp120. The infection often leads to the lower counts of CD4 cells, which makes it an effective biomarker to monitor the AIDS progression and treatment. Despite research over decades, quantitative assays for effective measurements of binding affinities of protein-protein (ligand-receptor, antigen-antibody) interactions remains highly sought. Solid-phase electrochemiluminescence (ECL) immunoassay has been commonly used to capture analytes from the solution for analysis, which involves immobilization of antibody on solid surfaces (micron-sized beads), but it cannot quantitatively measure binding affinities of molecular interactions. In this study, we have developed solution-phase ECL assay with a wide dynamic range (0–2 nM) and high sensitivity and specificity for quantitative analysis of CD4 at femtomolar level and their binding affinity with gp120 and monoclonal antibodies (MABs). We found that binding affinities of CD4 with gp120 and MAB (Q4120) are 9.5×108 and 1.2×109 M−1, respectively. The results also show that MAB (Q4120) of CD4 can completely block the binding of gp120 with CD4, while MAB (17b) of gp120 can only partially block their interaction. This study demonstrates that the solution-phase ECL assay can be used for ultrasensitive and quantitative analysis of binding affinities of protein-protein interactions in solution for better understating of protein functions and identification of effective therapies to block their interactions. PMID:23565071

  19. Two high-affinity ligand binding states of uterine estrogen receptor distinguished by modulation of hydrophobic environment

    SciTech Connect

    Hutchens, T.W.; Li, C.M.; Zamah, N.M.; Besch, P.K.

    1987-02-10

    The steroid binding function of soluble (cytosolic) estrogen receptors from calf uteri was evaluated under conditions known to modify the extent of hydrophobic interaction with receptor-associated proteins. Receptor preparations were equilibrated into 6 M urea buffers and control buffers by chromatography through small columns of Sephadex G-25 or by dialysis at 0.6 /sup 0/C. Equilibrium dissociation constants (K/sub d/) and binding capacities (n) of experimental and control receptor preparations were determined by 13-point Scatchard analyses using concentrations of 17..beta..-(/sup 3/H)estradiol from 0.05 to 10 nM. Nonspecific binding was determined at each concentration by parallel incubations with a 200-fold molar excess of the receptor-specific competitor diethylstilbestrol. The control receptor population was consistently found to be a single class of binding sites with a high affinity for estradiol which was unaffected by G-25 chromatography, by dialysis, by dilution, or by the presence of 0.4 M KCl. However, equilibration into 6 M urea induced a discrete (10-fold) reduction in receptor affinity to reveal a second, thermodynamically stable, high-affinity binding state. The presence of 0.4 M KCl did not significantly influence the discrete change in receptor affinity induced by urea. The effects of urea on both receptor affinity and binding capacity were reversible, suggesting a lack of covalent modification. These results demonstrate nonenzymatic means by which not only the binding capacity but also the affinity of receptor for estradiol can be reversibly controlled, suggesting that high concentrations of urea might be more effectively utilized during the physicochemical characterization and purification of steroid receptor proteins.

  20. The N-terminus of TDP-43 promotes its oligomerization and enhances DNA binding affinity

    SciTech Connect

    Chang, Chung-ke; Wu, Tzong-Huah; Wu, Chu-Ya; Chiang, Ming-hui; Toh, Elsie Khai-Woon; Hsu, Yin-Chih; Lin, Ku-Feng; Liao, Yu-heng; Huang, Tai-huang; Huang, Joseph Jen-Tse

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer The N-terminus of TDP-43 contains an independently folded structural domain (NTD). Black-Right-Pointing-Pointer The structural domains of TDP-43 are arranged in a beads-on-a-string fashion. Black-Right-Pointing-Pointer The NTD promotes TDP-43 oligomerization in a concentration-dependent manner. Black-Right-Pointing-Pointer The NTD may assist nucleic acid-binding activity of TDP-43. -- Abstract: TDP-43 is a DNA/RNA-binding protein associated with different neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-U). Here, the structural and physical properties of the N-terminus on TDP-43 have been carefully characterized through a combination of nuclear magnetic resonance (NMR), circular dichroism (CD) and fluorescence anisotropy studies. We demonstrate for the first time the importance of the N-terminus in promoting TDP-43 oligomerization and enhancing its DNA-binding affinity. An unidentified structural domain in the N-terminus is also disclosed. Our findings provide insights into the N-terminal domain function of TDP-43.

  1. Calculation of substrate binding affinities for a bacterial GH78 rhamnosidase through molecular dynamics simulations

    PubMed Central

    Grandits, Melanie; Michlmayr, Herbert; Sygmund, Christoph; Oostenbrink, Chris

    2013-01-01

    Ram2 from Pediococcus acidilactici is a rhamnosidase from the glycoside hydrolase family 78. It shows remarkable selectivity for rutinose rather than para-nitrophenyl-alpha-l-rhamnopyranoside (p-NPR). Molecular dynamics simulations were performed using a homology model of this enzyme, in complex with both substrates. Free energy calculations lead to predicted binding affinities of −34.4 and −30.6 kJ mol−1 respectively, agreeing well with an experimentally estimated relative free energy of 5.4 kJ mol−1. Further, the most relevant binding poses could be determined. While p-NPR preferably orients its rhamnose moiety toward the active site, rutinose interacts most strongly with its glucose moiety. A detailed hydrogen bond analysis confirms previously implicated residues in the active site (Asp217, Asp222, Trp226, Asp229 and Glu488) and quantifies the importance of individual residues for the binding. The most important amino acids are Asp229 and Phe339 which are involved in many interactions during the simulations. While Phe339 was observed in more simulations, Asp229 was involved in more persistent interactions (forming an average of at least 2 hydrogen bonds during the simulation). These analyses directly suggest mutations that could be used in a further experimental characterization of the enzyme. This study shows once more the strength of computer simulations to rationalize and guide experiments at an atomic level. PMID:23914137

  2. Determining force dependence of two-dimensional receptor-ligand binding affinity by centrifugation.

    PubMed Central

    Piper, J W; Swerlick, R A; Zhu, C

    1998-01-01

    Analyses of receptor-ligand interactions are important to the understanding of cellular adhesion. Traditional methods of measuring the three-dimensional (3D) dissociation constant (Kd) require at least one of the molecular species in solution and hence cannot be directly applied to the case of cell adhesion. We describe a novel method of measuring 2D binding characteristics of receptors and ligands that are attached to surfaces and whose bonds are subjected to forces. The method utilizes a common centrifugation assay to quantify adhesion. A model for the experiment has been formulated, solved exactly, and tested carefully. The model is stochastically based and couples the bond force to the binding affinity. The method was applied to examine tumor cell adherence to recombinant E-selectin. Satisfactory agreement was found between predictions and data. The estimated zero-force 2D Kd for E-selectin/carbohydrate ligand binding was approximately 5 x 10(3) microm(-2), and the bond interaction range was subangstrom. Our results also suggest that the number of bonds mediating adhesion was small (<5). PMID:9449350

  3. FMRFamide: low affinity inhibition of opioid binding to rabbit brain membranes

    SciTech Connect

    Zhu, X.Z.; Raffa, R.B.

    1986-03-05

    FMRFamide (Phe-Met-Arg-Phe-NH/sub 2/) was first isolated from the ganglia of molluscs by Price and Greenberg in 1977. The peptide was subsequently shown to have diverse actions on various types of molluscan and mammalian tissues. The presence of immunoreactive FMRFamide-like material (irFMRF) in multiple areas of rat brain, spinal cord, and gastrointestinal tract suggests that irFMRF may have a physiological role in mammals. Tang, Yang and Costa recently demonstrated that FMRFamide attenuates morphine antinociception in rats and postulated, based on this and several other lines of evidence, that irFMRF might be an endogenous opioid antagonist. In the present study, they tested the ability of FMRFamide to inhibit the binding of opioid receptor ligands to rabbit membrane preparations. FMRFamide inhibited the specific binding of both /sup 3/(H)-dihydromorphine and /sup 3/(H)-ethylketocyclazocine (IC/sub 50/ = 14 ..mu..M and 320 ..mu..M, respectively) in a dose-related manner, suggesting that FMRFamide may affect binding to at least two types of opioid receptors (mu and kappa). These data are consistent with the concept that irFMRF might act as an endogenous opioid antagonist. However, the low affinity of FMRFamide leaves open the possibility of another mechanism of opioid antagonism, such as neuromodulation.

  4. Influence of Linker Structure on the Anion Binding Affinity of Biscyclopeptides

    SciTech Connect

    Reyheller, Carsten; Hay, Benjamin; Kubik, Stefan

    2007-01-01

    A systematic analysis is presented on the influence of the linking unit between two cyclopeptide rings on the affinity of such biscyclopeptide-based anion receptors in aqueous solvent mixtures. Although the differences in the affinity and selectivity of these receptors toward a given anion are not very pronounced, there are profound differences in the thermodynamics of anion complexation. Enthalpic and entropic contributions both (1) play a role in determining the binding affinity and (2) show significant variation as the linking structure is changed. A decrease in conformational rigidity of the linker improves the entropic advantage for complex formation, but not necessarily the overall complex stability. This effect may be due, in part, to the fact that structural constraints within more rigid linkers might prevent efficient interactions between the host and guest. The optimal linker, which exhibits both favourable enthalpic and entropic contributions, was identified using de novo structure-based design methods as implemented in the HostDesigner software. The submitted manuscript has been authored by a contractor of the U. S. Government under contract No. DE-AC05-00OR22725. Accordingly, the U. S. Government retains a non-exclusive, royalty-free license to publish or reproduce the published form of this contribution, or allow others to do so, for the U. S. Government purposes. This research was sponsored by the following program of the U. S. Department of Energy, Office of Science: the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences (ORNL FWP No. ERKKC08. Oak Ridge National Laboratory is managed and operated by UT-Battelle, LLC under contract number DE-AC05-00OR22725 with the U. S. Department of Energy.

  5. Differences between high-affinity forskolin binding sites in dopamine-riche and other regions of rat brain

    SciTech Connect

    Poat, J.A.; Cripps, H.E.; Iversen, L.L.

    1988-05-01

    Forskolin labelled with (/sup 3/H) bound to high- and low-affinity sites in the rat brain. The high-affinity site was discretely located, with highest densities in the striatum, nucleus accumbens, olfactory tubercule, substantia nigra, hippocampus, and the molecular layers of the cerebellum. This site did not correlate well with the distribution of adenylate cyclase. The high-affinity striatal binding site may be associated with a stimulatory guanine nucleotide-binding protein. Thus, the number of sites was increased by the addition of Mg/sup 2 +/ and guanylyl imidodiphosphate. Cholera toxin stereotaxically injected into rat striatum increased the number of binding sites, and no further increase was noted following the subsequent addition of guanyl nucleotide. High-affinity forskolin binding sites in non-dopamine-rich brain areas (hippocampus and cerebullum) were modulated in a qualitatively different manner by guanyl nucleotides. In these areas the number of binding sites was significantly reduced by the addition of guanyl nucleotide. These results suggest that forskolin may have a potential role in identifying different functional/structural guanine nucleotide-binding proteins.

  6. Identification and properties of very high affinity brain membrane-binding sites for a neurotoxic phospholipase from the taipan venom

    SciTech Connect

    Lambeau, G.; Barhanin, J.; Schweitz, H.; Qar, J.; Lazdunski, M. )

    1989-07-05

    Four new monochain phospholipases were purified from the Oxyuranus scutellatus (taipan) venom. Three of them were highly toxic when injected into mice brain. One of these neurotoxic phospholipases, OS2, was iodinated and used in binding experiments to demonstrate the presence of two families of specific binding sites in rat brain synaptic membranes. The affinities were exceptionally high, Kd1 = 1.5 +/- 0.5 pM and Kd2 = 45 +/- 10 pM, and the maximal binding capacities were Bmax 1 = 1 +/- 0.4 and Bmax 2 = 3 +/- 0.5 pmol/mg of protein. Both binding sites were sensitive to proteolysis and demonstrated to be located on proteins of Mr 85,000-88,000 and 36,000-51,000 by cross-linking and photoaffinity labeling techniques. The binding of {sup 125}I-OS2 to synaptic membranes was dependent on Ca2+ ions and enhanced by Zn2+ ions which inhibit phospholipase activity. Competition experiments have shown that, except for beta-bungarotoxin, a number of known toxic snake or bee phospholipases have very high affinities for the newly identified binding sites. A good correlation (r = 0.80) was observed between toxicity and affinity but not between phospholipase activity and affinity.

  7. Inhibitory GTP binding protein G/sub i/ regulates US -adrenoceptor affinity towards US -agonists

    SciTech Connect

    Marbach, I.; Levitzki, A.

    1987-05-01

    Treatment of S-49 lymphoma cell membranes with pertussis toxin (PT) causes a three-fold reduction of US -adrenoceptor (US AR) affinity towards isoproterenol. A similar treatment with cholera toxin (CT) does not cause such a modulation. The effects were studied by the detailed analysis of SVI-cyanopindolol (CYP) binding curves in the absence and presence of increasing agonist concentrations. Thus, the authors were able to compare in detail the effects of G/sub s/ and G/sub i/ on the agonist-associated state of the US AR. In contrast to these findings, PT treatment does not have any effect on the displacement of SVI-CYP by (-)isoproterenol. These results demonstrate that the inhibitory GTP protein G/sub i/ modulates the US AR affinity towards US -agonists. This might be due to the association of G/sub i/ with the agonist-bound US AR x G/sub s/ x C complex within the membrane. This hypothesis, as well as others, is under investigation.

  8. Protein-Polyelectrolyte Coacervation: Morphology Diagram, Binding Affinity, and Protein Separation

    NASA Astrophysics Data System (ADS)

    Hoagland, David; Du, Xiaosong; Dubin, Paul

    2014-03-01

    For aqueous mixtures of negatively charged polysaccharide, hyaluronic acid (HA), and globular protein, either bovine serum albumin (BSA) or beta-lactoglobulin (BLG), a pH-ionic strength (I) morphology diagram, with regions of homogeneous solution, soluble complex, coacervation, precipitation, and redissolution, was developed by pH titrations performed at fixed I. The systems are models for coacervation, or liquid-liquid phase separation, between flexible and compact solutes of opposite charge. Protein charge here is tuned by pH, and titration keeps the mixtures close to equilibrium. At high I, only homogeneous solution is observed, as true at high and low pH. Diagrams for the proteins differ because HA affinity for BSA is higher than for BLG, traced to BSA's greater charge patchiness and higher net charge; isothermal solution titration calorimetry finds a factor of two difference in binding energy. Dependences of transition pH on protein charge Z and solution I offer additional insights into interactions underlying morphology transitions. At optimal conditions, the affinity disparity is sufficient to achieve highly selective BSA coacervation in a 1:1 protein mixture, suggesting coacervation to separate similar proteins under mild, non-denaturing conditions. Funding: NSF CBET-1133289, NSF (UMass MRSEC).

  9. Characterization of an Enterococcus hirae penicillin-binding protein 3 with low penicillin affinity.

    PubMed

    Piras, G; el Kharroubi, A; van Beeumen, J; Coeme, E; Coyette, J; Ghuysen, J M

    1990-12-01

    Enterococcus hirae S185, a clinical isolate from swine intestine, exhibits a relatively high resistance to penicillin and contains two 77-kDa penicillin-binding proteins 3 of high (PBP 3s) and low (PBP 3r) affinity to penicillin, respectively. A laboratory mutant S185r has been obtained which overproduces PBP 3r and has a highly increased resistance to penicillin. Peptide fragments specifically produced by trypsin and SV8 protease digestions of PBP 3r were isolated, and the amino acid sequences of their amino terminal regions were determined. On the basis of these sequences, oligonucleotides were synthesized and used as primers to generate, by polymerization chain reaction, a 233-bp DNA fragment the sequence of which translated into a 73-amino-acid peptide segment of PBP 3r. These structural data led to the conclusion that the E. hirae PBP 3r and the methicillin-resistant staphylococcal PBP 2' are members of the same class of high-Mr PBPs. As shown by immunological tests, PBP 3r is not related to PBP 3s but, in contrast, is related to the 71-kDa PBP 5 of low penicillin affinity which is responsible for penicillin resistance in E. hirae ATCC 9790 and R40. PMID:2254261

  10. Two Amino Acid Residues Confer Different Binding Affinities of Abelson Family Kinase Src Homology 2 Domains for Phosphorylated Cortactin*

    PubMed Central

    Gifford, Stacey M.; Liu, Weizhi; Mader, Christopher C.; Halo, Tiffany L.; Machida, Kazuya; Boggon, Titus J.; Koleske, Anthony J.

    2014-01-01

    The closely related Abl family kinases, Arg and Abl, play important non-redundant roles in the regulation of cell morphogenesis and motility. Despite similar N-terminal sequences, Arg and Abl interact with different substrates and binding partners with varying affinities. This selectivity may be due to slight differences in amino acid sequence leading to differential interactions with target proteins. We report that the Arg Src homology (SH) 2 domain binds two specific phosphotyrosines on cortactin, a known Abl/Arg substrate, with over 10-fold higher affinity than the Abl SH2 domain. We show that this significant affinity difference is due to the substitution of arginine 161 and serine 187 in Abl to leucine 207 and threonine 233 in Arg, respectively. We constructed Abl SH2 domains with R161L and S187T mutations alone and in combination and find that these substitutions are sufficient to convert the low affinity Abl SH2 domain to a higher affinity “Arg-like” SH2 domain in binding to a phospho-cortactin peptide. We crystallized the Arg SH2 domain for structural comparison to existing crystal structures of the Abl SH2 domain. We show that these two residues are important determinants of Arg and Abl SH2 domain binding specificity. Finally, we expressed Arg containing an “Abl-like” low affinity mutant Arg SH2 domain (L207R/T233S) and find that this mutant, although properly localized to the cell periphery, does not support wild type levels of cell edge protrusion. Together, these observations indicate that these two amino acid positions confer different binding affinities and cellular functions on the distinct Abl family kinases. PMID:24891505

  11. Somatostatin analogs. Dissociation of brain receptor binding affinities and pituitary actions in the rat.

    PubMed

    Srikant, C B; Patel, Y C

    1981-01-01

    We have recently demonstrated the presence of specific receptors for somatostatin (SRIF) in rat brain synaptosomal membranes which appear to mediate its action. Using this system as a radioreceptor assay, we have examined the ability of a wide range of SRIF analogs to interact with these receptors. Although structural modifications in the Trp8 moiety of SRIF resulted in significant enhancement of affinity for binding to the brain SRIF receptors, the different relative specificities of des AA1,2,4,5,12,13 D-Trp8 SRIF (oligo D-Trp8 SRIF), D-Trp8 SRIF and D-5-Br-Trp8 SRIF in the pituitary and the central nervous system (CNS) suggest that basic differences exist between SRIF receptors present in the brain and the pituitary.

  12. Mu/sub 1/: A very high affinity subtype of enkephalin binding sites in rat brain

    SciTech Connect

    Lutz, R.A.; Cruciani, R.A.; Munson, P.J.; Rodbard, D.

    1985-06-10

    Displacement studies of (/sup 3/H)-(D-Ala/sup 2/-MePhe/sup 4/-Gly-ol/sup 5/)-enkephalin ((/sup 3/H)-DAGO) and (/sup 3/H)-(D-Ala/sup 2/-D-Leu/sup 5/)-enkephalin ((/sup 3/H)-DADL) by the corresponding unlabeled ligands show that there are at least three classes of sites which bind these enkephalin analogs with high affinity. Using computer modeling, the introduction of the third site significantly improved the goodness of fit in ten consecutive experiments. These sites appear to correspond to the ..mu.., delta, and ..mu../sub 1/ sites, with mean dissociation constants of 11, 1.3 and 0.9 nM for DADL and 2.5, 300 and 0.3 nM for DAGO, respectively. 15 reference, 3 figures, 1 table.

  13. Packing density of glycolipid biosurfactant monolayers give a significant effect on their binding affinity toward immunoglobulin G.

    PubMed

    Imura, Tomohiro; Masuda, Yuma; Ito, Seya; Worakitkanchanakul, Wannasiri; Morita, Tomotake; Fukuoka, Tokuma; Sakai, Hideki; Abe, Masahiko; Kitamoto, Dai

    2008-01-01

    Mannosylerythritol lipid-A (MEL-A) is one of the most promising glycolipid biosurfactants, and abundantly produced by Pseudozyma yeasts. MEL-A gives not only excellent self-assembling properties but also a high binding affinity toward human immunoglobulin G (HIgG). In this study, three kinds of MEL-A were prepared from methyl myristate [MEL-A (m)], olive oil [MEL-A (o)], and soybean oil [MEL-A (s)], and the effect of interfacial properties of each MEL-A monolayer on the binding affinity toward HIgG was investigated using surface plasmon resonance (SPR) and the measurement of surface pressure (pi)-area (A) isotherms. Based on GC-MS analysis, the main fatty acids were C(8) and C(10) acids in all MEL-A, and the content of unsaturated fatty acids was 0% for MEL-A (m), 9.1% for MEL-A (o), 46.3% for MEL-A (s), respectively. Interestingly, the acid content significantly influenced on their binding affinity, and the monolayer of MEL-A (o) gave a higher binding affinity than that of MEL-A (m) and MEL-A (s). Moreover, the mixed MEL-A (o)/ MEL-A (s) monolayer prepared from 1/1 molar ratio, which comprised of 27.8% of unsaturated fatty acids, indicated the highest binding affinity. At the air/water interface, MEL-A (o) monolayer exhibited a phase transition at 13 degrees C from a liquid condensed monolayer to a liquid expanded monolayer, and the area per molecule significantly expanded above 13 degrees C, while the amount of HIgG bound to the liquid expanded monolayer was much higher than that bound to liquid condensed monolayer. The binding affinity of MEL-A toward HIgG is thus likely to closely relate to the monolayer packing density, and may be partly controlled by temperature. PMID:18622124

  14. Amide-mediated hydrogen bonding at organic crystal/water interfaces enables selective endotoxin binding with picomolar affinity.

    PubMed

    Vagenende, Vincent; Ching, Tim-Jang; Chua, Rui-Jing; Thirumoorthi, Navanita; Gagnon, Pete

    2013-05-22

    Since the discovery of endotoxins as the primary toxic component of Gram-negative bacteria, researchers have pursued the quest for molecules that detect, neutralize, and remove endotoxins. Selective removal of endotoxins is particularly challenging for protein solutions and, to this day, no general method is available. Here, we report that crystals of the purine-derived compound allantoin selectively adsorb endotoxins with picomolar affinity through amide-mediated hydrogen bonding in aqueous solutions. Atom force microscopy and chemical inhibition experiments indicate that endotoxin adsorption is largely independent from hydrophobic and ionic interactions with allantoin crystals and is mediated by hydrogen bonding with amide groups at flat crystal surfaces. The small size (500 nm) and large specific surface area of allantoin crystals results in a very high endotoxin-binding capacity (3 × 10(7) EU/g) which compares favorably with known endotoxin-binding materials. These results provide a proof-of-concept for hydrogen bond-based molecular recognition processes in aqueous solutions and establish a practical method for removing endotoxins from protein solutions.

  15. Arene Binding Affinities in [CpRu(nu6-arene)]+ Complexes: Models for the Adsorption of Arenes on Hydroesulferization Catalysts

    SciTech Connect

    Choi, M. G.; Ho, T. C.; Angelici, R.

    2008-02-29

    Product/reactant ratios (Y) were determined for the reactions CpRu({eta}{sup 6}-DBT){sup +} + L CpRu({eta}{sup 6}-L){sup +} + DBT (where DBT is dibenzothiophene and L is a homo- or heterocyclic arene), which were conducted under UV photolysis conditions. In the photostationary state, the Y values for the different arenes decrease in the following order: mesitylene (17) > toluene (13) > indole (9.1) > carbazole (6.7) > benzene (5.9) > fluorene (5.1) > biphenyl (3.9) > DBT (1.0) > phenanthrene (0.65) > naphthalene (0.35). In general, alkyl-substituted arenes have a higher binding affinity than the parent arene, except for tert-butyl groups, which decrease the Y values. These trends in {eta}{sup 6}-arene binding to CpRu{sup +} provide a basis for understanding competitive adsorption of arenes on metal sites of hydrotreating catalysts. Such arene components in petroleum feedstocks reduce the rates of hydrodesulfurization of dibenzothiophenes.

  16. Structure-Affinity Properties of a High-Affinity Ligand of FKBP12 Studied by Molecular Simulations of a Binding Intermediate

    PubMed Central

    Olivieri, Lilian; Gardebien, Fabrice

    2014-01-01

    With a view to explaining the structure-affinity properties of the ligands of the protein FKBP12, we characterized a binding intermediate state between this protein and a high-affinity ligand. Indeed, the nature and extent of the intermolecular contacts developed in such a species may play a role on its stability and, hence, on the overall association rate. To find the binding intermediate, a molecular simulation protocol was used to unbind the ligand by gradually decreasing the biasing forces introduced. The intermediate was subsequently refined with 17 independent stochastic boundary molecular dynamics simulations that provide a consistent picture of the intermediate state. In this state, the core region of the ligand remains stable, notably because of the two anchoring oxygen atoms that correspond to recurrent motifs found in all FKBP12 ligand core structures. Besides, the non-core regions participate in numerous transient intermolecular and intramolecular contacts. The dynamic aspect of most of the contacts seems important both for the ligand to retain at least a part of its configurational entropy and for avoiding a trapped state along the binding pathway. Since the transient and anchoring contacts contribute to increasing the stability of the intermediate, as a corollary, the dissociation rate constant of this intermediate should be decreased, resulting in an increase of the affinity constant . The present results support our previous conclusions and provide a coherent rationale for explaining the prevalence in high-affinity ligands of (i) the two oxygen atoms found in carbonyl or sulfonyl groups of dissimilar core structures and of (ii) symmetric or pseudo-symmetric mobile groups of atoms found as non-core moieties. Another interesting aspect of the intermediate is the distortion of the flexible 80 s loop of the protein, mainly in its tip region, that promotes the accessibility to the bound state. PMID:25502559

  17. Dynamic Multi-Component Covalent Assembly for the Reversible Binding of Secondary Alcohols and Chirality Sensing

    PubMed Central

    You, Lei; Berman, Jeffrey S.; Anslyn, Eric V.

    2011-01-01

    Reversible covalent bonding is often employed for the creation of novel supramolecular structures, multi-component assemblies, and sensing ensembles. In spite of remarkable success of dynamic covalent systems, the reversible binding of a mono-alcohol with high strength is challenging. Here we show that a strategy of carbonyl activation and hemiaminal ether stabilization can be embodied in a four-component reversible assembly that creates a tetradentate ligand and incorporates secondary alcohols with exceptionally high affinity. Evidence is presented that the intermediate leading to binding and exchange of alcohols is an iminium ion. Further, to demonstrate the use of this assembly process we explored chirality sensing and enantiomeric excess determinations. An induced twist in the ligand by a chiral mono-ol results in large Cotton effects in the circular dichroism spectra indicative of the alcohol’s handedness. The strategy revealed in this study should prove broadly applicable for the incorporation of alcohols into supramolecular architecture construction. PMID:22109274

  18. Nonlinear scoring functions for similarity-based ligand docking and binding affinity prediction.

    PubMed

    Brylinski, Michal

    2013-11-25

    A common strategy for virtual screening considers a systematic docking of a large library of organic compounds into the target sites in protein receptors with promising leads selected based on favorable intermolecular interactions. Despite a continuous progress in the modeling of protein-ligand interactions for pharmaceutical design, important challenges still remain, thus the development of novel techniques is required. In this communication, we describe eSimDock, a new approach to ligand docking and binding affinity prediction. eSimDock employs nonlinear machine learning-based scoring functions to improve the accuracy of ligand ranking and similarity-based binding pose prediction, and to increase the tolerance to structural imperfections in the target structures. In large-scale benchmarking using the Astex/CCDC data set, we show that 53.9% (67.9%) of the predicted ligand poses have RMSD of <2 Å (<3 Å). Moreover, using binding sites predicted by recently developed eFindSite, eSimDock models ligand binding poses with an RMSD of 4 Å for 50.0-39.7% of the complexes at the protein homology level limited to 80-40%. Simulations against non-native receptor structures, whose mean backbone rearrangements vary from 0.5 to 5.0 Å Cα-RMSD, show that the ratio of docking accuracy and the estimated upper bound is at a constant level of ∼0.65. Pearson correlation coefficient between experimental and predicted by eSimDock Ki values for a large data set of the crystal structures of protein-ligand complexes from BindingDB is 0.58, which decreases only to 0.46 when target structures distorted to 3.0 Å Cα-RMSD are used. Finally, two case studies demonstrate that eSimDock can be customized to specific applications as well. These encouraging results show that the performance of eSimDock is largely unaffected by the deformations of ligand binding regions, thus it represents a practical strategy for across-proteome virtual screening using protein models. eSimDock is freely

  19. Nonlinear scoring functions for similarity-based ligand docking and binding affinity prediction.

    PubMed

    Brylinski, Michal

    2013-11-25

    A common strategy for virtual screening considers a systematic docking of a large library of organic compounds into the target sites in protein receptors with promising leads selected based on favorable intermolecular interactions. Despite a continuous progress in the modeling of protein-ligand interactions for pharmaceutical design, important challenges still remain, thus the development of novel techniques is required. In this communication, we describe eSimDock, a new approach to ligand docking and binding affinity prediction. eSimDock employs nonlinear machine learning-based scoring functions to improve the accuracy of ligand ranking and similarity-based binding pose prediction, and to increase the tolerance to structural imperfections in the target structures. In large-scale benchmarking using the Astex/CCDC data set, we show that 53.9% (67.9%) of the predicted ligand poses have RMSD of <2 Å (<3 Å). Moreover, using binding sites predicted by recently developed eFindSite, eSimDock models ligand binding poses with an RMSD of 4 Å for 50.0-39.7% of the complexes at the protein homology level limited to 80-40%. Simulations against non-native receptor structures, whose mean backbone rearrangements vary from 0.5 to 5.0 Å Cα-RMSD, show that the ratio of docking accuracy and the estimated upper bound is at a constant level of ∼0.65. Pearson correlation coefficient between experimental and predicted by eSimDock Ki values for a large data set of the crystal structures of protein-ligand complexes from BindingDB is 0.58, which decreases only to 0.46 when target structures distorted to 3.0 Å Cα-RMSD are used. Finally, two case studies demonstrate that eSimDock can be customized to specific applications as well. These encouraging results show that the performance of eSimDock is largely unaffected by the deformations of ligand binding regions, thus it represents a practical strategy for across-proteome virtual screening using protein models. eSimDock is freely

  20. Pharmacologically distinct phenotypes of α1B-adrenoceptors: variation in binding and functional affinities for antagonists

    PubMed Central

    Yoshiki, Hatsumi; Uwada, Junsuke; Anisuzzaman, Abu Syed Md; Umada, Hidenori; Hayashi, Ryoji; Kainoh, Mie; Masuoka, Takayoshi; Nishio, Matomo; Muramatsu, Ikunobu

    2014-01-01

    Background and Purpose The pharmacological properties of particular receptors have recently been suggested to vary under different conditions. We compared the pharmacological properties of the α1B-adrenoceptor subtype in various tissue preparations and under various conditions. Experimental Approach [3H]-prazosin binding to α1B-adrenoceptors in rat liver (segments, dispersed hepatocytes and homogenates) was assessed and the pharmacological profiles were compared with the functional and binding profiles in rat carotid artery and recombinant α1B-adrenoceptors. Key Results In association and saturation-binding experiments with rat liver, binding affinity for [3H]-prazosin varied significantly between preparations (KD value approximately ten times higher in segments than in homogenates). The binding profile for various drugs in liver segments also deviated from the representative α1B-adrenoceptor profile observed in liver homogenates and recombinant receptors. L-765,314 and ALS-77, selective antagonists of α1B-adrenoceptors, showed high binding and antagonist affinities in liver homogenates and recombinant α1B-adrenoceptors. However, binding affinities for both ligands in the segments of rat liver and carotid artery were 10 times lower, and the antagonist potencies in α1B-adrenoceptor-mediated contractions of carotid artery were more than 100 times lower than the representative α1B-adrenoceptor profile. Conclusions and Implications In contrast to the consistent profile of recombinant α1B-adrenoceptors, the pharmacological profile of native α1B-adrenoceptors of rat liver and carotid artery varied markedly under various receptor environments, showing significantly different binding properties between intact tissues and homogenates, and dissociation between functional and binding affinities. In addition to conventional ‘subtype’ characterization, ‘phenotype’ pharmacology must be considered in native receptor evaluations in vivo and in future

  1. Comparison of Performance of Docking, LIE, Metadynamics and QSAR in Predicting Binding Affinity of Benzenesulfonamides.

    PubMed

    Raškevičius, Vytautas; Kairys, Visvaldas

    2015-01-01

    The design of inhibitors specific for one relevant carbonic anhydrase isozyme is the major challenge in the new therapeutic agents development. Comparative computational chemical structure and biological activity relationship studies on a series of carbonic anhydrase II inhibitors, benzenesulfonamide derivatives, bearing pyrimidine moieties are reported in this paper using docking, Linear Interaction Energy (LIE), Metadynamics and Quantitative Structure Activity Relationship (QSAR) methods. The computed binding affinities were compared with the experimental data with the goal to explore strengths and weaknesses of various approaches applied to the investigated carbonic anhydrase/inhibitor system. From the tested methods initially only QSAR showed promising results (R2=0.83-0.89 between experimentally determined versus predicted pKd values.). Possible reasons for this performance were discussed. A modification of the LIE method was suggested which used an alternative LIE-like equation yielding significantly improved results (R2 between the experimentally determined versus the predicted ΔG(bind) improved from 0.24 to 0.50).

  2. Evolved Streptavidin Mutants Reveal Key Role of Loop Residue in High-affinity Binding

    SciTech Connect

    M Magalhaes; C Melo Czekster; R Guan; V Malashkevich; S Almo; M Levy

    2011-12-31

    We have performed a detailed analysis of streptavidin variants with altered specificity towards desthiobiotin. In addition to changes in key residues which widen the ligand binding pocket and accommodate the more structurally flexible desthiobiotin, the data revealed the role of a key, non-active site mutation at the base of the flexible loop (S52G) which slows dissociation of this ligand by approximately sevenfold. Our data suggest that this mutation results in the loss of a stabilizing contact which keeps this loop open and accessible in the absence of ligand. When this mutation was introduced into the wild-type protein, destabilization of the opened loop conferred a {approx}10-fold decrease in both the on-rate and off-rate for the ligand biotin-4-fluoroscein. A similar effect was observed when this mutation was added to a monomeric form of this protein. Our results provide key insight into the role of the streptavidin flexible loop in ligand binding and maintaining high affinity interactions.

  3. Quantification of helix–helix binding affinities in micelles and lipid bilayers

    PubMed Central

    Lomize, Andrei L.; Pogozheva, I.D.; Mosberg, H.I.

    2004-01-01

    A theoretical approach for estimating association free energies of α-helices in nonpolar media has been developed. The parameters of energy functions have been derived from ΔΔG values of mutants in water-soluble proteins and partitioning of organic solutes between water and nonpolar solvents. The proposed approach was verified successfully against three sets of published data: (1) dissociation constants of α-helical oligomers formed by 27 hydrophobic peptides; (2) stabilities of 22 bacteriorhodopsin mutants, and (3) protein-ligand binding affinities in aqueous solution. It has been found that coalescence of helices is driven exclusively by van der Waals interactions and H-bonds, whereas the principal destabilizing contributions are represented by side-chain conformational entropy and transfer energy of atoms from a detergent or lipid to the protein interior. Electrostatic interactions of α-helices were relatively weak but important for reproducing the experimental data. Immobilization free energy, which originates from restricting rotational and translational rigid-body movements of molecules during their association, was found to be less than 1 kcal/mole. The energetics of amino acid substitutions in bacteriorhodopsin was complicated by specific binding of lipid and water molecules to cavities created in certain mutants. PMID:15340167

  4. Ligand affinity chromatography, an indispensable method for the purification of soluble cytokine receptors and binding proteins.

    PubMed

    Novick, Daniela; Rubinstein, Menachem

    2012-01-01

    Ligand affinity chromatography separation is based on unique interaction between the target analyte and a ligand, which is coupled covalently to a resin. It is a simple, rapid, selective, and efficient purification procedure of proteins providing tens of thousands fold purification in one step. The biological activity of the isolated proteins is retained in most cases thus function is revealed concomitantly with the isolation. Prior to the completion of the genome project this method facilitated rapid and reliable cloning of the corresponding gene. Upon completion of this project, a partial protein sequence is enough for retrieving its complete mRNA and hence its complete protein sequence. This method is indispensable for the isolation of both expected (e.g. receptors) but mainly unexpected, unpredicted and very much surprising binding proteins. No other approach would yield the latter. This chapter provides examples for both the expected target proteins, isolated from rich sources of human proteins, as well as the unexpected binding proteins, found by serendipity. PMID:22131033

  5. Specific cell components of Bacteroides gingivalis mediate binding and degradation of human fibrinogen

    SciTech Connect

    Lantz, M.S.; Allen, R.D.; Vail, T.A.; Switalski, L.M.; Hook, M. )

    1991-01-01

    Bacteroides (Porphyromonas) gingivalis, which has been implicated as an etiologic agent in human periodontal diseases, has been shown to bind and degrade human fibrinogen. B. gingivalis strains bind fibrinogen reversibly and with high affinity and bind to a specific region of the fibrinogen molecule that appears to be located between the D and E domains. The authors now report that human fibrinogen is bound and then degraded by specific B. gingivalis components that appear to be localized at the cell surface. Fibrinogen binding to bacterial cells occurred at 4, 22, and 37{degree}C. A functional fibrinogen-binding component (M{sub r}, 150 000) was identified when sodium dodecyl sulfate-solubilized bacteria were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed with {sup 125}I-fibrinogen. Fibrinogen degradation did not occur at 4{degree}C but did occur at 22 and 37{degree}C. When bacteria and iodinated fibrinogen were incubated at 37{degree}C, two major fibrinogen fragments (M{sub r}, 97 000 and 50 000) accumulated in incubation mixture supernatant fractions. Two major fibrinogen-degrading components (M{sub r}, 120 000 and 150 000) have been identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in substrate-containing gels. Fibrinogen degradation by the M{sub r}-120 000 and -150 000 proteases was enhanced by reducing agents, completely inhibited by N-{alpha}-p-tosyl-L-lysyl chloromethyl ketone, and partially inhibited by n-ethyl maleimide, suggesting that these enzymes are thiol-dependent proteases with trypsinlike substrate specificity. The fibrinogen-binding component could be separated from the fibrinogen-degrading components by selective solubilization of bacteria in sodium deoxycholate.

  6. Specific cell components of Bacteroides gingivalis mediate binding and degradation of human fibrinogen.

    PubMed Central

    Lantz, M S; Allen, R D; Vail, T A; Switalski, L M; Hook, M

    1991-01-01

    Bacteroides (Porphyromonas) gingivalis, which has been implicated as an etiologic agent in human periodontal diseases, has been shown to bind and degrade human fibrinogen. B. gingivalis strains bind fibrinogen reversibly and with high affinity and bind to a specific region of the fibrinogen molecule that appears to be located between the D and E domains (M. S. Lantz, R. D. Allen, P. Bounelis, L. M. Switalski, and M. Hook, J. Bacteriol. 172:716-726, 1990). We now report that human fibrinogen is bound and then degraded by specific B. gingivalis components that appear to be localized at the cell surface. Fibrinogen binding to bacterial cells occurred at 4, 22, and 37 degrees C. A functional fibrinogen-binding component (Mr, 150,000) was identified when sodium dodecyl sulfate-solubilized bacteria were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed with 125I-fibrinogen. Fibrinogen degradation did not occur at 4 degrees C but did occur at 22 and 37 degrees C. When bacteria and iodinated fibrinogen were incubated at 37 degrees C, two major fibrinogen fragments (Mr, 97,000 and 50,000) accumulated in incubation mixture supernatant fractions. Two major fibrinogen-degrading components (Mr, 120,000 and 150,000) have been identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in substrate-containing gels. Fibrinogen degradation by the Mr-120,000 and -150,000 proteases was enhanced by reducing agents, completely inhibited by N-alpha-p-tosyl-L-lysyl chloromethyl ketone, and partially inhibited by n-ethyl maleimide, suggesting that these enzymes are thiol-dependent proteases with trypsinlike substrate specificity. The fibrinogen-binding component could be separated from the fibrinogen-degrading components by selective solubilization of bacteria in sodium deoxycholate. Images PMID:1987144

  7. High affinity ( sup 3 H)glibenclamide binding sites in rat neuronal and cardiac tissue: Localization and developmental characteristics

    SciTech Connect

    Miller, J.A.; Velayo, N.L.; Dage, R.C.; Rampe, D. )

    1991-01-01

    We examined the binding of the antidiabetic sulfonylurea (3H) glibenclamide to rat brain and heart membranes. High affinity binding was observed in adult rat forebrain (Kd = 137.3 pM, maximal binding site density = 91.8 fmol/mg of protein) and ventricle (Kd = 77.1 pM, maximal binding site density = 65.1 fmol/mg of protein). Binding site density increased approximately 250% in forebrain membranes during postnatal development but was constant in ventricular membranes. Quantitative autoradiography was used to examine the regional distribution of (3H) glibenclamide binding sites in sections from rat brain, spinal cord and heart. The greatest density of binding in adult brain was found in the substantia nigra and globus pallidus, whereas the other areas displayed heterogenous binding. In agreement with the membrane binding studies, 1-day-old rat brain had significantly fewer (3H)glibenclamide binding sites than adult brain. Additionally, the pattern of distribution of these sites was qualitatively different from that of the adult. In adult rat spinal cord, moderate binding densities were observed in spinal cord gray and displayed a rostral to caudal gradient. In adult rat heart, moderate binding densities were observed and the sites were distributed homogeneously. In conclusion, significant development of (3H)glibenclamide binding sites was seen in the brain but not the heart during postnatal maturation. Furthermore, a heterogeneous distribution of binding sites was observed in both the brain and spinal cord of adult rats.

  8. 5-Carboxamide tryptamine, a compound with high affinity for 5-hydroxytryptamine1 binding sites, dilates arterioles and constricts arteriovenous anastomoses.

    PubMed Central

    Saxena, P. R.; Verdouw, P. D.

    1985-01-01

    The effects of 5-carboxamide tryptamine, which activates non-5-hydroxytryptamine2-'atypical' receptors for 5-hydroxytryptamine (5-HT) in the dog saphenous vein, was studied on the complete distribution of cardiac output and common carotid blood flow in anaesthetized pigs. The drug was infused for 10 min at the rate of 0.025, 0.1 and 0.4 micrograms kg-1 min-1 either intravenously (cardiac output distribution) or intra-arterially (carotid distribution). 5-Carboxamide tryptamine decreased arterial blood pressure due to a reduction of cardiac output. This reduction was confined to its arteriovenous anastomotic component; the component used for the tissue perfusion (nutrient part) in fact increased. Similar changes were observed in the carotid blood flow distribution. Vasodilation was observed in several tissues, but the skin, ears and stomach responded most prominently. The effects of 5-carboxamide tryptamine on the carotid distribution were not significantly modified by cyproheptadine (1 mg kg-1). It is concluded that, like 5-HT, 5-carboxamide tryptamine constricts arteriovenous anastomoses and dilates arterioles by activating non-5-HT2-'atypical' receptors. These 'atypical' 5-HT receptors appear to be of the 5-HT1 type since both 5-carboxamide tryptamine and BEA 1654, a new piperazine derivative, produced similar vascular effects in the carotid bed of the pig and also showed a high and selective affinity for the 5-HT1 binding sites. PMID:3978321

  9. Omega-conotoxin GVIA binding to a high-affinity receptor in brain: characterization, calcium sensitivity, and solubilization

    SciTech Connect

    Wagner, J.A.; Snowman, A.M.; Biswas, A.; Olivera, B.M.; Snyder, S.H.

    1988-09-01

    We describe unique, high-affinity binding sites for omega(/sup 125/I)conotoxin GVIA in membranes from rat brain and rabbit sympathetic ganglia which appear to be primarily associated with N-type voltage-dependent calcium channels. The dissociation constant (KD) for the toxin in rat brain membranes is 60 pM. Physiologic extracellular concentrations of calcium inhibit toxin binding noncompetitively (IC50 = 0.2 mM). The regional distribution of the binding sites in rat brain differs markedly from that of dihydropyridine calcium antagonist receptors associated with L-type calcium channels. In detergent-solubilized brain membranes, toxin binding retains the same affinity, specificity, and ionic sensitivity as in particulate preparations.

  10. Sperm in poor quality semen from bulls during heat stress have a lower affinity for binding hydrogen-3 heparin

    SciTech Connect

    Ax, R.L.; Gilbert, G.R.; Shook, G.E.

    1987-01-01

    Binding assays with (/sup 3/H) heparin were performed using spermatozoa collected prior to, during, and following summer heat stress to dairy bulls. Ejaculates collected in August 1983 after a period of ambient temperatures exceeding 29.4/sup 0/C exhibited a high frequency of abnormal sperm, and motility was reduced in some samples. Sperm in samples collected during heat stress possessed dissociation constants for binding (/sup 3/H) heparin ranging from 134.5 to 163.2 nmol. In contrast, sperm in semen collected prior to and after heat stress had significantly lower dissociation constants (higher affinity) for (/sup 3/H)heparin, 12.9 to 56.4 nmol. The number of binding sites for (/sup 3/H) heparin on sperm did not change among collection periods. It was concluded that the binding affinity for (/sup 3/H) heparin may reflect membrane integrity of bull sperm.

  11. Disease-associated mutation in SRSF2 misregulates splicing by altering RNA-binding affinities.

    PubMed

    Zhang, Jian; Lieu, Yen K; Ali, Abdullah M; Penson, Alex; Reggio, Kathryn S; Rabadan, Raul; Raza, Azra; Mukherjee, Siddhartha; Manley, James L

    2015-08-25

    Serine/arginine-rich splicing factor 2 (SRSF2) is an RNA-binding protein that plays important roles in splicing of mRNA precursors. SRSF2 mutations are frequently found in patients with myelodysplastic syndromes and certain leukemias, but how these mutations affect SRSF2 function has only begun to be examined. We used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease to introduce the P95H mutation to SRSF2 in K562 leukemia cells, generating an isogenic model so that splicing alterations can be attributed solely to mutant SRSF2. We found that SRSF2 (P95H) misregulates 548 splicing events (<1% of total). Of these events, 374 involved the inclusion of cassette exons, and the inclusion was either increased (206) or decreased (168). We detected a specific motif (UCCA/UG) enriched in the more-included exons and a distinct motif (UGGA/UG) in the more-excluded exons. RNA gel shift assays showed that a mutant SRSF2 derivative bound more tightly than its wild-type counterpart to RNA sites containing UCCAG but bound less tightly to UGGAG sites. Thus in most cases the pattern of exon inclusion or exclusion correlated with stronger or weaker RNA binding, respectively. We further show that the P95H mutation does not affect other functions of SRSF2, i.e., protein-protein interactions with key splicing factors. Our results thus demonstrate that the P95H mutation positively or negatively alters the binding affinity of SRSF2 for cognate RNA sites in target transcripts, leading to misregulation of exon inclusion. Our findings shed light on the mechanism of the disease-associated SRSF2 mutation in splicing regulation and also reveal a group of misspliced mRNA isoforms for potential therapeutic targeting.

  12. Affinity Labeling of Hepatitis C Virus Replicase with a Nucleotide Analog: Identification of binding site

    PubMed Central

    Manvar, Dinesh; Singh, Kamlendra; Pandey, Virendra N.

    2013-01-01

    We have used an ATP analog, 5′-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA), to modify HCV replicase in order to identify ATP binding site in the enzyme. FSBA inactivates HCV replicase activity in a concentration dependent manner with a binding stoichiometry of 2 moles of FSBA per mole of enzyme. The enzyme activity is protected from FSBA in the presence of rNTP substrates or double stranded RNA template primers that do not support ATP as the incoming nucleotide but not in the presence of polyrU.rA26. The HPLC analysis of tryptic peptides of FSBA-modified enzyme revealed the presence of two distinct peptides eluted at 23 min and 36 min; these were absent in the control. Further we noted that both the peptides were protected from FSBA modification in the presence of Mg.ATP. The LC/MS/MS analysis of the affinity-labeled tryptic peptides purified from HPLC, identified two major modification sites at positions 382 (Tyr), and 491 (Lys) and a minor site at position 38 (Tyr). To validate the functional significance of Tyr38, Tyr382 and Lys491 in catalysis, we individually substituted these residues by alanine and examined their ability to catalyze RdRp activity. We found that both Y382A and K491A mutants were significantly affected in their ability to catalyze RdRp activity while Y38A remained unaffected. We further observed that both Y382A and K491A mutants were not affected in their ability to bind template primer but significantly affected in their ability to photo-crosslink ATP in the absence or presence of template primer. PMID:23268692

  13. The MM/PBSA and MM/GBSA methods to estimate ligand-binding affinities

    PubMed Central

    Genheden, Samuel; Ryde, Ulf

    2015-01-01

    Introduction: The molecular mechanics energies combined with the Poisson–Boltzmann or generalized Born and surface area continuum solvation (MM/PBSA and MM/GBSA) methods are popular approaches to estimate the free energy of the binding of small ligands to biological macromolecules. They are typically based on molecular dynamics simulations of the receptor–ligand complex and are therefore intermediate in both accuracy and computational effort between empirical scoring and strict alchemical perturbation methods. They have been applied to a large number of systems with varying success. Areas covered: The authors review the use of MM/PBSA and MM/GBSA methods to calculate ligand-binding affinities, with an emphasis on calibration, testing and validation, as well as attempts to improve the methods, rather than on specific applications. Expert opinion: MM/PBSA and MM/GBSA are attractive approaches owing to their modular nature and that they do not require calculations on a training set. They have been used successfully to reproduce and rationalize experimental findings and to improve the results of virtual screening and docking. However, they contain several crude and questionable approximations, for example, the lack of conformational entropy and information about the number and free energy of water molecules in the binding site. Moreover, there are many variants of the method and their performance varies strongly with the tested system. Likewise, most attempts to ameliorate the methods with more accurate approaches, for example, quantum-mechanical calculations, polarizable force fields or improved solvation have deteriorated the results. PMID:25835573

  14. Disease-associated mutation in SRSF2 misregulates splicing by altering RNA-binding affinities

    PubMed Central

    Zhang, Jian; Lieu, Yen K.; Ali, Abdullah M.; Penson, Alex; Reggio, Kathryn S.; Rabadan, Raul; Raza, Azra; Mukherjee, Siddhartha; Manley, James L.

    2015-01-01

    Serine/arginine-rich splicing factor 2 (SRSF2) is an RNA-binding protein that plays important roles in splicing of mRNA precursors. SRSF2 mutations are frequently found in patients with myelodysplastic syndromes and certain leukemias, but how these mutations affect SRSF2 function has only begun to be examined. We used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease to introduce the P95H mutation to SRSF2 in K562 leukemia cells, generating an isogenic model so that splicing alterations can be attributed solely to mutant SRSF2. We found that SRSF2 (P95H) misregulates 548 splicing events (<1% of total). Of these events, 374 involved the inclusion of cassette exons, and the inclusion was either increased (206) or decreased (168). We detected a specific motif (UCCA/UG) enriched in the more-included exons and a distinct motif (UGGA/UG) in the more-excluded exons. RNA gel shift assays showed that a mutant SRSF2 derivative bound more tightly than its wild-type counterpart to RNA sites containing UCCAG but bound less tightly to UGGAG sites. Thus in most cases the pattern of exon inclusion or exclusion correlated with stronger or weaker RNA binding, respectively. We further show that the P95H mutation does not affect other functions of SRSF2, i.e., protein–protein interactions with key splicing factors. Our results thus demonstrate that the P95H mutation positively or negatively alters the binding affinity of SRSF2 for cognate RNA sites in target transcripts, leading to misregulation of exon inclusion. Our findings shed light on the mechanism of the disease-associated SRSF2 mutation in splicing regulation and also reveal a group of misspliced mRNA isoforms for potential therapeutic targeting. PMID:26261309

  15. Affinity labeling of hepatitis C virus replicase with a nucleotide analogue: identification of binding site.

    PubMed

    Manvar, Dinesh; Singh, Kamlendra; Pandey, Virendra N

    2013-01-15

    We have used an ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA) to modify HCV replicase in order to identify the ATP binding site in the enzyme. FSBA inactivates HCV replicase activity in a concentration-dependent manner with a binding stoichiometry of 2 moles of FSBA per mole of enzyme. The enzyme activity is protected from FSBA in the presence of rNTP substrates or double-stranded RNA template primers that do not support ATP as the incoming nucleotide but not in the presence of polyrU.rA(26). HPLC analysis of tryptic peptides of FSBA-modified enzyme revealed the presence of two distinct peptides eluted at 23 and 36 min; these were absent in the control. Further we noted that both peptides were protected from FSBA modification in the presence of Mg·ATP. The LC/MS/MS analysis of the affinity-labeled tryptic peptides purified from HPLC, identified two major modification sites at positions 382 (Tyr), and 491 (Lys) and a minor site at position 38 (Tyr). To validate the functional significance of Tyr38, Tyr382, and Lys491 in catalysis, we individually substituted these residues by alanine and examined their ability to catalyze RdRp activity. We found that both Y382A and K491A mutants were significantly affected in their ability to catalyze RdRp activity while Y38A remained unaffected. We further observed that both Y382A and K491A mutants were not affected in their ability to bind template primer but were significantly affected in their ability to photo-cross-link ATP in the absence or presence of template primer. PMID:23268692

  16. Recombinant human nerve growth factor is biologically active and labels novel high-affinity binding sites in rat brain

    SciTech Connect

    Altar, C.A.; Burton, L.E.; Bennett, G.L.; Dugich-Djordjevic, M. )

    1991-01-01

    Iodinated recombinant human nerve growth factor (125I-rhNGF) stimulated neurite formation in PC12 cell cultures with a half-maximal potency of 35-49 pg/ml, compared with 39-52 pg/ml for rhNGF. In quantitative ligand autoradiography, the in vitro equilibrium binding of 125I-rhNGF to brain sections showed a 10-fold regional variation in density and was saturable, reversible, and specifically displaced by up to 74% with rhNGF or murine NGF (muNGF). At equilibrium, 125I-rhNGF bound to these sites with high affinity and low capacity (Bmax less than or equal to 13.2 fmol/mg of protein). Calculation of 125I-rhNGF binding affinity by kinetic methods gave average Kd values of 24 and 31 pM. Computer-generated maps revealed binding in brain regions not identified previously with 125I-muNGF, including hippocampus; dentate gyrus; amygdala; paraventricular thalamus; frontal, parietal, occipital, and cingulate cortices; nucleus accumbens; olfactory tubercle; subiculum; pineal gland; and medial geniculate nucleus. NGF binding sites were distributed in a 2-fold increasing medial-lateral gradient in the caudate-putamen and a 2-fold lateral-medial gradient in the nucleus accumbens. 125I-rhNGF binding sites were also found in most areas labeled by 125I-muNGF, including the interpedunucular nucleus, cerebellum, forebrain cholinergic nuclei, caudoventral caudate-putamen, and trigeminal nerve nucleus. 125I-rhNGF binding sites were absent from areas replete with low-affinity NGF binding sites, including circumventricular organs, myelinated fiber bundles, and choroid plexus. The present analysis provides an anatomical differentiation of high-affinity 125I-rhNGF binding sites and greatly expands the number of brain structures that may respond to endogenous NGF or exogenously administered rhNGF.

  17. Receptor binding profiles and quantitative structure-affinity relationships of some 5-substituted-N,N-diallyltryptamines.

    PubMed

    Cozzi, Nicholas V; Daley, Paul F

    2016-02-01

    N,N-Diallyltryptamine (DALT) and 5-methoxy-N,N-diallyltryptamine (5-MeO-DALT) are two tryptamines synthesized and tested by Alexander Shulgin. In self-experiments, 5-MeO-DALT was reported to be psychoactive in the 12-20mg range, while the unsubstituted compound DALT had few discernible effects in the 42-80 mg range. Recently, 5-MeO-DALT has been used in nonmedical settings for its psychoactive effects, but these effects have been poorly characterized and little is known of its pharmacological properties. We extended the work of Shulgin by synthesizing additional 5-substituted-DALTs. We then compared them to DALT and 5-MeO-DALT for their binding affinities at 45 cloned receptors and transporter proteins. Based on in vitro binding affinity, we identified 27 potential receptor targets for the 5-substituted-DALT compounds. Five of the DALT compounds had affinity in the 10-80 nM range for serotonin 5-HT1A and 5-HT2B receptors, while the affinity of DALT itself at 5-HT1A receptors was slightly lower at 100 nM. Among the 5-HT2 subtypes, the weakest affinity was at 5-HT2A receptors, spanning 250-730 nM. Five of the DALT compounds had affinity in the 50-400 nM range for serotonin 5-HT1D, 5-HT6, and 5-HT7 receptors; again, it was the unsubstituted DALT that had the weakest affinity at all three subtypes. The test drugs had even weaker affinity for 5-HT1B, 5-HT1E, and 5-HT5A subtypes and little or no affinity for the 5-HT3 subtype. These compounds also had generally nanomolar affinities for adrenergic α2A, α2B, and α2C receptors, sigma receptors σ1 and σ2, histamine H1 receptors, and norepinephrine and serotonin uptake transporters. They also bound to other targets in the nanomolar-to-low micromolar range. Based on these binding results, it is likely that multiple serotonin receptors, as well as several nonserotonergic sites are important for the psychoactive effects of DALT drugs. To learn whether any quantitative structure-affinity relationships existed, we evaluated

  18. Identification and affinity of very high affinity binding sites for the phenylalkylamine series of Ca/sup +/ channel blockers in the Drosophila nervous system

    SciTech Connect

    Pauron, D.; Qar, J.; Barhanin, J.; Fournier, D.; Cuany, A.; Pralavorio, M.; Berge, J.B.; Lazdunski, M.

    1987-10-06

    The interaction of putative Ca/sup 2 +/ channels of Drosophila head membranes with molecules of the phenylalkylamine series was studied from binding experiments using (-)-(/sup 3/H)D888 and (+/-)-(/sup 3/H)verapamil. These ligands recognize a single class of very high affinity binding sites. The most potent molecule in the phenylalkylamine series was (-)-verapamil with a K/sub d/ value as exceptional low as 4.7 pM. Molecules in the benzothiazepine and diphenylbutylpiperidine series of Ca/sup 2 +/ channel blockers as well as bepridil inhibited (-)-(/sup 3/H)D888 binding in a competitive way with K/sub d/ values between 12 and 190 nM, suggesting a close correlation, as in the mammalian system, between these receptor sites and those recognizing phenylalkylamines. A tritiated (arylazido)phenylalkylamine with high affinity for the Drosophila head membranes, phenylalkylamine receptor was used in photoaffinity experiments. A protein of M/sub r/ 135,000 +/- 5000 was specifically labeled after ultraviolet irradiation.

  19. Identification by affinity chromatography of the eukaryotic ribosomal proteins that bind to 5.8 S ribosomal ribonucleic acid.

    PubMed

    Ulbrich, N; Lin, A; Wool, I G

    1979-09-10

    The proteins that bind to rat liver 5.8 S ribosomal ribonucleic acid were identified by affinity chromatography. The nucleic acid was oxidized with periodate and coupled by its 3'-terminus to Sepharose 4B through and adipic acid dihydrazide spacer. The ribosomal proteins that associate with the immobilized 5.8 S rRNA were identified by polyacrylamide gel electrophoresiss: they were L19, L8, and L6 from the 60 S subunit; and S13 and S9 from the small subparticle. Small amounts of L14, L17', L18, L27/L27', and L35', and of S11, S15, S23/S24, and S26 also were bound to the affinity column, but whether they associate directly and specifically with 5.8 S rRNA is not known. Escherichia coli ribosomal proteins did not bind to the rat liver 5.8 S rRNA affinity column. PMID:468846

  20. Complementary DNA display selection of high-affinity peptides binding the vacuolating toxin (VacA) of Helicobacter pylori.

    PubMed

    Hayakawa, Yumiko; Matsuno, Mitsuhiro; Tanaka, Makoto; Wada, Akihiro; Kitamura, Koichiro; Takei, Osamu; Sasaki, Ryuzo; Mizukami, Tamio; Hasegawa, Makoto

    2015-09-01

    Artificial peptides designed for molecular recognition of a bacterial toxin have been developed. Vacuolating cytotoxin A protein (VacA) is a major virulence factor of Helicobacter pylori, a gram-negative microaerophilic bacterium inhabiting the upper gastrointestinal tract, particularly the stomach. This study attempted to identify specific peptide sequences with high affinity for VacA using systematic directed evolution in vitro, a cDNA display method. A surface plasmon resonance-based biosensor and fluorescence correlation spectroscopy to examine binding of peptides with VacA identified a peptide (GRVNQRL) with high affinity. Cyclization of the peptide by attaching cysteine residues to both termini improved its binding affinity to VacA, with a dissociation constant (Kd ) of 58 nm. This study describes a new strategy for the development of artificial functional peptides, which are promising materials in biochemical analyses and medical applications.

  1. Metal-ligand binding affinity vs reactivity: qualitative studies in Rh(I)-catalyzed asymmetric ring-opening reactions.

    PubMed

    Tsui, Gavin Chit; Dougan, Patrick; Lautens, Mark

    2013-06-01

    Rh(I)-catalyzed asymmetric ring opening (ARO) of oxabenzonorbornadiene is used as a model system to qualitatively study reactions involving multiple metal-ligand interactions. The key feature of this approach is the use of product ee as an indicator to quickly gain important information such as the relative ligand binding affinity and relative reactivity of catalysts.

  2. Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding affibody molecule.

    PubMed

    Friedman, Mikaela; Orlova, Anna; Johansson, Eva; Eriksson, Tove L J; Höidén-Guthenberg, Ingmarie; Tolmachev, Vladimir; Nilsson, Fredrik Y; Ståhl, Stefan

    2008-03-01

    The epidermal growth factor receptor 1 (EGFR) is overexpressed in various malignancies and is associated with a poor patient prognosis. A small, receptor-specific, high-affinity imaging agent would be a useful tool in diagnosing malignant tumors and in deciding upon treatment and assessing the response to treatment. We describe here the affinity maturation procedure for the generation of Affibody molecules binding with high affinity and specificity to EGFR. A library for affinity maturation was constructed by rerandomization of selected positions after the alignment of first-generation binding variants. New binders were selected with phage display technology, using a single oligonucleotide in a single-library effort, and the best second-generation binders had an approximately 30-fold improvement in affinity (K(d)=5-10 nM) for the soluble extracellular domain of EGFR in biospecific interaction analysis using Biacore. The dissociation equilibrium constant, K(d), was also determined for the Affibody with highest affinity using EGFR-expressing A431 cells in flow cytometric analysis (K(d)=2.8 nM). A retained high specificity for EGFR was verified by a dot blot assay showing staining only of EGFR proteins among a panel of serum proteins and other EGFR family member proteins (HER2, HER3, and HER4). The EGFR-binding Affibody molecules were radiolabeled with indium-111, showing specific binding to EGFR-expressing A431 cells and successful targeting of the A431 tumor xenografts with 4-6% injected activity per gram accumulated in the tumor 4 h postinjection. PMID:18207161

  3. Topography of the high-affinity lysine binding site of plasminogen as defined with a specific antibody probe

    SciTech Connect

    Miles, L.A.; Plow, E.F.

    1986-11-04

    An antibody population that reacted with the high-affinity lysine binding site of human plasminogen was elicited by immunizing rabbits with an elastase degradation product containing kringles 1-3 (EDP I). This antibody was immunopurified by affinity chromatography on plasminogen-Sepharose and elution with 0.2 M 6-aminohexanoic acid. The eluted antibodies bound (/sup 125/I)EDP I, (/sup 125/I)Glu-plasminogen, and (/sup 125/I)Lys-plasminogen in radioimmunoassays, and binding of each ligand was at least 99% inhibited by 0.2 M 6-aminohexanoic acid. The concentrations for 50% inhibition of (/sup 125/I)EDP I binding by tranexamic acid, 6-aminohexanoic acid, and lysine were 2.6, 46, and l730 ..mu..M, respectively. Similar values were obtained with plasminogen and suggested that an unoccupied high-affinity lysine binding site was required for antibody recognition. The antiserum reacted exclusively with plasminogen derivatives containing the EDP I region and did not react with those lacking an EDP I region, or with tissue plasminogen activator or prothrombin, which also contains kringles. By immunoblotting analyses, a chymotryptic degradation product of M/sub r/ 20,000 was derived from EDP I that retained reactivity with the antibody. ..cap alpha../sub 2/-Antiplasmin inhibited the binding of radiolabeled EDP I, Glu-plasminogen, or Lys-plasminogen by the antiserum, suggesting that the recognized site is involved in the noncovalent interaction of the inhibitor with plasminogen. The binding of (/sup 125/I)EDP I to fibrin was also inhibited by the antiserum. The observations provide independent evidence for the role of the high-affinity lysine binding site in the functional interactions of plasminogen with its primary substrate and inhibitor.

  4. Characterization of the sources of protein-ligand affinity: 1-sulfonato-8-(1')anilinonaphthalene binding to intestinal fatty acid binding protein.

    PubMed Central

    Kirk, W R; Kurian, E; Prendergast, F G

    1996-01-01

    1-Sulfonato-8-(1')anilinonaphthalene (1,8-ANS) was employed as a fluorescent probe of the fatty acid binding site of recombinant rat intestinal fatty acid binding protein (1-FABP). The enhancement of fluorescence upon binding allowed direct determination of binding affinity by fluorescence titration experiments, and measurement of the effects on that affinity of temperature, pH, and ionic strength. Solvent isotope effects were also determined. These data were compared to results from isothermal titration calorimetry. We obtained values for the enthalpy and entropy of this interaction at a variety of temperatures, and hence determined the change in heat capacity of the system consequent upon binding. The ANS-1-FABP is enthalpically driven; above approximately 14 degrees C it is entropically opposed, but below this temperature the entropy makes a positive contribution to the binding. The changes we observe in both enthalpy and entropy of binding with temperature can be derived from the change in heat capacity upon binding by integration, which demonstrates the internal consistency of our results. Bound ANS is displaced by fatty acids and can itself displace fatty acids bound to I-FABP. The binding site for ANS appears to be inside the solvent-containing cavity observed in the x-ray crystal structure, the same cavity occupied by fatty acid. From the fluorescence spectrum and from an inversion of the Debye-Hueckel formula for the activity coefficients as a function of added salt, we inferred that this cavity is fairly polar in character, which is in keeping with inferences drawn from the x-ray structure. The binding affinity of ANS is considered to be a consequence of both electrostatic and conditional hydrophobic effects. We speculate that the observed change in heat capacity is produced mainly by the displacement of strongly hydrogen-bonded waters from the protein cavity. PMID:8770188

  5. Affinity binding of antibodies to supermacroporous cryogel adsorbents with immobilized protein A for removal of anthrax toxin protective antigen.

    PubMed

    Ingavle, Ganesh C; Baillie, Les W J; Zheng, Yishan; Lis, Elzbieta K; Savina, Irina N; Howell, Carol A; Mikhalovsky, Sergey V; Sandeman, Susan R

    2015-05-01

    Polymeric cryogels are efficient carriers for the immobilization of biomolecules because of their unique macroporous structure, permeability, mechanical stability and different surface chemical functionalities. The aim of the study was to demonstrate the potential use of macroporous monolithic cryogels for biotoxin removal using anthrax toxin protective antigen (PA), the central cell-binding component of the anthrax exotoxins, and covalent immobilization of monoclonal antibodies. The affinity ligand (protein A) was chemically coupled to the reactive hydroxyl and epoxy-derivatized monolithic cryogels and the binding efficiencies of protein A, monoclonal antibodies to the cryogel column were determined. Our results show differences in the binding capacity of protein A as well as monoclonal antibodies to the cryogel adsorbents caused by ligand concentrations, physical properties and morphology of surface matrices. The cytotoxicity potential of the cryogels was determined by an in vitro viability assay using V79 lung fibroblast as a model cell and the results reveal that the cryogels are non-cytotoxic. Finally, the adsorptive capacities of PA from phosphate buffered saline (PBS) were evaluated towards a non-glycosylated, plant-derived human monoclonal antibody (PANG) and a glycosylated human monoclonal antibody (Valortim(®)), both of which were covalently attached via protein A immobilization. Optimal binding capacities of 108 and 117 mg/g of antibody to the adsorbent were observed for PANG attached poly(acrylamide-allyl glycidyl ether) [poly(AAm-AGE)] and Valortim(®) attached poly(AAm-AGE) cryogels, respectively, This indicated that glycosylation status of Valortim(®) antibody could significantly increase (8%) its binding capacity relative to the PANG antibody on poly(AAm-AGE)-protien-A column (p < 0.05). The amounts of PA which remained in the solution after passing PA spiked PBS through PANG or Valortim bound poly(AAm-AGE) cryogel were significantly (p < 0

  6. Affinity binding of antibodies to supermacroporous cryogel adsorbents with immobilized protein A for removal of anthrax toxin protective antigen.

    PubMed

    Ingavle, Ganesh C; Baillie, Les W J; Zheng, Yishan; Lis, Elzbieta K; Savina, Irina N; Howell, Carol A; Mikhalovsky, Sergey V; Sandeman, Susan R

    2015-05-01

    Polymeric cryogels are efficient carriers for the immobilization of biomolecules because of their unique macroporous structure, permeability, mechanical stability and different surface chemical functionalities. The aim of the study was to demonstrate the potential use of macroporous monolithic cryogels for biotoxin removal using anthrax toxin protective antigen (PA), the central cell-binding component of the anthrax exotoxins, and covalent immobilization of monoclonal antibodies. The affinity ligand (protein A) was chemically coupled to the reactive hydroxyl and epoxy-derivatized monolithic cryogels and the binding efficiencies of protein A, monoclonal antibodies to the cryogel column were determined. Our results show differences in the binding capacity of protein A as well as monoclonal antibodies to the cryogel adsorbents caused by ligand concentrations, physical properties and morphology of surface matrices. The cytotoxicity potential of the cryogels was determined by an in vitro viability assay using V79 lung fibroblast as a model cell and the results reveal that the cryogels are non-cytotoxic. Finally, the adsorptive capacities of PA from phosphate buffered saline (PBS) were evaluated towards a non-glycosylated, plant-derived human monoclonal antibody (PANG) and a glycosylated human monoclonal antibody (Valortim(®)), both of which were covalently attached via protein A immobilization. Optimal binding capacities of 108 and 117 mg/g of antibody to the adsorbent were observed for PANG attached poly(acrylamide-allyl glycidyl ether) [poly(AAm-AGE)] and Valortim(®) attached poly(AAm-AGE) cryogels, respectively, This indicated that glycosylation status of Valortim(®) antibody could significantly increase (8%) its binding capacity relative to the PANG antibody on poly(AAm-AGE)-protien-A column (p < 0.05). The amounts of PA which remained in the solution after passing PA spiked PBS through PANG or Valortim bound poly(AAm-AGE) cryogel were significantly (p < 0

  7. Specific high-affinity binding sites for a synthetic gliadin heptapeptide of human peripheral blood lymphocytes

    SciTech Connect

    Payan, D.G.; Horvath, K.; Graf, L.

    1987-03-23

    The synthetic peptide containing residues 43-49 of ..cap alpha..-gliadin, the major protein component of gluten, has previously been shown to inhibit the production of lymphokine activities by mononuclear leukocytes. The authors demonstrate using radiolabeled ..cap alpha..-gliadin(43-49) that human peripheral blood lymphocytes express approximately 20,000-25,000 surface receptors for this peptide, with a dissociation constant (K/sub D/) of 20 nM. In addition, binding is inhibited by naloxone and an enkephalin analog, thus confirming the functional correlate which demonstrates inhibition by these agents of ..cap alpha..-gliadin(43-49) functional effects. Furthermore, B-lymphocytes bind specifically a greater amount of (/sup 125/I)..cap alpha..-gliadin(43-49) than T-lymphocytes. The lymphocyte ..cap alpha..-gliadin(43-49) receptor may play an important role in mediating the immunological response to ..cap alpha..-gliadin. 16 references, 4 figures.

  8. The predicted 3D structure of the human D2 dopamine receptor and the binding site and binding affinities for agonists and antagonists

    NASA Astrophysics Data System (ADS)

    Kalani, M. Yashar S.; Vaidehi, Nagarajan; Hall, Spencer E.; Trabanino, Rene J.; Freddolino, Peter L.; Kalani, Maziyar A.; Floriano, Wely B.; Tak Kam, Victor Wai; Goddard, William A., III

    2004-03-01

    Dopamine neurotransmitter and its receptors play a critical role in the cell signaling process responsible for information transfer in neurons functioning in the nervous system. Development of improved therapeutics for such disorders as Parkinson's disease and schizophrenia would be significantly enhanced with the availability of the 3D structure for the dopamine receptors and of the binding site for dopamine and other agonists and antagonists. We report here the 3D structure of the long isoform of the human D2 dopamine receptor, predicted from primary sequence using first-principles theoretical and computational techniques (i.e., we did not use bioinformatic or experimental 3D structural information in predicting structures). The predicted 3D structure is validated by comparison of the predicted binding site and the relative binding affinities of dopamine, three known dopamine agonists (antiparkinsonian), and seven known antagonists (antipsychotic) in the D2 receptor to experimentally determined values. These structures correctly predict the critical residues for binding dopamine and several antagonists, identified by mutation studies, and give relative binding affinities that correlate well with experiments. The predicted binding site for dopamine and agonists is located between transmembrane (TM) helices 3, 4, 5, and 6, whereas the best antagonists bind to a site involving TM helices 2, 3, 4, 6, and 7 with minimal contacts to TM helix 5. We identify characteristic differences between the binding sites of agonists and antagonists.

  9. Epidermal transformation leads to increased perlecan synthesis with heparin-binding-growth-factor affinity.

    PubMed Central

    Tapanadechopone, P; Tumova, S; Jiang, X; Couchman, J R

    2001-01-01

    Perlecan, a proteoglycan of basement membrane and extracellular matrices, has important roles in both normal biological and pathological processes. As a result of its ability to store and protect growth factors, perlecan may have crucial roles in tumour-cell growth and invasion. Since the biological functions of different types of glycosaminoglycan vary with cellular origin and structural modifications, we analysed the expression and biological functions of perlecan produced by a normal epidermal cell line (JB6) and its transformed counterpart (RT101). Expression of perlecan in tumorigenic cells was significantly increased in both mRNA and protein levels. JB6 perlecan was exclusively substituted with heparan sulphate, whereas that of RT101 contained some additional chondroitin sulphate. Detailed structural analysis of the heparan sulphate (HS) chains from perlecan of both cell types revealed that their overall sulphation and chain length were similar (approximately 60 kDa), but the HS chains of tumour-cell-derived perlecan were less sulphated. This resulted from reduced 2-O- and 6-O-sulphation, but not N-sulphation, and an increase in the proportion of unsulphated disaccharides. Despite this, the heparan sulphate of RT101- and JB6-derived perlecan bound fibroblast growth factor-1, -2, -4 and -7 and heparin-binding epidermal growth factor with similar affinity. Therefore abundant tumour-derived perlecan may support the angiogenic responses seen in vivo and be a key player in tumorigenesis. PMID:11284741

  10. Energetics of ligand-receptor binding affinity on endothelial cells: An in vitro model.

    PubMed

    Fotticchia, Iolanda; Guarnieri, Daniela; Fotticchia, Teresa; Falanga, Andrea Patrizia; Vecchione, Raffaele; Giancola, Concetta; Netti, Paolo Antonio

    2016-08-01

    Targeted therapies represent a challenge in modern medicine. In this contest, we propose a rapid and reliable methodology based on Isothermal Titration Calorimetry (ITC) coupled with confluent cell layers cultured around biocompatible templating microparticles to quantify the number of overexpressing receptors on cell membrane and study the energetics of receptor-ligand binding in near-physiological conditions. In the in vitro model here proposed we used the bEnd3 cell line as brain endothelial cells to mimic the blood brain barrier (BBB) cultured on dextran microbeads ranging from 67μm to 80μm in size (Cytodex) and the primary human umbilical vein cells (HUVEC) for comparison. The revealed affinity between transferrin (Tf) and transferrin receptor (TfR) in both systems is very high, Kd values are in the order of nM. Conversely, the value of TfRs/cell reveals a 100-fold increase in the number of TfRs per bEnd3 cells compared to HUVEC cells. The presented methodology can represent a novel and helpful strategy to identify targets, to address drug design and selectively deliver therapeutics that can cross biological barriers such as the blood brain barrier.

  11. Energetics of ligand-receptor binding affinity on endothelial cells: An in vitro model.

    PubMed

    Fotticchia, Iolanda; Guarnieri, Daniela; Fotticchia, Teresa; Falanga, Andrea Patrizia; Vecchione, Raffaele; Giancola, Concetta; Netti, Paolo Antonio

    2016-08-01

    Targeted therapies represent a challenge in modern medicine. In this contest, we propose a rapid and reliable methodology based on Isothermal Titration Calorimetry (ITC) coupled with confluent cell layers cultured around biocompatible templating microparticles to quantify the number of overexpressing receptors on cell membrane and study the energetics of receptor-ligand binding in near-physiological conditions. In the in vitro model here proposed we used the bEnd3 cell line as brain endothelial cells to mimic the blood brain barrier (BBB) cultured on dextran microbeads ranging from 67μm to 80μm in size (Cytodex) and the primary human umbilical vein cells (HUVEC) for comparison. The revealed affinity between transferrin (Tf) and transferrin receptor (TfR) in both systems is very high, Kd values are in the order of nM. Conversely, the value of TfRs/cell reveals a 100-fold increase in the number of TfRs per bEnd3 cells compared to HUVEC cells. The presented methodology can represent a novel and helpful strategy to identify targets, to address drug design and selectively deliver therapeutics that can cross biological barriers such as the blood brain barrier. PMID:27100851

  12. Peptide specificity of protein prenyltransferases is determined mainly by reactivity rather than binding affinity.

    PubMed

    Hartman, Heather L; Hicks, Katherine A; Fierke, Carol A

    2005-11-22

    Protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type I (GGTase I) catalyze the attachment of lipid groups from farnesyl diphosphate and geranylgeranyl diphosphate, respectively, to a cysteine near the C-terminus of protein substrates. FTase and GGTase I modify several important signaling and regulatory proteins with C-terminal CaaX sequences ("C" refers to the cysteine residue that becomes prenylated, "a" refers to any aliphatic amino acid, and "X" refers to any amino acid). In the CaaX paradigm, the C-terminal X-residue of the protein/peptide confers specificity for FTase or GGTase I. However, some proteins, such as K-Ras, RhoB, and TC21, are substrates for both FTase and GGTase I. Here we demonstrate that the C-terminal amino acid affects the binding affinity of K-Ras4B-derived hexapeptides (TKCVIX) to FTase and GGTase I modestly. In contrast, reactivity, as indicated by transient and steady-state kinetics, varies significantly and correlates with hydrophobicity, volume, and structure of the C-terminal amino acid. The reactivity of FTase decreases as the hydrophobicity of the C-terminal amino acid increases whereas the reactivity of GGTase I increases with the hydrophobicity of the X-group. Therefore, the hydrophobicity, as well as the structure of the X-group, determines whether peptides are specific for farnesylation, geranylgeranylation, or dual prenylation.

  13. A human β-III-spectrin spinocerebellar ataxia type 5 mutation causes high-affinity F-actin binding

    PubMed Central

    Avery, Adam W.; Crain, Jonathan; Thomas, David D.; Hays, Thomas S.

    2016-01-01

    Spinocerebellar ataxia type 5 (SCA5) is a human neurodegenerative disease that stems from mutations in the SPTBN2 gene encoding the protein β-III-spectrin. Here we investigated the molecular consequence of a SCA5 missense mutation that results in a L253P substitution in the actin-binding domain (ABD) of β-III-spectrin. We report that the L253P substitution in the isolated β-III-spectrin ABD causes strikingly high F-actin binding affinity (Kd = 75.5 nM) compared to the weak F-actin binding affinity of the wild-type ABD (Kd = 75.8 μM). The mutation also causes decreased thermal stability (Tm = 44.6 °C vs 59.5 °C). Structural analyses indicate that leucine 253 is in a loop at the interface of the tandem calponin homology (CH) domains comprising the ABD. Leucine 253 is predicted to form hydrophobic contacts that bridge the CH domains. The decreased stability of the mutant indicates that these bridging interactions are probably disrupted, suggesting that the high F-actin binding affinity of the mutant is due to opening of the CH domain interface. These results support a fundamental role for leucine 253 in regulating opening of the CH domain interface and binding of the ABD to F-actin. This study indicates that high-affinity actin binding of L253P β-III-spectrin is a likely driver of neurodegeneration. PMID:26883385

  14. Evaluation of adhesion force and binding affinity of phytohemagglutinin erythroagglutinating to EGF receptor on human lung cancer cells.

    PubMed

    Kuo, W-T; Dong, G-C; Yao, C-H; Huang, J-Y; Lin, F-H

    2013-01-01

    PHA-E is a natural product extracted from red kidney beans, and it has been reported to induce cell apoptosis by blocking EGFR in lung cancer cells. Because EGF is the major in vivo competitor to PHA-E in clinical application, PHA-E must be proved that has better affinity to EGFR than EGF. This study would focus on how PHA-E tightly bind to EGFR and the results would compare with EGF. The adhesion force, measured by AFM, between EGFR and PHA-E was 207.14±74.42 pN that was higher than EGF (183.65±86.93 pN). The equilibrium dissociation constant of PHA-E and EGF to EGFR was 2.4 10(-9)±1.4 10(-9) and 7.3 10(-8)±2.7 10(-8), respectively, that could evaluate binding affinity. The result showed that binding affinity of PHA-E to EGFR was one order higher than EGF to EGFR. In the results of flow cytometer and confocal microscope, we found binding efficiency of EGF to EGFR was decrease as the concentration of PHA-E increased. In the analysis of Western blot, treatment of A-549 cells with PHA-E resulted in a dose-dependent decrease in EGFR phosphorylation. In conclusion, we found that PHA-E had better adhesion force and binding affinity to EGFR than that of the EGF. The interaction between PHA-E and EGFR could block EGF binding and then inhibit EGFR phosphorylation. PHA-E could be developed into a new target molecule for lung cancer treatment that could be immobilized on the drug carrier to guide therapeutic particles to the tumor site. PMID:23394551

  15. Binding affinity of hydrolyzable tannins to parotid saliva and to proline-rich proteins derived from it.

    PubMed

    Bacon, J R; Rhodes, M J

    2000-03-01

    Proline-rich proteins (PRP) in human parotid saliva have a high affinity for dietary polyphenolic compounds (tannins), forming stable complexes that may modulate the biological and nutritional properties of the tannin. The formation of such complexes may also have an important role in the modulation or promotion of the sensation of oral astringency perceived when tannin-rich foods and beverages are consumed. The major classes of PRP (acidic, basic, and glycosylated) have been isolated from human saliva, and the relative binding affinities of a series of hydrolyzable tannins, which are found in a number of plant-derived foods and beverages, to these PRP classes have been determined using a competition assay. All of the classes of PRP have a high capacity for hydrolyzable tannins. Within the narrow range of binding affinities exhibited, structure/binding relationships with the levels of tannin galloylation, hexahydroxydiphenoyl esterification, and degree of polymerization were identified. No individual class of human salivary PRP appears to have an exclusive affinity for a particular type of hydrolyzable tannin.

  16. Binding affinity of hydrolyzable tannins to parotid saliva and to proline-rich proteins derived from it.

    PubMed

    Bacon, J R; Rhodes, M J

    2000-03-01

    Proline-rich proteins (PRP) in human parotid saliva have a high affinity for dietary polyphenolic compounds (tannins), forming stable complexes that may modulate the biological and nutritional properties of the tannin. The formation of such complexes may also have an important role in the modulation or promotion of the sensation of oral astringency perceived when tannin-rich foods and beverages are consumed. The major classes of PRP (acidic, basic, and glycosylated) have been isolated from human saliva, and the relative binding affinities of a series of hydrolyzable tannins, which are found in a number of plant-derived foods and beverages, to these PRP classes have been determined using a competition assay. All of the classes of PRP have a high capacity for hydrolyzable tannins. Within the narrow range of binding affinities exhibited, structure/binding relationships with the levels of tannin galloylation, hexahydroxydiphenoyl esterification, and degree of polymerization were identified. No individual class of human salivary PRP appears to have an exclusive affinity for a particular type of hydrolyzable tannin. PMID:10725160

  17. Design of Bcl-2 and Bcl-xL Inhibitors with Subnanomolar Binding Affinities Based upon a New Scaffold

    SciTech Connect

    Zhou, Haibin; Chen, Jianfang; Meagher, Jennifer L.; Yang, Chao-Yie; Aguilar, Angelo; Liu, Liu; Bai, Longchuan; Cong, Xin; Cai, Qian; Fang, Xueliang; Stuckey, Jeanne A.; Wang, Shaomeng

    2014-10-02

    Employing a structure-based strategy, we have designed a new class of potent small-molecule inhibitors of the anti-apoptotic proteins Bcl-2 and Bcl-xL. An initial lead compound with a new scaffold was designed based upon the crystal structure of Bcl-xL and U.S. Food and Drug Administration (FDA) approved drugs and was found to have an affinity of 100 {micro}M for both Bcl-2 and Bcl-xL. Linking this weak lead to another weak-affinity fragment derived from Abbott's ABT-737 led to an improvement of the binding affinity by a factor of >10,000. Further optimization ultimately yielded compounds with subnanomolar binding affinities for both Bcl-2 and Bcl-xL and potent cellular activity. The best compound (21) binds to Bcl-xL and Bcl-2 with K{sub i} < 1 nM, inhibits cell growth in the H146 and H1417 small-cell lung cancer cell lines with IC{sub 50} values of 60-90 nM, and induces robust cell death in the H146 cancer cell line at 30-100 nM.

  18. Critical factors governing the difference in antizyme-binding affinities between human ornithine decarboxylase and antizyme inhibitor.

    PubMed

    Liu, Yen-Chin; Liu, Yi-Liang; Su, Jia-Yang; Liu, Guang-Yaw; Hung, Hui-Chih

    2011-01-01

    Both ornithine decarboxylase (ODC) and its regulatory protein, antizyme inhibitor (AZI), can bind with antizyme (AZ), but the latter has a higher AZ-binding affinity. The results of this study clearly identify the critical amino acid residues governing the difference in AZ-binding affinities between human ODC and AZI. Inhibition experiments using a series of ODC mutants suggested that residues 125 and 140 may be the key residues responsible for the differential AZ-binding affinities. The ODC_N125K/M140K double mutant demonstrated a significant inhibition by AZ, and the IC(50) value of this mutant was 0.08 µM, three-fold smaller than that of ODC_WT. Furthermore, the activity of the AZ-inhibited ODC_N125K/M140K enzyme was hardly rescued by AZI. The dissociation constant (K(d)) of the [ODC_N125K/M140K]-AZ heterodimer was approximately 0.02 µM, which is smaller than that of WT_ODC by approximately 10-fold and is very close to the K(d) value of AZI_WT, suggesting that ODC_N125K/M140K has an AZ-binding affinity higher than that of ODC_WT and similar to that of AZI. The efficiency of the AZI_K125N/K140M double mutant in the rescue of AZ-inhibited ODC enzyme activity was less than that of AZI_WT. The K(d) value of [AZI_K125N/K140M]-AZ was 0.18 µM, nine-fold larger than that of AZI_WT and close to the K(d) value of ODC_WT, suggesting that AZI_K125N/K140M has an AZ-binding affinity lower than that of AZI_WT and similar to that of ODC. These data support the hypothesis that the differences in residues 125 and 140 in ODC and AZI are responsible for the differential AZ-binding affinities. PMID:21552531

  19. Spectroscopic detection of etoposide binding to chromatin components: The role of histone proteins

    NASA Astrophysics Data System (ADS)

    Chamani, Elham; Rabbani-Chadegani, Azra; Zahraei, Zohreh

    2014-12-01

    Chromatin has been introduced as a main target for most anticancer drugs. Etoposide is known as a topoisomerase II inhibitor, but its effect on chromatin components is unknown. This report, for the first time, describes the effect of etoposide on DNA, histones and DNA-histones complex in the structure of nucleosomes employing thermal denaturation, fluorescence, UV absorbance and circular dichroism spectroscopy techniques. The results showed that the binding of etoposide decreased UV absorbance and fluorescence emission intensity, altered secondary structure of chromatin and hypochromicity was occurred in thermal denaturation profiles. The drug exhibited higher affinity to chromatin compared to DNA. Quenching of drug chromophores with tyrosine residues of histones indicated that globular domain of histones is the site of etoposide binding. Moreover, the binding of etoposide to histones altered their secondary structure accompanied with hypochromicity revealing compaction of histones in the presence of the drug. From the results it is concludes that apart from topoisomerase II, chromatin components especially its protein moiety can be introduced as a new site of etoposide binding and histone proteins especially H1 play a fundamental role in this process and anticancer activity of etoposide.

  20. A fullerene C60-based ligand in a stationary phase for affine chromatography of membrane porphyrin-binding proteins

    NASA Astrophysics Data System (ADS)

    Amirshakhi, N.; Alyautdin, R. N.; Orlov, A. P.; Poloznikov, A. A.; Kuznetsov, D. A.

    2008-11-01

    A new affine chromatography technique is suggested for the purification of porphyrin-binding proteins (PBP) from mammal cell membranes. The procedure uses new fullerene-porphyrin ligands immobilized on agarose and bound to the polysaccharide matrix via the epoxycyclohexyl residue. A selective PBP stationary phase was used in a single-column chromatography run for the complete purification of a monomeric protein (17.6 kDa) from mitochondrial membranes of rat myocardium. This protein was characterized by high affinity for porphyrin-related structures. To separate it from other nonspecifically sorbed membrane proteins, synchronous linear pH and ionic strength gradients were used.

  1. Linear Interaction Energy Based Prediction of Cytochrome P450 1A2 Binding Affinities with Reliability Estimation

    PubMed Central

    Capoferri, Luigi; Verkade-Vreeker, Marlies C. A.; Buitenhuis, Danny; Commandeur, Jan N. M.; Pastor, Manuel; Vermeulen, Nico P. E.; Geerke, Daan P.

    2015-01-01

    Prediction of human Cytochrome P450 (CYP) binding affinities of small ligands, i.e., substrates and inhibitors, represents an important task for predicting drug-drug interactions. A quantitative assessment of the ligand binding affinity towards different CYPs can provide an estimate of inhibitory activity or an indication of isoforms prone to interact with the substrate of inhibitors. However, the accuracy of global quantitative models for CYP substrate binding or inhibition based on traditional molecular descriptors can be limited, because of the lack of information on the structure and flexibility of the catalytic site of CYPs. Here we describe the application of a method that combines protein-ligand docking, Molecular Dynamics (MD) simulations and Linear Interaction Energy (LIE) theory, to allow for quantitative CYP affinity prediction. Using this combined approach, a LIE model for human CYP 1A2 was developed and evaluated, based on a structurally diverse dataset for which the estimated experimental uncertainty was 3.3 kJ mol-1. For the computed CYP 1A2 binding affinities, the model showed a root mean square error (RMSE) of 4.1 kJ mol-1 and a standard error in prediction (SDEP) in cross-validation of 4.3 kJ mol-1. A novel approach that includes information on both structural ligand description and protein-ligand interaction was developed for estimating the reliability of predictions, and was able to identify compounds from an external test set with a SDEP for the predicted affinities of 4.6 kJ mol-1 (corresponding to 0.8 pKi units). PMID:26551865

  2. Transmembrane-truncated alphavbeta3 integrin retains high affinity for ligand binding: evidence for an 'inside-out' suppressor?

    PubMed Central

    Mehta, R J; Diefenbach, B; Brown, A; Cullen, E; Jonczyk, A; Güssow, D; Luckenbach, G A; Goodman, S L

    1998-01-01

    The molecular mechanisms of alphavbeta3 integrin affinity regulation have important biological implications in tumour development, wound repair and angiogenesis. We expressed, purified and characterized recombinant forms of human alphavbeta3 (r-alphavbeta3) and compared the activation state of these with alphavbeta3 in its cellular environment. The ligand specificity and selectivity of recombinant full-length and double transmembrane truncations of r-alphavbeta3 cloned in BacPAK6 vectors and expressed in Sf9 and High Five insect cells were compared with those of native placental alphavbeta3 and the receptor in situ on the cell surface. r-alphavbeta3 integrins were purified by affinity chromatography from detergent extracts of cells (full-length), and from the culture medium of cells expressing double-truncated r-alphavbeta3. r-alphavbeta3 had the same epitopes, ligand-binding specificities, bivalent cation requirements and susceptibility to RGD-containing peptides as native alphavbeta3. On M21-L4 melanoma cells, alphavbeta3 mediated binding to vitronectin, but not to fibrinogen unless activated with Mn2+. Non-activated alphaIIbbeta3 integrin as control in M21-L-IIb cells had the opposite profile, mediating binding to fibrinogen, but not to vitronectin unless activated with Mn2+. Thus these receptors had moderate to low ligand affinity. In marked contrast, purified alphavbeta3 receptors, with or without transmembrane and cytoplasmic domains, were constitutively of high affinity and able to bind strongly to vitronectin, fibronectin and fibrinogen under physiological conditions. Our data suggest that, in contrast with the positive regulation of alphaIIbbeta3 in situ, intracellular controls lower the affinity of alphavbeta3, and the cytoplasmic domains may act as a target for negative regulators of alphavbeta3 activity. PMID:9480902

  3. New ursane triterpenoids from Ficus pandurata and their binding affinity for human cannabinoid and opioid receptors.

    PubMed

    Khedr, Amgad I M; Ibrahim, Sabrin R M; Mohamed, Gamal A; Ahmed, Hany E A; Ahmad, Amany S; Ramadan, Mahmoud A; El-Baky, Atef E Abd; Yamada, Koji; Ross, Samir A

    2016-07-01

    Phytochemical investigation of Ficus pandurata Hance (Moraceae) fruits has led to the isolation of two new triterpenoids, ficupanduratin A [1β-hydroxy-3β-acetoxy-11α-methoxy-urs-12-ene] (11) and ficupanduratin B [21α-hydroxy-3β-acetoxy-11α-methoxy-urs-12-ene] (17), along with 20 known compounds: α-amyrin acetate (1), α-amyrin (2), 3β-acetoxy-20-taraxasten-22-one (3), 3β-acetoxy-11α-methoxy-olean-12-ene (4), 3β-acetoxy-11α-methoxy-12-ursene (5), 11-oxo-α-amyrin acetate (6), 11-oxo-β-amyrin acetate (7), palmitic acid (8), stigmast-4,22-diene-3,6-dione (9), stigmast-4-ene-3,6-dione (10), stigmasterol (12), β-sitosterol (13), stigmast-22-ene-3,6-dione (14), stigmastane-3,6-dione (15), 3β,21β-dihydroxy-11α-methoxy-olean-12-ene (16), 3β-hydroxy-11α-methoxyurs-12-ene (18), 6-hydroxystigmast-4,22-diene-3-one (19), 6-hydroxystigmast-4-ene-3-one (20), 11α,21α-dihydroxy-3β-acetoxy-urs-12-ene (21), and β-sitosterol-3-O-β-D-glucopyranoside (22). Compound 21 is reported for the first time from a natural source. The structures of the 20 compounds were elucidated on the basis of IR, 1D ((1)H and (13)C), 2D ((1)H-(1)H COSY, HSQC, HMBC and NOESY) NMR and MS spectroscopic data, in addition to comparison with literature data. The isolated compounds were evaluated for their anti-microbial, anti-malarial, anti-leishmanial, and cytotoxic activities. In addition, their radioligand displacement affinity on opioid and cannabinoid receptors was assessed. Compounds 4, 11, and 15 exhibited good affinity towards the CB2 receptor, with displacement values of 69.7, 62.5 and 86.5 %, respectively. Furthermore, the binding mode of the active compounds in the active site of the CB2 cannabinoid receptors was investigated through molecular modelling. PMID:27350550

  4. Vasoactive intestinal peptide (VIP) binds to guinea pig peritoneal eosinophils: A single class of binding sites with low affinity and high capacity

    SciTech Connect

    Sakakibara, H.; Shima, K. Takamatsu, J.; Said, S.I. )

    1990-02-26

    VIP binds to specific receptors on lymphocytes and mononuclear cells and exhibits antiinflammatory properties. Eosinophils (Eos) contribute to inflammatory reactions but the regulation of Eos function is incompletely understood. The authors examined the binding of monoradioiodinated VIP, (Tyr({sup 125}I){sup 10}) VIP ({sup 125}I-VIP), to Eos in guinea pigs. The interaction of {sup 125}i-VIP with Eos was rapid, reversible, saturable and linearly dependent on the number of cells. At equilibrium the binding was competitively inhibited by native peptide or by the related peptide helodermin. Scatchard analysis suggested the presence of a single class of VIP binding sites with a low affinity and a high capacity. In the presence of isobutyl-methylxanthine, VIP, PHI or helodermin did not stimulate cyclic AMP accumulation in intact Eos, while PGE{sub 2} or 1-isoproterenol did. VIP also did not inhibit superoxide anion generation from Eos stimulated by phorbol myristate acetate. The authors conclude that: (1) VIP binds to low-affinity, specific sites on guinea pig peritoneal eosinophils; (2) this binding is not coupled to stimulation of adenylate cyclase; and (3) the possible function of these binding sites is at present unknown.

  5. High-affinity consensus binding of target RNAs by the STAR/GSG proteins GLD-1, STAR-2 and Quaking

    PubMed Central

    2010-01-01

    Background STAR/GSG proteins regulate gene expression in metazoans by binding consensus sites in the 5' or 3' UTRs of target mRNA transcripts. Owing to the high degree of homology across the STAR domain, most STAR proteins recognize similar RNA consensus sequences. Previously, the consensus for a number of well-characterized STAR proteins was defined as a hexameric sequence, referred to as the SBE, for STAR protein binding element. C. elegans GLD-1 and mouse Quaking (Qk-1) are two representative STAR proteins that bind similar consensus hexamers, which differ only in the preferred nucleotide identities at certain positions. Earlier reports also identified partial consensus elements located upstream or downstream of a canonical consensus hexamer in target RNAs, although the relative contribution of these sequences to the overall binding energy remains less well understood. Additionally, a recently identified STAR protein called STAR-2 from C. elegans is thought to bind target RNA consensus sites similar to that of GLD-1 and Qk-1. Results Here, a combination of fluorescence-polarization and gel mobility shift assays was used to demonstrate that STAR-2 binds to a similar RNA consensus as GLD-1 and Qk-1. These assays were also used to further delineate the contributions of each hexamer consensus nucleotide to high-affinity binding by GLD-1, Qk-1 and STAR-2 in a variety of RNA contexts. In addition, the effects of inserting additional full or partial consensus elements upstream or downstream of a canonical hexamer in target RNAs were also measured to better define the sequence elements and RNA architecture recognized by different STAR proteins. Conclusions The results presented here indicate that a single hexameric consensus is sufficient for high-affinity RNA binding by STAR proteins, and that upstream or downstream partial consensus elements may alter binding affinities depending on the sequence and spacing. The general requirements determined for high-affinity RNA

  6. Mixed-model QSAR at the glucocorticoid receptor: predicting the binding mode and affinity of psychotropic drugs.

    PubMed

    Spreafico, Morena; Ernst, Beat; Lill, Markus A; Smiesko, Martin; Vedani, Angelo

    2009-01-01

    The glucocorticoid receptor (GR) is a member of the nuclear receptor superfamily that affects immune response, development, and metabolism in target tissues. Glucocorticoids are widely used to treat diverse pathophysiological conditions, but their clinical applicability is limited by side effects. A prediction of the binding affinity toward the GR would be beneficial for identifying glucocorticoid-mediated adverse effects triggered by drugs or chemicals. By identifying the binding mode to the GR using flexible docking (software Yeti) and quantifying the binding affinity through multidimensional QSAR (software Quasar), we validated a model family based on 110 compounds, representing four different chemical classes. The correlation with the experimental data (cross-validated r(2)=0.702; predictive r(2)=0.719) suggests that our approach is suited for predicting the binding affinity of related compounds toward the GR. After challenging the model by a series of scramble tests, a consensus approach (software Raptor), and a prediction set, it was incorporated into our VirtualToxLab and used to simulate and quantify the interaction of 24 psychotropic drugs with the GR.

  7. Molecular Weight, Protein Binding Affinity and Methane Mitigation of Condensed Tannins from Mangosteen-peel (Garcinia mangostana L).

    PubMed

    Paengkoum, P; Phonmun, T; Liang, J B; Huang, X D; Tan, H Y; Jahromi, M F

    2015-10-01

    The objectives of this study were to determine the molecular weight of condensed tannins (CT) extracted from mangosteen (Garcinia mangostana L) peel, its protein binding affinity and effects on fermentation parameters including total gas, methane (CH4) and volatile fatty acids (VFA) production. The average molecular weight (Mw) of the purified CT was 2,081 Da with a protein binding affinity of 0.69 (the amount needed to bind half the maximum bovine serum albumin). In vitro gas production declined by 0.409, 0.121, and 0.311, respectively, while CH4 production decreased by 0.211, 0.353, and 0.549, respectively, with addition of 10, 20, and 30 mg CT/500 mg dry matter (DM) compared to the control (p<0.05). The effects of CT from mangosteen-peel on in vitro DM degradability (IVDMD) and in vitro N degradability was negative and linear (p<0.01). Total VFA, concentrations of acetic, propionic, butyric and isovaleric acids decreased linearly with increasing amount of CT. The aforementioned results show that protein binding affinity of CT from mangosteen-peel is lower than those reported for Leucaena forages, however, the former has stronger negative effect on IVDMD. Therefore, the use of mangosteen-peel as protein source and CH4 mitigating agent in ruminant feed requires further investigations.

  8. Molecular Weight, Protein Binding Affinity and Methane Mitigation of Condensed Tannins from Mangosteen-peel (Garcinia mangostana L)

    PubMed Central

    Paengkoum, P.; Phonmun, T.; Liang, J. B.; Huang, X. D.; Tan, H. Y.; Jahromi, M. F.

    2015-01-01

    The objectives of this study were to determine the molecular weight of condensed tannins (CT) extracted from mangosteen (Garcinia mangostana L) peel, its protein binding affinity and effects on fermentation parameters including total gas, methane (CH4) and volatile fatty acids (VFA) production. The average molecular weight (Mw) of the purified CT was 2,081 Da with a protein binding affinity of 0.69 (the amount needed to bind half the maximum bovine serum albumin). In vitro gas production declined by 0.409, 0.121, and 0.311, respectively, while CH4 production decreased by 0.211, 0.353, and 0.549, respectively, with addition of 10, 20, and 30 mg CT/500 mg dry matter (DM) compared to the control (p<0.05). The effects of CT from mangosteen-peel on in vitro DM degradability (IVDMD) and in vitro N degradability was negative and linear (p<0.01). Total VFA, concentrations of acetic, propionic, butyric and isovaleric acids decreased linearly with increasing amount of CT. The aforementioned results show that protein binding affinity of CT from mangosteen-peel is lower than those reported for Leucaena forages, however, the former has stronger negative effect on IVDMD. Therefore, the use of mangosteen-peel as protein source and CH4 mitigating agent in ruminant feed requires further investigations. PMID:26323400

  9. Peptides from the Plasmodium falciparum STEVOR putative protein bind with high affinity to normal human red blood cells.

    PubMed

    García, Javier E; Puentes, Alvaro; Curtidor, Hernando; Vera, Ricardo; Rodriguez, Luis; Valbuena, John; López, Ramses; Ocampo, Marisol; Cortés, Jimena; Vanegas, Magnolia; Rosas, Jaiver; Reyes, Claudia; Patarroyo, Manuel E

    2005-07-01

    Synthetic 20-mer long non-overlapped peptides, from STEVOR protein, were tested in RBC binding assays for identifying STEVOR protein regions having high RBC binding activity and evaluating whether these regions inhibit Plasmodium falciparum in vitro invasion. Affinity constants, binding site number per cell and Hill coefficients were determined by saturation assay with high activity binding peptides (HABPs). HABP binding assays using RBCs previously treated with enzymes were carried out to study the nature of the receptor. The molecular weight of RBC surface proteins interacting with HABPs was determined by cross-linking assays and SDS-PAGE analysis. RBC binding assays revealed that peptides 30561 (41MKSRRLAEIQLPKCPHYNND60), 30562 (61PELKKIIDKLNEERIKKYIE80) and 30567 (161ASCCKVHDNYLDNLKKGCFG180) bound saturably and with high binding activity, presenting nanomolar affinity constants. HABP binding activity to RBCs previously treated with neuraminidase and trypsin decreased, suggesting that these peptides bound to RBC surface proteins and that such binding could be sialic acid dependent. Cross-linking and SDS-PAGE assays showed that the three HABPs specifically bound to 30 and 40 kDa molecular weight RBC membrane proteins. Peptides 30561, 30562 and 30567 inhibited P. falciparum in vitro invasion of red blood cells in a concentration-dependent way. Goat sera having STEVOR protein polymeric peptides antibodies inhibit parasite in vitro invasion depending on concentration. Three peptides localized in STEVOR N-terminal and central regions had high, saturable, binding activity to 30 and 40 kDa RBC membrane proteins. These peptides inhibited the parasite's in vitro invasion, suggesting that STEVOR protein regions are involved in P. falciparum invasion processes during intra-erythrocyte stage.

  10. Human IgA-binding peptides selected from random peptide libraries: affinity maturation and application in IgA purification.

    PubMed

    Hatanaka, Takaaki; Ohzono, Shinji; Park, Mirae; Sakamoto, Kotaro; Tsukamoto, Shogo; Sugita, Ryohei; Ishitobi, Hiroyuki; Mori, Toshiyuki; Ito, Osamu; Sorajo, Koichi; Sugimura, Kazuhisa; Ham, Sihyun; Ito, Yuji

    2012-12-14

    Phage display system is a powerful tool to design specific ligands for target molecules. Here, we used disulfide-constrained random peptide libraries constructed with the T7 phage display system to isolate peptides specific to human IgA. The binding clones (A1-A4) isolated by biopanning exhibited clear specificity to human IgA, but the synthetic peptide derived from the A2 clone exhibited a low specificity/affinity (K(d) = 1.3 μm). Therefore, we tried to improve the peptide using a partial randomized phage display library and mutational studies on the synthetic peptides. The designed Opt-1 peptide exhibited a 39-fold higher affinity (K(d) = 33 nm) than the A2 peptide. An Opt-1 peptide-conjugated column was used to purify IgA from human plasma. However, the recovered IgA fraction was contaminated with other proteins, indicating nonspecific binding. To design a peptide with increased binding specificity, we examined the structural features of Opt-1 and the Opt-1-IgA complex using all-atom molecular dynamics simulations with explicit water. The simulation results revealed that the Opt-1 peptide displayed partial helicity in the N-terminal region and possessed a hydrophobic cluster that played a significant role in tight binding with IgA-Fc. However, these hydrophobic residues of Opt-1 may contribute to nonspecific binding with other proteins. To increase binding specificity, we introduced several mutations in the hydrophobic residues of Opt-1. The resultant Opt-3 peptide exhibited high specificity and high binding affinity for IgA, leading to successful isolation of IgA without contamination.

  11. Identification of 14-3-3 Proteins Phosphopeptide-Binding Specificity Using an Affinity-Based Computational Approach.

    PubMed

    Li, Zhao; Tang, Jijun; Guo, Fei

    2016-01-01

    The 14-3-3 proteins are a highly conserved family of homodimeric and heterodimeric molecules, expressed in all eukaryotic cells. In human cells, this family consists of seven distinct but highly homologous 14-3-3 isoforms. 14-3-3σ is the only isoform directly linked to cancer in epithelial cells, which is regulated by major tumor suppressor genes. For each 14-3-3 isoform, we have 1,000 peptide motifs with experimental binding affinity values. In this paper, we present a novel method for identifying peptide motifs binding to 14-3-3σ isoform. First, we propose a sampling criteria to build a predictor for each new peptide sequence. Then, we select nine physicochemical properties of amino acids to describe each peptide motif. We also use auto-cross covariance to extract correlative properties of amino acids in any two positions. Finally, we consider elastic net to predict affinity values of peptide motifs, based on ridge regression and least absolute shrinkage and selection operator (LASSO). Our method tests on the 1,000 known peptide motifs binding to seven 14-3-3 isoforms. On the 14-3-3σ isoform, our method has overall pearson-product-moment correlation coefficient (PCC) and root mean squared error (RMSE) values of 0.84 and 252.31 for N-terminal sublibrary, and 0.77 and 269.13 for C-terminal sublibrary. We predict affinity values of 16,000 peptide sequences and relative binding ability across six permutated positions similar with experimental values. We identify phosphopeptides that preferentially bind to 14-3-3σ over other isoforms. Several positions on peptide motifs are in the same amino acid category with experimental substrate specificity of phosphopeptides binding to 14-3-3σ. Our method is fast and reliable and is a general computational method that can be used in peptide-protein binding identification in proteomics research. PMID:26828594

  12. Positive modulation of synaptic and extrasynaptic GABAA receptors by an antagonist of the high affinity benzodiazepine binding site.

    PubMed

    Middendorp, Simon J; Maldifassi, Maria C; Baur, Roland; Sigel, Erwin

    2015-08-01

    GABAA receptors are the major inhibitory neurotransmitter receptors in the brain and are the target for many clinically important drugs such as the benzodiazepines. Benzodiazepines act at the high-affinity binding site at the α+/γ- subunit interface. Previously, an additional low affinity binding site for diazepam located in the transmembrane (TM) domain has been described. The compound SJM-3 was recently identified in a prospective screening of ligands for the benzodiazepine binding site and investigated for its site of action. We determined the binding properties of SJM-3 at GABAA receptors recombinantly expressed in HEK-cells using radioactive ligand binding assays. Impact on function was assessed in Xenopus laevis oocytes with electrophysiological experiments using the two-electrode voltage clamp method. SJM-3 was shown to act as an antagonist at the α+/γ- site. At the same time it strongly potentiated GABA currents via the binding site for diazepam in the transmembrane domain. Mutation of a residue in M2 of the α subunit strongly reduced receptor modulation by SJM-3 and a homologous mutation in the β subunit abolished potentiation. SJM-3 acts as a more efficient modulator than diazepam at the site in the trans-membrane domain. In contrast to low concentrations of benzodiazepines, SJM-3 modulates both synaptic and extrasynaptic receptors. A detailed exploration of the membrane site may provide the basis for the design and identification of subtype-selective modulatory drugs.

  13. Metals affect the structure and activity of human plasminogen activator inhibitor-1. II. Binding affinity and conformational changes

    PubMed Central

    Thompson, Lawrence C; Goswami, Sumit; Peterson, Cynthia B

    2011-01-01

    Human plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor with a metastable active conformation. The lifespan of the active form of PAI-1 is modulated via interaction with the plasma protein, vitronectin, and various metal ions. These metal ions fall into two categories: Type I metals, including calcium, magnesium, and manganese, stabilize PAI-1 in the absence of vitronectin, whereas Type II metals, including cobalt, copper, and nickel, destabilize PAI-1 in the absence of vitronectin, but stabilize PAI-1 in its presence. To provide a mechanistic basis for understanding the unusual modulation of PAI-1 structure and activity, the binding characteristics and conformational effects of these two types of metals were further evaluated. Steady-state binding measurements using surface plasmon resonance indicated that both active and latent PAI-1 exhibit a dissociation constant in the low micromolar range for binding to immobilized nickel. Stopped-flow measurements of approach-to-equilibrium changes in intrinsic protein fluorescence indicated that the Type I and Type II metals bind in different modes that induce distinct conformational effects on PAI-1. Changes in the observed rate constants with varying concentrations of metal allowed accurate determination of binding affinities for cobalt, nickel, and copper, yielding dissociation constants of ∼40, 30, and 0.09 μM, respectively. Competition experiments that tested effects on PAI-1 stability were consistent with these measurements of affinity and indicate that copper binds tightly to PAI-1. PMID:21280128

  14. Molecular dynamics simulations reveal fundamental role of water as factor determining affinity of binding of beta-blocker nebivolol to beta(2)-adrenergic receptor.

    PubMed

    Kaszuba, Karol; Róg, Tomasz; Bryl, Krzysztof; Vattulainen, Ilpo; Karttunen, Mikko

    2010-07-01

    The beta-adrenergic antagonists (beta-blockers) constitute a class of drugs that have well-established roles in treatments of various cardiovascular diseases. Despite a 50 year history, there are two clinically important subtypes of beta-adrenergic receptors (betaARs) called beta(1)AR and beta(2)AR that still are promising drug targets. Our study maps the interactions between nebivolol-one of the most efficient beta-blocking agents-and the beta(2)-adrenergic receptor by simulating two optical isomers of nebivolol: ssss-nebivolol and srrr-nebivolol. The srrr-configuration binds preferentially to beta(1)AR and beta(2)AR. The ssss-form has much lower binding affinity to both of them. Our work indicates that water is a very important component of the binding site of the beta(2)AR receptor. We found that the higher stereoselectivity of the srrr-configuration is due to interactions with water molecules, which extensively hydrate the binding site of beta(2)AR. By lowering the energy of binding, water enhanced the affinity of the srrr-form to beta(2)AR. We also address the problem of beta(1)AR/beta(2)AR selectivity. At higher concentrations, all beta-blocking agents lose their specificity and bind nonselectively, causing many adverse effects. Our simulations indicate that PHE194, TYR308, and ILE309 of the beta(2)AR and the corresponding residues of the beta(1)AR receptor may be important determinants of beta(1)AR versus beta(2)AR selectivity.

  15. High affinity binding of /sup 125/I-labeled mouse interferon to a specific cell surface receptor. II. Analysis of binding properties

    SciTech Connect

    Aguet, M.; Blanchard, B.

    1981-12-01

    Direct ligand-binding studies with highly purified /sup 125/I-labeled virus-induced mouse interferon on mouse lymphoma L 1210 cells revealed a direct correlation of specific high-affinity binding with the biologic response to interferon. Neutralization of the antiviral effect by anti-interferon gamma globulin occurred at the same antibody concentration as the inhibition of specific binding. These results suggest that specific high-affinity binding of /sup 125/I-interferon occurred at a biologically functional interferon receptor. Competitive inhibition experiments using /sup 125/I- and /sup 127/I-labeled interferon provided strong evidence that the fraction of /sup 125/I-interferon inactivated upon labeling did not bind specifically. Scatchard analysis of the binding data yielded linear plots and thus suggested that interferon binds to homogeneous noncooperative receptor sites. In contrast to a characteristic property of several peptide hormone systems, binding of /sup 125/I-interferon to its specific receptor did not induce subsequent ligand degradation. At 37/sup o/ bound interferon was rapidly released in a biologically active form without evidence for molecular degradation. The expression of interferon receptors was not modified by treatment with interferon. Trypsin treatment of target cells and inhibition of protein synthesis abolished the specific binding of /sup 125/I-interferon. Three major molecular weight species of Newcastle disease virus-induced mouse C 243 cell interferon were isolated, separated, and identified as mouse ..cap alpha.. and ..beta.. interferons. These interferons were shown to inhibit competitively the specific binding of the highly purified labeled starting material thus providing evidence for a common receptor site for mouse interferon.

  16. Chemical Affinity between Tannin Size and Salivary Protein Binding Abilities: Implications for Wine Astringency.

    PubMed

    Ma, Wen; Waffo-Teguo, Pierre; Jourdes, Michael; Li, Hua; Teissedre, Pierre-Louis

    2016-01-01

    Astringency perception, as an essential parameter for high-quality red wine, is principally elicited by condensed tannins in diversified chemical structures. Condensed tannins, which are also known as proanthocyanidins (PAs), belong to the flavonoid class of polyphenols and are incorporated by multiple flavan-3-ols units according to their degree of polymerization (DP). However, the influence of DP size of PAs on astringency perception remains unclear for decades. This controversy was mainly attributed to the lack of efficient strategies to isolate the PAs in non-galloylated forms and with individual degree size from grape/wine. In the present study, the astringency intensity of purified and identified grape oligomeric tannins (DP ranged from 1 to 5) was firstly explored. A novel non-solid phase strategy was used to rapidly exclude the galloylated PAs from the non-galloylated PAs and fractionate the latter according to their DP size. Then, a series of PAs with individual DP size and galloylation were purified by an approach of preparative hydrophilic interaction chromatography. Furthermore, purified compounds were identified by both normal phase HPLC-FLD and reverse phase UHPLC-ESI-Q-TOF. Finally, the contribution of the astringency perception of the individual purified tannins was examined with a salivary protein binding ability test. The results were observed by HPLC-FLD and quantified by changes in PA concentration remaining in the filtrate. In summary, a new approach without a solid stationary phase was developed to isolate PAs according to their DP size. And a positive relationship between the DP of PAs and salivary protein affinity was revealed. PMID:27518822

  17. Chemical Affinity between Tannin Size and Salivary Protein Binding Abilities: Implications for Wine Astringency

    PubMed Central

    Ma, Wen; Waffo-Teguo, Pierre; Jourdes, Michael; Li, Hua

    2016-01-01

    Astringency perception, as an essential parameter for high-quality red wine, is principally elicited by condensed tannins in diversified chemical structures. Condensed tannins, which are also known as proanthocyanidins (PAs), belong to the flavonoid class of polyphenols and are incorporated by multiple flavan-3-ols units according to their degree of polymerization (DP). However, the influence of DP size of PAs on astringency perception remains unclear for decades. This controversy was mainly attributed to the lack of efficient strategies to isolate the PAs in non-galloylated forms and with individual degree size from grape/wine. In the present study, the astringency intensity of purified and identified grape oligomeric tannins (DP ranged from 1 to 5) was firstly explored. A novel non-solid phase strategy was used to rapidly exclude the galloylated PAs from the non-galloylated PAs and fractionate the latter according to their DP size. Then, a series of PAs with individual DP size and galloylation were purified by an approach of preparative hydrophilic interaction chromatography. Furthermore, purified compounds were identified by both normal phase HPLC-FLD and reverse phase UHPLC-ESI-Q-TOF. Finally, the contribution of the astringency perception of the individual purified tannins was examined with a salivary protein binding ability test. The results were observed by HPLC-FLD and quantified by changes in PA concentration remaining in the filtrate. In summary, a new approach without a solid stationary phase was developed to isolate PAs according to their DP size. And a positive relationship between the DP of PAs and salivary protein affinity was revealed. PMID:27518822

  18. Mapping of barley alpha-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft.

    PubMed

    Kandra, Lili; Hachem, Maher Abou; Gyémánt, Gyöngyi; Kramhøft, Birte; Svensson, Birte

    2006-09-18

    Subsite affinity maps of long substrate binding clefts in barley alpha-amylases, obtained using a series of maltooligosaccharides of degree of polymerization of 3-12, revealed unfavorable binding energies at the internal subsites -3 and -5 and at subsites -8 and +3/+4 defining these subsites as binding barriers. Barley alpha-amylase 1 mutants Y105A and T212Y at subsite -6 and +4 resulted in release or anchoring of bound substrate, thus modifying the affinities of other high-affinity subsites (-2 and +2) and barriers. The double mutant Y105A-T212Y displayed a hybrid subsite affinity profile, converting barriers to binding areas. These findings highlight the dynamic binding energy distribution and the versatility of long maltooligosaccharide derivatives in mapping extended binding clefts in alpha-amylases.

  19. Ion Interactions in the High-Affinity Binding Locus of a Voltage-Gated Ca2+ Channel

    PubMed Central

    Cloues, Robin K.; Cibulsky, Susan M.; Sather, William A.

    2000-01-01

    The selectivity filter of voltage-gated Ca2+ channels is in part composed of four Glu residues, termed the EEEE locus. Ion selectivity in Ca2+ channels is based on interactions between permeant ions and the EEEE locus: in a mixture of ions, all of which can pass through the pore when present alone, those ions that bind weakly are impermeant, those that bind more strongly are permeant, and those that bind more strongly yet act as pore blockers as a consequence of their low rate of unbinding from the EEEE locus. Thus, competition among ion species is a determining feature of selectivity filter function in Ca2+ channels. Previous work has shown that Asp and Ala substitutions in the EEEE locus reduce ion selectivity by weakening ion binding affinity. Here we describe for wild-type and EEEE locus mutants an analysis at the single channel level of competition between Cd2+, which binds very tightly within the EEEE locus, and Ba2+ or Li+, which bind less tightly and hence exhibit high flux rates: Cd2+ binds to the EEEE locus ∼104× more tightly than does Ba2+, and ∼108× more tightly than does Li+. For wild-type channels, Cd2+ entry into the EEEE locus was 400× faster when Li+ rather than Ba2+ was the current carrier, reflecting the large difference between Ba2+ and Li+ in affinity for the EEEE locus. For the substitution mutants, analysis of Cd2+ block kinetics shows that their weakened ion binding affinity can result from either a reduction in blocker on rate or an enhancement of blocker off rate. Which of these rate effects underlay weakened binding was not specified by the nature of the mutation (Asp vs. Ala), but was instead determined by the valence and affinity of the current-carrying ion (Ba2+ vs. Li+). The dependence of Cd2+ block kinetics upon properties of the current-carrying ion can be understood by considering the number of EEEE locus oxygen atoms available to interact with the different ion pairs. PMID:11004206

  20. Engineered Cystine-Knot Peptides That Bind αvβ3 Integrin With Antibody-Like Affinities

    PubMed Central

    Silverman, Adam P.; Levin, Aron M.; Lahti, Jennifer L.; Cochran, Jennifer R.

    2010-01-01

    The αvβ3 integrin receptor is an important cancer target due to its overexpression on many solid tumors and the tumor neovasculature, and its role in metastasis and angiogenesis. We used a truncated form of the Agouti-related protein (AgRP), a 4 kDa cystine-knot peptide with four disulfide bonds and four solvent-exposed loops, as a scaffold for engineering peptides that bound to αvβ3 integrins with high affinity and specificity. A yeast-displayed cystine-knot peptide library was generated by substituting a 6-amino acid loop of AgRP with a 9-amino acid loop containing the Arg-Gly-Asp (RGD) integrin recognition motif and randomized flanking residues. Mutant cystine-knot peptides were screened in a high-throughput manner by fluorescence-activated cell sorting (FACS) to identify clones with high affinity to detergent-solubilized αvβ3 integrin receptor. Select integrin-binding peptides were expressed recombinantly in Pichia pastoris and were tested for their ability to bind to human cancer cells expressing various integrin receptors. These studies showed that the engineered AgRP peptides bound to cells expressing αvβ3 integrins with affinities ranging from 15 nM to 780 pM. Furthermore, the engineered peptides were shown bind specifically to αvβ3 integrins, and had only minimal or no binding to αvβ5, α5β1, and αiibβ3 integrins. The engineered AgRP peptides were also shown to inhibit cell adhesion to the extracellular matrix protein vitronectin, which is a naturally-occurring ligand for αvβ3 and other integrins. Next, to evaluate whether the other three loops of AgRP could modulate integrin specificity, we made second generation libraries by individually randomizing these loops in one of the high affinity integrin-binding variants. Screening of these loop-randomized libraries against αvβ3 integrins resulted in peptides that retained high affinities for αvβ3 and had increased specificities for αvβ3 over αiibβ3 integrins. Collectively, these data

  1. Afi-Chip: An Equipment-Free, Low-Cost, and Universal Binding Ligand Affinity Evaluation Platform.

    PubMed

    Song, Yanling; Shi, Yuanzhi; Li, Xingrui; Ma, Yanli; Gao, Mingxuan; Liu, Dan; Mao, Yu; Zhu, Zhi; Lin, Hui; Yang, Chaoyong

    2016-08-16

    Binding affinity characterization is of great importance for aptamer screening because the dissociation constant (Kd) value is a key parameter for evaluating molecular interaction. However, conventional methods often require sophisticated equipment and time-consuming processing. Here, we present a portable device, Afi-Chip, as an equipment-free, rapid, low-cost, and universal platform for evaluation of the aptamer affinity. The Afi-Chip displays a distance readout based on the reaction of an enzyme catalyzing the decomposition of H2O2 for gas generation to push the movement of ink bar. Taking advantage of translating the recognition signal to distance signal and realizing the regents mixing and quantitative readout on the chip, we successfully monitored the aptamer evolution process and characterized binding affinity of aptamers against multiple types of targets, including small molecule glucose, cancer biomarker protein EpCAM, and tumor cell SW620. We also applied the Afi-Chip for rapid characterization of the affinity between anti-HCG and HCG to demonstrate the generality for the molecular interaction study. All of the Kd values obtained are comparable to those reported in the literature or obtained by sophisticated instruments such as a flow cytometer. The Afi-Chip offers a new approach for equipment-free investigation of molecular interactions, such as aptamer identification, ligand selection monitoring, and drug screening. PMID:27454185

  2. Afi-Chip: An Equipment-Free, Low-Cost, and Universal Binding Ligand Affinity Evaluation Platform.

    PubMed

    Song, Yanling; Shi, Yuanzhi; Li, Xingrui; Ma, Yanli; Gao, Mingxuan; Liu, Dan; Mao, Yu; Zhu, Zhi; Lin, Hui; Yang, Chaoyong

    2016-08-16

    Binding affinity characterization is of great importance for aptamer screening because the dissociation constant (Kd) value is a key parameter for evaluating molecular interaction. However, conventional methods often require sophisticated equipment and time-consuming processing. Here, we present a portable device, Afi-Chip, as an equipment-free, rapid, low-cost, and universal platform for evaluation of the aptamer affinity. The Afi-Chip displays a distance readout based on the reaction of an enzyme catalyzing the decomposition of H2O2 for gas generation to push the movement of ink bar. Taking advantage of translating the recognition signal to distance signal and realizing the regents mixing and quantitative readout on the chip, we successfully monitored the aptamer evolution process and characterized binding affinity of aptamers against multiple types of targets, including small molecule glucose, cancer biomarker protein EpCAM, and tumor cell SW620. We also applied the Afi-Chip for rapid characterization of the affinity between anti-HCG and HCG to demonstrate the generality for the molecular interaction study. All of the Kd values obtained are comparable to those reported in the literature or obtained by sophisticated instruments such as a flow cytometer. The Afi-Chip offers a new approach for equipment-free investigation of molecular interactions, such as aptamer identification, ligand selection monitoring, and drug screening.

  3. Mutations within Helix I of Twist1 Result in Distinct Limb Defects and Variation of DNA-Binding Affinities

    PubMed Central

    Firulli, Beth A.; Redick, Bradley A.; Conway, Simon J.; Firulli, Anthony B.

    2008-01-01

    Twist1 is a basic helix-loop-helix (bHLH) factor that plays an important role in limb development. Haploinsufficiency of Twist1 results in polydactyly via the inability of Twist1 to antagonistically regulate the related factor Hand2. The mechanism modulating Twist1-Hand2 antagonism is via phosphoregulation of conserved threonine and serine residues in helix I of the bHLH domain. Phosphoregulation alters the dimerization affinities for both proteins. Here we show that the expression of Twist1 and Twist1 phosphoregulation mutants result in distinct limb phenotypes in mice. In addition to dimer regulation, Twist1 phosphoregulation affects the DNA-binding affinities of Twist1 in a partner dependent and cis-element dependent manner. In order to gain a better understanding of the specific Twist1 transcriptional complexes that function during limb morphogensis, we employ a series of Twist1-tethered dimers that include the known Twist1 partners, E12 and Hand2, as well as a tethered Twist1 homodimer. We show that these dimers behave in a manner similar to monomerically expressed bHLH factors and result in distinct limb phenotypes that correlate well with those observed from the limb expression of Twist1 and Twist1 phosphoregulation mutants. Taken together, this study shows that the Twist1 dimer affinity for a given partner can modulate the DNA binding affinity and that Twist1 dimer choice determines phenotypic outcome during limb development. PMID:17652084

  4. Automethylation of SUV39H2, an oncogenic histone lysine methyltransferase, regulates its binding affinity to substrate proteins

    PubMed Central

    Piao, Lianhua; Nakakido, Makoto; Suzuki, Takehiro; Dohmae, Naoshi; Nakamura, Yusuke; Hamamoto, Ryuji

    2016-01-01

    We previously reported that the histone lysine methyltransferase SUV39H2, which is overexpressed in various types of human cancer, plays a critical role in the DNA repair after double strand breakage, and possesses oncogenic activity. Although its biological significance in tumorigenesis has been elucidated, the regulatory mechanism of SUV39H2 activity through post-translational modification is not well known. In this study, we demonstrate in vitro and in vivo automethylation of SUV39H2 at lysine 392. Automethylation of SUV39H2 led to impairment of its binding affinity to substrate proteins such as histone H3 and LSD1. Furthermore, we observed that hyper-automethylated SUV39H2 reduced methylation activities to substrates through affecting the binding affinity to substrate proteins. Our finding unveils a novel autoregulatory mechanism of SUV39H2 through lysine automethylation. PMID:26988914

  5. Structural basis for high substrate-binding affinity and enantioselectivity of 3-quinuclidinone reductase AtQR

    SciTech Connect

    Hou, Feng; Miyakawa, Takuya; Kataoka, Michihiko; Takeshita, Daijiro; Kumashiro, Shoko; Uzura, Atsuko; Urano, Nobuyuki; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2014-04-18

    Highlights: • Crystal structure of AtQR has been determined at 1.72 Å. • NADH binding induces the formation of substrate binding site. • AtQR possesses a conserved hydrophobic wall for stereospecific binding of substrate. • Additional Glu197 residue is critical to the high binding affinity. - Abstract: (R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (>100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of AtQR at 1.72 Å. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity.

  6. Lysine 114 of antithrombin is of crucial importance for the affinity and kinetics of heparin pentasaccharide binding.

    PubMed

    Arocas, V; Bock, S C; Raja, S; Olson, S T; Bjork, I

    2001-11-23

    Lys(114) of the plasma coagulation proteinase inhibitor, antithrombin, has been implicated in binding of the glycosaminoglycan activator, heparin, by previous mutagenesis studies and by the crystal structure of antithrombin in complex with the active pentasaccharide unit of heparin. In the present work, substitution of Lys(114) by Ala or Met was shown to decrease the affinity of antithrombin for heparin and the pentasaccharide by approximately 10(5)-fold at I 0.15, corresponding to a reduction in binding energy of approximately 50%. The decrease in affinity was due to the loss of two to three ionic interactions, consistent with Lys(114) and at least one other basic residue of the inhibitor binding cooperatively to heparin, as well as to substantial nonionic interactions. The mutation minimally affected the initial, weak binding of the two-step mechanism of pentasaccharide binding to antithrombin but appreciably (>40-fold) decreased the forward rate constant of the conformational change in the second step and greatly (>1000-fold) increased the reverse rate constant of this step. Lys(114) is thus of greater importance for the affinity of heparin binding than any of the other antithrombin residues investigated so far, viz. Arg(47), Lys(125), and Arg(129). It contributes more than Arg(47) and Arg(129) to increasing the rate of induction of the activating conformational change, a role presumably exerted by interactions with the nonreducing end trisaccharide unit of the heparin pentasaccharide. However, its major effect, also larger than that of these two residues, is in maintaining antithrombin in the activated state by interactions that most likely involve the reducing end disaccharide unit. PMID:11567021

  7. Affinity of the heparin binding motif of Noggin1 to heparan sulfate and its visualization in the embryonic tissues.

    PubMed

    Nesterenko, Alexey M; Orlov, Eugeny E; Ermakova, Galina V; Ivanov, Igor A; Semenyuk, Pavel I; Orlov, Victor N; Martynova, Natalia Y; Zaraisky, Andrey G

    Heparin binding motifs were found in many secreted proteins and it was suggested that they are responsible for retardation of the protein diffusion within the intercellular space due to the binding to heparan sulfate proteoglycanes (HSPG). Here we used synthetic FITC labeled heparin binding motif (HBM peptide) of the Xenopus laevis secreted BMP inhibitor Noggin1 to study its diffusion along the surface of the heparin beads by FRAP method. As a result, we have found out that diffusivity of HBM-labeled FITC was indeed much lesser than those predicted by theoretical calculations even for whole protein of the Noggin size. We also compared by isothermal titration calorimetry the binding affinity of HBM and the control oligolysine peptide to several natural polyanions including heparan sulfate (HS), heparin, the bacterial dextran sulfate and salmon sperm DNA, and demonstrated that HBM significantly exceeds oligolysine peptide in the affinity to HS, heparin and DNA. By contrast, oligolysine peptide bound with higher affinity to dextran sulfate. We speculate that such a difference may ensure specificity of the morphogen binding to HSPG and could be explained by steric constrains imposed by different distribution of the negative charges along a given polymeric molecule. Finally, by using EGFP-HBM recombinant protein we have visualized the natural pattern of the Noggin1 binding sites within the X. laevis gastrula and demonstrated that these sites forms a dorsal-ventral concentration gradient, with a maximum in the dorsal blastopore lip. In sum, our data provide a quantitative basis for modeling the process of Noggin1 diffusion in embryonic tissues, considering its interaction with HSPG.

  8. Irreversible blockade of the high and low affinity ( sup 3 H) naloxone binding sites by C-6 derivatives of morphinane-6-ones

    SciTech Connect

    Krizsan, D. ); Varga, E.; Benyhe, S.; Szucs, M.; Borsodi, A. ); Hosztafi, S. )

    1991-01-01

    C-6 derivatives-hydrazones, phenylhydrazones, dinitrophenylhydrazones, oximes and semicarbazones - of morphinane-6-ones were synthesized and their binding characteristics were studied on rat brain membranes. The dihydromorphinone and oxymorphone derivatives compete for the ({sup 3}H)naloxone binding sites with high affinity, while the dihydrocodeinone and oxycodone derivatives are less potent. The affinity of the new compounds is decreased for the delta sites as compared to the parent ligands. The ligands bearing bulky substituents also bind with low affinity to the kappa sites. The modification decreased the Na{sup +}-index of compounds indicating their mixed agonist-antagonist character. The dihydromorphinone derivatives are all capable to block irreversibly the high affinity binding site of ({sup 3}H)naloxone, whereas the dihydrocodeinone derivatives block irreversibly the low affinity site. A possible mechanism for the inhibition is suggested.

  9. Affinity-defining domains in the Na-Cl cotransporter: a different location for Cl- and thiazide binding.

    PubMed

    Moreno, Erika; Cristóbal, Pedro San; Rivera, Manuel; Vázquez, Norma; Bobadilla, Norma A; Gamba, Gerardo

    2006-06-23

    The thiazide-sensitive Na+-Cl- cotransporter (NCC) is the major pathway for salt reabsorption in the distal convoluted tubule, serves as a receptor for thiazide-type diuretics, and is involved in inherited diseases associated with abnormal blood pressure. Little is known regarding the structure-function relationship in this cotransporter. Previous studies from our group reveal that mammalian NCC exhibits higher affinity for ions and thiazides than teleost NCC and suggest a role for glycosylation upon thiazide affinity. Here we have constructed a series of chimeric and mutant cDNAs between rat and flounder NCC to define the role of glycosylation status, the amino-terminal domain, the carboxyl-terminal domain, the extracellular glycosylated loop, and the transmembrane segments upon affinity for Na+, Cl-, and metolazone. Xenopus laevis oocytes were used as the heterologous expression system. We observed that elimination of glycosylation sites in flounder NCC did not affect the affinity of the cotransporter for metolazone. Also, swapping the amino-terminal domain, the carboxyl-terminal domain, the glycosylation sites, or the entire extracellular glycosylation loop between rat and flounder NCC had no effect upon ions or metolazone affinity. In contrast, interchanging transmembrane regions between rat and flounder NCC revealed that affinity-modifying residues for chloride are located within the transmembrane 1-7 region and for thiazides are located within the transmembrane 8-12 region, whereas both segments seem to be implicated in defining sodium affinity. These observations strongly suggest that binding sites for chloride and thiazide in NCC are different. PMID:16624820

  10. Multipurpose ligand, DAKLI (Dynorphin A-analogue Kappa LIgand), with high affinity and selectivity for dynorphin (. kappa. opioid) binding sites

    SciTech Connect

    Goldstein, A.; Nestor, J.J. Jr.; Naidu, A.; Newman, S.R. )

    1988-10-01

    The authors describe a synthetic ligand, DALKI (Dynorphin A-analogue Kappa LIgand), related to the opioid peptide dynorphin A. A single reactive amino group at the extended carboxyl terminus permits various reporter groups to be attached, such as {sup 125}I-labeled Bolton-Hunter reagent, fluorescein isothiocyanate, or biotin. These derivatives have high affinity and selectivity for the dynorphin ({kappa} opioid) receptor. An incidental finding is that untreated guinea pig brain membranes have saturable avidin binding sites.

  11. High-affinity binding by the periplasmic iron-binding protein from Haemophilus influenzae is required for acquiring iron from transferrin

    PubMed Central

    Khan, Ali G.; Shouldice, Stephen R.; Kirby, Shane D.; Yu, Rong-hua; Tari, Leslie W.; Schryvers, Anthony B.

    2007-01-01

    The periplasmic iron-binding protein, FbpA (ferric-ion-binding protein A), performs an essential role in iron acquisition from transferrin in Haemophilus influenzae. A series of site-directed mutants in the metal-binding amino acids of FbpA were prepared to determine their relative contribution to iron binding and transport. Structural studies demonstrated that the mutant proteins crystallized in an open conformation with the iron atom associated with the C-terminal domain. The iron-binding properties of the mutant proteins were assessed by several assays, including a novel competitive iron-binding assay. The relative ability of the proteins to compete for iron was pH dependent, with a rank order at pH 6.5 of wild-type, Q58L, H9Q>H9A, E57A>Y195A, Y196A. The genes encoding the mutant FbpA were introduced into H. influenzae and the resulting strains varied in the level of ferric citrate required to support growth on iron-limited medium, suggesting a rank order for metal-binding affinities under physiological conditions comparable with the competitive binding assay at pH 6.5 (wild-type=Q58L>H9Q>H9A, E57A>Y195A, Y196A). Growth dependence on human transferrin was only obtained with cells expressing wild-type, Q58L or H9Q FbpAs, proteins with stability constants derived from the competition assay >2.0×1018 M−1. These results suggest that a relatively high affinity of iron binding by FbpA is required for removal of iron from transferrin and its transport across the outer membrane. PMID:17313366

  12. Binding modes of noncompetitive GABA-channel blockers revisited using engineered affinity-labeling reactions combined with new docking studies.

    PubMed

    Charon, Sébastien; Taly, Antoine; Rodrigo, Jordi; Perret, Philippe; Goeldner, Maurice

    2011-04-13

    The binding modes of noncompetitive GABA(A)-channel blockers were re-examined taking into account the recent description of the 3D structure of prokaryotic pentameric ligand-gated ion channels, which provided access to new mammalian or insect GABA receptor models, emphasizing their transmembrane portion. Two putative binding modes were deciphered for this class of compounds, including the insecticide fipronil, located nearby either the intra- or the extracellular part of the membrane, respectively. These results are in full agreement with previously described affinity-labeling reactions performed with GABA(A) noncompetitive blockers (Perret et al. J. Biol. Chem.1999, 274, 25350-25354).

  13. Processivity and Subcellular Localization of Glycogen Synthase Depend on a Non-catalytic High Affinity Glycogen-binding Site*

    PubMed Central

    Díaz, Adelaida; Martínez-Pons, Carlos; Fita, Ignacio; Ferrer, Juan C.; Guinovart, Joan J.

    2011-01-01

    Glycogen synthase, a central enzyme in glucose metabolism, catalyzes the successive addition of α-1,4-linked glucose residues to the non-reducing end of a growing glycogen molecule. A non-catalytic glycogen-binding site, identified by x-ray crystallography on the surface of the glycogen synthase from the archaeon Pyrococcus abyssi, has been found to be functionally conserved in the eukaryotic enzymes. The disruption of this binding site in both the archaeal and the human muscle glycogen synthases has a large impact when glycogen is the acceptor substrate. Instead, the catalytic efficiency remains essentially unchanged when small oligosaccharides are used as substrates. Mutants of the human muscle enzyme with reduced affinity for glycogen also show an altered intracellular distribution and a marked decrease in their capacity to drive glycogen accumulation in vivo. The presence of a high affinity glycogen-binding site away from the active center explains not only the long-recognized strong binding of glycogen synthase to glycogen but also the processivity and the intracellular localization of the enzyme. These observations demonstrate that the glycogen-binding site is a critical regulatory element responsible for the in vivo catalytic efficiency of GS. PMID:21464127

  14. Competition effects in cation binding to humic acid: Conditional affinity spectra for fixed total metal concentration conditions

    NASA Astrophysics Data System (ADS)

    David, Calin; Mongin, Sandrine; Rey-Castro, Carlos; Galceran, Josep; Companys, Encarnació; Garcés, José Luis; Salvador, José; Puy, Jaume; Cecilia, Joan; Lodeiro, Pablo; Mas, Francesc

    2010-09-01

    Information on the Pb and Cd binding to a purified Aldrich humic acid (HA) is obtained from the influence of different fixed total metal concentrations on the acid-base titrations of this ligand. NICA (Non-Ideal Competitive Adsorption) isotherm has been used for a global quantitative description of the binding, which has then been interpreted by plotting the Conditional Affinity Spectra of the H + binding at fixed total metal concentrations (CAScTM). This new physicochemical tool, here introduced, allows the interpretation of binding results in terms of distributions of proton binding energies. A large increase in the acidity of the phenolic sites as the total metal concentration increases, especially in presence of Pb, is revealed from the shift of the CAScTM towards lower affinities. The variance of the CAScTM distribution, which can be used as a direct measure of the heterogeneity, also shows a significant dependence on the total metal concentration. A discussion of the factors that influence the heterogeneity of the HA under the conditions of each experiment is provided, so that the smoothed pattern exhibited by the titration curves can be justified.

  15. Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity

    PubMed Central

    Fleetwood, Filippa; Klint, Susanne; Hanze, Martin; Gunneriusson, Elin; Frejd, Fredrik Y.; Ståhl, Stefan; Löfblom, John

    2014-01-01

    Angiogenesis plays an important role in cancer and ophthalmic disorders such as age-related macular degeneration and diabetic retinopathy. The vascular endothelial growth factor (VEGF) family and corresponding receptors are regulators of angiogenesis and have been much investigated as therapeutic targets. The aim of this work was to generate antagonistic VEGFR2-specific affinity proteins having adjustable pharmacokinetic properties allowing for either therapy or molecular imaging. Two antagonistic Affibody molecules that were cross-reactive for human and murine VEGFR2 were selected by phage and bacterial display. Surprisingly, although both binders independently blocked VEGF-A binding, competition assays revealed interaction with non-overlapping epitopes on the receptor. Biparatopic molecules, comprising the two Affibody domains, were hence engineered to potentially increase affinity even further through avidity. Moreover, an albumin-binding domain was included for half-life extension in future in vivo experiments. The best-performing of the biparatopic constructs demonstrated up to 180-fold slower dissociation than the monomers. The new Affibody constructs were also able to specifically target VEGFR2 on human cells, while simultaneously binding to albumin, as well as inhibit VEGF-induced signaling. In summary, we have generated small antagonistic biparatopic Affibody molecules with high affinity for VEGFR2, which have potential for both future therapeutic and diagnostic purposes in angiogenesis-related diseases. PMID:25515662

  16. Tripartite ATP-independent Periplasmic (TRAP) Transporters Use an Arginine-mediated Selectivity Filter for High Affinity Substrate Binding*

    PubMed Central

    Fischer, Marcus; Hopkins, Adam P.; Severi, Emmanuele; Hawkhead, Judith; Bawdon, Daniel; Watts, Andrew G.; Hubbard, Roderick E.; Thomas, Gavin H.

    2015-01-01

    Tripartite ATP-independent periplasmic (TRAP) transporters are secondary transporters that have evolved an obligate dependence on a substrate-binding protein (SBP) to confer unidirectional transport. Different members of the DctP family of TRAP SBPs have binding sites that recognize a diverse range of organic acid ligands but appear to only share a common electrostatic interaction between a conserved arginine and a carboxylate group in the ligand. We investigated the significance of this interaction using the sialic acid-specific SBP, SiaP, from the Haemophilus influenzae virulence-related SiaPQM TRAP transporter. Using in vitro, in vivo, and structural methods applied to SiaP, we demonstrate that the coordination of the acidic ligand moiety of sialic acid by the conserved arginine (Arg-147) is essential for the function of the transporter as a high affinity scavenging system. However, at high substrate concentrations, the transporter can function in the absence of Arg-147 suggesting that this bi-molecular interaction is not involved in further stages of the transport cycle. As well as being required for high affinity binding, we also demonstrate that the Arg-147 is a strong selectivity filter for carboxylate-containing substrates in TRAP transporters by engineering the SBP to recognize a non-carboxylate-containing substrate, sialylamide, through water-mediated interactions. Together, these data provide biochemical and structural support that TRAP transporters function predominantly as high affinity transporters for carboxylate-containing substrates. PMID:26342690

  17. High affinity and covalent-binding microtubule stabilizing agents show activity in chemotherapy-resistant acute myeloid leukemia cells

    PubMed Central

    Pera, Benet; Calvo-Vidal, M. Nieves; Ambati, Srikanth; Jordi, Michel; Kahn, Alissa; Díaz, J. Fernando; Fang, Weishuo; Altmann, Karl-Heinz; Cerchietti, Leandro; Moore, Malcolm A.S.

    2016-01-01

    Treatment failure in acute myeloid leukemia (AML) is frequently due to the persistence of a cell population resistant to chemotherapy through different mechanisms, in which drug efflux via ATP-binding cassette (ABC) proteins, specifically P-glycoprotein, is one of the most recognized. However, disappointing results from clinical trials employing inhibitors for these transporters have demonstrated the need to adopt different strategies. We hypothesized that microtubule targeting compounds presenting high affinity or covalent binding could overcome the effect of ABC transporters. We therefore evaluated the activity of the high-affinity paclitaxel analog CTX-40 as well as the covalent binder zampanolide (ZMP) in AML cells. Both molecules were active in chemosensitive as well as in chemoresistant cell lines overexpressing P-glycoprotein. Moreover, ZMP or CTX-40 in combination with daunorubicin showed synergistic killing without increased in vitro hematopoietic toxicity. In a primary AML sample, we further demonstrated that ZMP and CTX-40 are active in progenitor and differentiated leukemia cell populations. In sum, our data indicate that high affinity and covalent-binding anti-microtubule agents are active in AML cells otherwise chemotherapy resistant. PMID:26277539

  18. Voltage-sensor conformation shapes the intra-membrane drug binding site that determines gambierol affinity in Kv channels.

    PubMed

    Kopljar, Ivan; Grottesi, Alessandro; de Block, Tessa; Rainier, Jon D; Tytgat, Jan; Labro, Alain J; Snyders, Dirk J

    2016-08-01

    Marine ladder-shaped polyether toxins are implicated in neurological symptoms of fish-borne food poisonings. The toxin gambierol, produced by the marine dinoflagellate Gambierdiscus toxicus, belongs to the group of ladder-shaped polyether toxins and inhibits Kv3.1 channels with nanomolar affinity through a mechanism of gating modification. Binding determinants for gambierol localize at the lipid-exposed interface of the pore forming S5 and S6 segments, suggesting that gambierol binds outside of the permeation pathway. To explore a possible involvement of the voltage-sensing domain (VSD), we made different chimeric channels between Kv3.1 and Kv2.1, exchanging distinct parts of the gating machinery. Our results showed that neither the electro-mechanical coupling nor the S1-S3a region of the VSD affect gambierol sensitivity. In contrast, the S3b-S4 part of the VSD (paddle motif) decreased gambierol sensitivity in Kv3.1 more than 100-fold. Structure determination by homology modeling indicated that the position of the S3b-S4 paddle and its primary structure defines the shape and∖or the accessibility of the binding site for gambierol, explaining the observed differences in gambierol affinity between the channel chimeras. Furthermore, these findings explain the observed difference in gambierol affinity for the closed and open channel configurations of Kv3.1, opening new possibilities for exploring the VSDs as selectivity determinants in drug design. PMID:26956727

  19. Synthesis and Binding Affinity of Novel Mono- and Bivalent Morphinan Ligands for κ, μ and δ Opioid Receptors

    PubMed Central

    Zhang, Bin; Zhang, Tangzhi; Sromek, Anna W.; Scrimale, Thomas; Bidlack, Jean M.

    2011-01-01

    A novel series of homo- and heterodimeric ligands containing κ/μ agonist and μ agonist/antagonist pharmacophores joined by a 10-carbon ester linker chain were synthesized and evaluated for their in vitro binding affinity at κ, μ, and δ opioid receptors and their functional activities were determined at κ and μ receptors in [35S]GTPγS functional assays. Most of these compounds had high binding affinity at μ and κ receptors (Ki values less than 1 nM). Compound 15b, which contains butorphan (1) at one end of linking chain and butorphanol (5) at the other end, was the most potent ligand in this series with binding affinity Ki values of 0.089 nM at the μ receptor and 0.073 nM at the κ receptor. All of the morphinan-derived ligands were found to be partial κ and μ agonists; ATPM-derived ligands 12 and 11 were found to be full κ agonists and partial μ agonists. PMID:21482470

  20. AlGaN/GaN high electron mobility transistors for protein-peptide binding affinity study.

    PubMed

    Huang, Chih-Cheng; Lee, Geng-Yen; Chyi, Jen-Inn; Cheng, Hui-Teng; Hsu, Chen-Pin; Hsu, You-Ren; Hsu, Chia-Hsien; Huang, Yu-Fen; Sun, Yuh-Chang; Chen, Chih-Chen; Li, Sheng-Shian; Yeh, J Andrew; Yao, Da-Jeng; Ren, Fan; Wang, Yu-Lin

    2013-03-15

    Antibody-immobilized AlGaN/GaN high electron mobility transistors (HEMTs) were used to detect a short peptide consisting of 20 amino acids. One-binding-site model and two-binding-site model were used for the analysis of the electrical signals, revealing the number of binding sites on an antibody and the dissociation constants between the antibody and the short peptide. In the binding-site models, the surface coverage ratio of the short peptide on the sensor surface is relevant to the electrical signals resulted from the peptide-antibody binding on the HEMTs. Two binding sites on an antibody were observed and two dissociation constants, 4.404×10(-11) M and 1.596×10(-9) M, were extracted from the binding-site model through the analysis of the surface coverage ratio of the short peptide on the sensor surface. We have also shown that the conventional method to extract the dissociation constant from the linear regression of curve-fitting with Langmuir isotherm equation may lead to an incorrect information if the receptor has more than one binding site for the ligand. The limit of detection (LOD) of the sensor observed in the experimental result (~10 pM of the short peptide) is very close to the LOD (around 2.7-3.4 pM) predicted from the value of the smallest dissociation constants. The sensitivity of the sensor is not only dependent on the transistors, but also highly relies on the affinity of the ligand-receptor pair. The results demonstrate that the AlGaN/GaN HEMTs cannot only be used for biosensors, but also for the biological affinity study.

  1. Nucleotide binding by the widespread high-affinity cyclic di-GMP receptor MshEN domain

    PubMed Central

    Wang, Yu-Chuan; Chin, Ko-Hsin; Tu, Zhi-Le; He, Jin; Jones, Christopher J.; Sanchez, David Zamorano; Yildiz, Fitnat H.; Galperin, Michael Y.; Chou, Shan-Ho

    2016-01-01

    C-di-GMP is a bacterial second messenger regulating various cellular functions. Many bacteria contain c-di-GMP-metabolizing enzymes but lack known c-di-GMP receptors. Recently, two MshE-type ATPases associated with bacterial type II secretion system and type IV pilus formation were shown to specifically bind c-di-GMP. Here we report crystal structure of the MshE N-terminal domain (MshEN1-145) from Vibrio cholerae in complex with c-di-GMP at a 1.37 Å resolution. This structure reveals a unique c-di-GMP-binding mode, featuring a tandem array of two highly conserved binding motifs, each comprising a 24-residue sequence RLGxx(L/V/I)(L/V/I)xxG(L/V/I)(L/V/I)xxxxLxxxLxxQ that binds half of the c-di-GMP molecule, primarily through hydrophobic interactions. Mutating these highly conserved residues markedly reduces c-di-GMP binding and biofilm formation by V. cholerae. This c-di-GMP-binding motif is present in diverse bacterial proteins exhibiting binding affinities ranging from 0.5 μM to as low as 14 nM. The MshEN domain contains the longest nucleotide-binding motif reported to date. PMID:27578558

  2. Nucleotide binding by the widespread high-affinity cyclic di-GMP receptor MshEN domain.

    PubMed

    Wang, Yu-Chuan; Chin, Ko-Hsin; Tu, Zhi-Le; He, Jin; Jones, Christopher J; Sanchez, David Zamorano; Yildiz, Fitnat H; Galperin, Michael Y; Chou, Shan-Ho

    2016-01-01

    C-di-GMP is a bacterial second messenger regulating various cellular functions. Many bacteria contain c-di-GMP-metabolizing enzymes but lack known c-di-GMP receptors. Recently, two MshE-type ATPases associated with bacterial type II secretion system and type IV pilus formation were shown to specifically bind c-di-GMP. Here we report crystal structure of the MshE N-terminal domain (MshEN1-145) from Vibrio cholerae in complex with c-di-GMP at a 1.37 Å resolution. This structure reveals a unique c-di-GMP-binding mode, featuring a tandem array of two highly conserved binding motifs, each comprising a 24-residue sequence RLGxx(L/V/I)(L/V/I)xxG(L/V/I)(L/V/I)xxxxLxxxLxxQ that binds half of the c-di-GMP molecule, primarily through hydrophobic interactions. Mutating these highly conserved residues markedly reduces c-di-GMP binding and biofilm formation by V. cholerae. This c-di-GMP-binding motif is present in diverse bacterial proteins exhibiting binding affinities ranging from 0.5 μM to as low as 14 nM. The MshEN domain contains the longest nucleotide-binding motif reported to date. PMID:27578558

  3. Nucleotide binding by the widespread high-affinity cyclic di-GMP receptor MshEN domain.

    PubMed

    Wang, Yu-Chuan; Chin, Ko-Hsin; Tu, Zhi-Le; He, Jin; Jones, Christopher J; Sanchez, David Zamorano; Yildiz, Fitnat H; Galperin, Michael Y; Chou, Shan-Ho

    2016-01-01

    C-di-GMP is a bacterial second messenger regulating various cellular functions. Many bacteria contain c-di-GMP-metabolizing enzymes but lack known c-di-GMP receptors. Recently, two MshE-type ATPases associated with bacterial type II secretion system and type IV pilus formation were shown to specifically bind c-di-GMP. Here we report crystal structure of the MshE N-terminal domain (MshEN1-145) from Vibrio cholerae in complex with c-di-GMP at a 1.37 Å resolution. This structure reveals a unique c-di-GMP-binding mode, featuring a tandem array of two highly conserved binding motifs, each comprising a 24-residue sequence RLGxx(L/V/I)(L/V/I)xxG(L/V/I)(L/V/I)xxxxLxxxLxxQ that binds half of the c-di-GMP molecule, primarily through hydrophobic interactions. Mutating these highly conserved residues markedly reduces c-di-GMP binding and biofilm formation by V. cholerae. This c-di-GMP-binding motif is present in diverse bacterial proteins exhibiting binding affinities ranging from 0.5 μM to as low as 14 nM. The MshEN domain contains the longest nucleotide-binding motif reported to date.

  4. A New Method for Navigating Optimal Direction for Pulling Ligand from Binding Pocket: Application to Ranking Binding Affinity by Steered Molecular Dynamics.

    PubMed

    Vuong, Quan Van; Nguyen, Tin Trung; Li, Mai Suan

    2015-12-28

    In this paper we present a new method for finding the optimal path for pulling a ligand from the binding pocket using steered molecular dynamics (SMD). Scoring function is defined as the steric hindrance caused by a receptor to ligand movement. Then the optimal path corresponds to the minimum of this scoring function. We call the new method MSH (Minimal Steric Hindrance). Contrary to existing navigation methods, our approach takes into account the geometry of the ligand while other methods including CAVER only consider the ligand as a sphere with a given radius. Using three different target + receptor sets, we have shown that the rupture force Fmax and nonequilibrium work Wpull obtained based on the MSH method show a much higher correlation with experimental data on binding free energies compared to CAVER. Furthermore, Wpull was found to be a better indicator for binding affinity than Fmax. Thus, the new MSH method is a reliable tool for obtaining the best direction for ligand exiting from the binding site. Its combination with the standard SMD technique can provide reasonable results for ranking binding affinities using Wpull as a scoring function. PMID:26595261

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

    SciTech Connect

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

    2010-04-07

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

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

    PubMed

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

    2010-03-16

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

  7. Biochemical characterization of ThiT from Lactococcus lactis: a thiamin transporter with picomolar substrate binding affinity.

    PubMed

    Erkens, Guus B; Slotboom, Dirk Jan

    2010-04-13

    The putative thiamin transporter ThiT from Lactococcus lactis was overproduced in the membrane of lactococcal cells. In vivo transport assays using radiolabeled thiamin demonstrated that ThiT indeed was involved in thiamin transport. The protein was solubilized from the membranes and purified in detergent solution. Size exclusion chromatography coupled to static light scattering, refractive index, and UV absorbance measurements (SEC-MALLS) showed that ThiT is a monomer of 22.7 kDa in detergent solution. When the cells overexpressing ThiT had been cultivated in complex growth medium, all binding sites of the purified protein were occupied with substrate, which had copurified with the protein. MALDI-TOF mass spectrometry analysis confirmed that the copurified substance was thiamin. Substrate-depleted ThiT was obtained by expressing the protein in cells that were cultivated in chemically defined growth medium without thiamin. The intrinsic tryptophan fluorescence of substrate-depleted ThiT was strongly quenched upon thiamin binding. The quenching of the fluorescence was used to determine dissociation constants for thiamin and related compounds. ThiT had an unusually high affinity for thiamin (K(D) = 122 +/- 13 pM) and bound the substrate with a 1:1 (protein:ligand) stoichiometry. TPP, TMP, and pyrithiamin bound to ThiT with nanomolar affinity. A multiple sequence alignment of ThiT homologues revealed that well-conserved residues were clustered in a tryptophan-rich stretch comprising the loop between the predicted membrane spanning segments 5 and 6. Mutational analysis of the conserved residues in this region combined with binding assays of thiamin and related compounds was used to build a model of the high-affinity binding site. The model was compared with thiamin binding sites of other proteins and interpreted in terms of the transport mechanism.

  8. Fluorescence Quenching Property of C-Phycocyanin from Spirulina platensis and its Binding Efficacy with Viable Cell Components.

    PubMed

    Paswan, Meenakshi B; Chudasama, Meghna M; Mitra, Madhusree; Bhayani, Khushbu; George, Basil; Chatterjee, Shruti; Mishra, Sandhya

    2016-03-01

    Phycocyanin is a natural brilliant blue colored, fluorescent protein, which is commonly present in cyanobacteria. In this study, C-phycocyanin was extracted and purified from Spirulina platensis, which are multicellular and filamentous cyanobacteria of greater importance because of its various biological and pharmacological potential. It was analyzed for its binding affinity towards blood cells, algal cells, genomic DNA of microalgae, and bacteria at different temperature and incubation time. It showed good binding affinity with these components even at low concentration of 2.5 μM. The purpose of this study was to evaluate the applicability of C-phycocyanin as a green fluorescent dye substituting carcinogenic chemical dyes. PMID:26678758

  9. A likelihood-based index of protein protein binding affinities with application to influenza HA escape from antibodies.

    PubMed

    Watabe, Teruaki; Kishino, Hirohisa; de Oliveira Martins, Leonardo; Kitazoe, Yasuhiro

    2007-08-01

    In many biological systems, proteins interact with other organic molecules to produce indispensable functions, in which molecular recognition phenomena are essential. Proteins have kept or gained their functions during molecular evolution. Their functions seem to be flexible, and a few amino acid substitutions sometimes cause drastic changes in function. In order to monitor and predict such drastic changes in the early stages in target populations, we need to identify patterns of structural changes during molecular evolution causing decreases or increases in the binding affinity of protein complexes. In previous work, we developed a likelihood-based index to quantify the degree to which a sequence fits a given structure. This index was named the sequence-structure fitness (SSF) and is calculated empirically based on amino acid preferences and pairwise interactions in the structural environment present in template structures. In the present work, we used the SSF to develop an index to measure the binding affinity of protein-protein complexes defined as the log likelihood ratio, contrasting the fitness of the sequences to the structure of the complex and that of the uncomplexed proteins. We applied the developed index to the complexes formed between influenza A hemagglutinin (HA) and four antibodies. The antibody-antigen binding region of HA is under strong selection pressure by the host immune system. Hence, examination of the long-term adaptation of HA to the four antibodies could reveal the strategy of the molecular evolution of HA. Two antibodies cover the HA receptor-binding region, while the other two bind away from the receptor-binding region. By focusing on branches with a significant decline in binding ability, we could detect key amino acid replacements and investigate the mechanism via conditional probabilities. The contrast between the adaptations to the two types of antibodies suggests that the virus adapts to the immune system at the cost of structural

  10. Structural mechanisms underlying sequence-dependent variations in GAG affinities of decorin binding protein A, a Borrelia burgdorferi adhesin.

    PubMed

    Morgan, Ashli M; Wang, Xu

    2015-05-01

    Decorin-binding protein A (DBPA) is an important surface adhesin of the bacterium Borrelia burgdorferi, the causative agent of Lyme disease. DBPA facilitates the bacteria's colonization of human tissue by adhering to glycosaminoglycan (GAG), a sulfated polysaccharide. Interestingly, DBPA sequence variation among different strains of Borrelia spirochetes is high, resulting in significant differences in their GAG affinities. However, the structural mechanisms contributing to these differences are unknown. We determined the solution structures of DBPAs from strain N40 of B. burgdorferi and strain PBr of Borrelia garinii, two DBPA variants whose GAG affinities deviate significantly from strain B31, the best characterized version of DBPA. Our structures revealed that significant differences exist between PBr DBPA and B31/N40 DBPAs. In particular, the C-terminus of PBr DBPA, unlike C-termini from B31 and N40 DBPAs, is positioned away from the GAG-binding pocket and the linker between helices one and two of PBr DBPA is highly structured and retracted from the GAG-binding pocket. The repositioning of the C-terminus allowed the formation of an extra GAG-binding epitope in PBr DBPA and the retracted linker gave GAG ligands more access to the GAG-binding epitopes than other DBPAs. Characterization of GAG ligands' interactions with wild-type (WT) PBr and mutants confirmed the importance of the second major GAG-binding epitope and established the fact that the two epitopes are independent of one another and the new epitope is as important to GAG binding as the traditional epitope.

  11. Identification of high-affinity VEGFR3-binding peptides through a phage-displayed random peptide library

    PubMed Central

    Wu, Yan; Li, Cai-Yun

    2015-01-01

    Objective Vascular endothelial growth factor (VEGF) interaction with its receptor, VEGFR-3/Flt-4, regulates lymphangiogenesis. VEGFR-3/Flt-4 expression in cancer cells has been correlated with clinical stage, lymph node metastasis, and lymphatic invasion. The objective of this study is to identify a VEGFR-3/Flt-4-interacting peptide that could be used to inhibit VEGFR-3 for ovarian cancer therapy. Methods The extracellular fragment of recombinant human VEGFR-3/Flt-4 (rhVEGFR-3/Flt-4) fused with coat protein pIII was screened against a phage-displayed random peptide library. Using affinity enrichment and enzyme-linked immunosorbent assay (ELISA) screening, positive clones of phages were amplified. Three phage clones were selected after four rounds of biopanning, and the specific binding of the peptides to rhVEGFR-3 was detected by ELISA and compared with that of VEGF-D. Immunohistochemistry and immunofluorescence analyses of ovarian cancer tissue sections was undertaken to demonstrate the specificity of the peptides. Results After four rounds of biopanning, ELISA confirmed the specificity of the enriched bound phage clones for rhVEGFR-3. Sequencing and translation identified three different peptides. Non-competitive ELISA revealed that peptides I, II, and III had binding affinities for VEGFR-3 with Kaff (affinity constant) of 16.4±8.6 µg/mL (n=3), 9.2±2.1 µg/mL (n=3), and 174.8±31.1 µg/mL (n=3), respectively. In ovarian carcinoma tissue sections, peptide III (WHWLPNLRHYAS), which had the greatest binding affinity, also co-localized with VEGFR-3 in endothelial cells lining lymphatic vessels; its labeling of ovarian tumors in vivo was also confirmed. Conclusion These finding showed that peptide III has high specificity and activity and, therefore, may represent a potential therapeutic approach to target VEGF-VEGFR-3 signaling for the treatment or diagnosis of ovarian cancer. PMID:26197772

  12. Isolation of the alpha subunits of GTP-binding regulatory proteins by affinity chromatography with immobilized beta gamma subunits.

    PubMed Central

    Pang, I H; Sternweis, P C

    1989-01-01

    Immobilized beta gamma subunits of GTP-binding regulatory proteins (G proteins) were used to isolate alpha subunits from solubilized membranes of bovine tissues and to separate specific alpha subunits based on their differential affinities for beta gamma subunits. The beta gamma subunits were cross-linked to omega-aminobutyl agarose. Up to 7 nmol of alpha subunit could bind to each milliliter of beta gamma-agarose and be recovered by elution with AIF4-. This affinity resin effectively separated the alpha subunits of Gi1 and Gi2 from "contaminating" alpha subunits of Go, the most abundant G protein in bovine brain, by taking advantage of the apparent lower affinity of the alpha subunits of Go for beta gamma subunits. The beta gamma-agarose was also used to isolate mixtures of alpha subunits from cholate extracts of membranes from different bovine tissues. alpha subunits of 39-41 kDa (in various ratios) as well as the alpha subunits of Gs were purified. The yields from extracts exceeded 60% for all alpha subunits examined and apparently represented the relative content of alpha subunits in the tissues. This technique can rapidly isolate and identify, from a small amount of sample, the endogenous G proteins in various tissues and cells. So far, only polypeptides in the range of 39-52 kDa have been detected with this approach. If other GTP-binding proteins interact with these beta gamma subunits, the interaction is either of low affinity or mechanistically unique from the alpha subunits isolated in this study. Images PMID:2510152

  13. Tuning the Binding Affinities and Reversion Kinetics of a Light Inducible Dimer Allows Control of Transmembrane Protein Localization.

    PubMed

    Zimmerman, Seth P; Hallett, Ryan A; Bourke, Ashley M; Bear, James E; Kennedy, Matthew J; Kuhlman, Brian

    2016-09-20

    Inducible dimers are powerful tools for controlling biological processes through colocalizing signaling molecules. To be effective, an inducible system should have a dissociation constant in the "off" state that is greater (i.e., weaker affinity) than the concentrations of the molecules that are being controlled, and in the "on" state a dissociation constant that is less (i.e., stronger affinity) than the relevant protein concentrations. Here, we reengineer the interaction between the light inducible dimer, iLID, and its binding partner SspB, to better control proteins present at high effective concentrations (5-100 μM). iLID contains a light-oxygen-voltage (LOV) domain that undergoes a conformational change upon activation with blue light and exposes a peptide motif, ssrA, that binds to SspB. The new variant of the dimer system contains a single SspB point mutation (A58V), and displays a 42-fold change in binding affinity when activated with blue light (from 3 ± 2 μM to 125 ± 40 μM) and allows for light-activated colocalization of transmembrane proteins in neurons, where a higher affinity switch (0.8-47 μM) was less effective because more colocalization was seen in the dark. Additionally, with a point mutation in the LOV domain (N414L), we lengthened the reversion half-life of iLID. This expanded suite of light induced dimers increases the variety of cellular pathways that can be targeted with light.

  14. Tuning the Binding Affinities and Reversion Kinetics of a Light Inducible Dimer Allows Control of Transmembrane Protein Localization.

    PubMed

    Zimmerman, Seth P; Hallett, Ryan A; Bourke, Ashley M; Bear, James E; Kennedy, Matthew J; Kuhlman, Brian

    2016-09-20

    Inducible dimers are powerful tools for controlling biological processes through colocalizing signaling molecules. To be effective, an inducible system should have a dissociation constant in the "off" state that is greater (i.e., weaker affinity) than the concentrations of the molecules that are being controlled, and in the "on" state a dissociation constant that is less (i.e., stronger affinity) than the relevant protein concentrations. Here, we reengineer the interaction between the light inducible dimer, iLID, and its binding partner SspB, to better control proteins present at high effective concentrations (5-100 μM). iLID contains a light-oxygen-voltage (LOV) domain that undergoes a conformational change upon activation with blue light and exposes a peptide motif, ssrA, that binds to SspB. The new variant of the dimer system contains a single SspB point mutation (A58V), and displays a 42-fold change in binding affinity when activated with blue light (from 3 ± 2 μM to 125 ± 40 μM) and allows for light-activated colocalization of transmembrane proteins in neurons, where a higher affinity switch (0.8-47 μM) was less effective because more colocalization was seen in the dark. Additionally, with a point mutation in the LOV domain (N414L), we lengthened the reversion half-life of iLID. This expanded suite of light induced dimers increases the variety of cellular pathways that can be targeted with light. PMID:27529180

  15. Comparison of Relative Binding Affinities for Trout and Human Estrogen Receptor Based upon Different Competitive Binding Assays, oral

    EPA Science Inventory

    The US EPA has been mandated to screen industrial chemicals and pesticides for potential endocrine activity. To evaluate the potential for chemicals to cause endocrine disruption in fish we have previously measured the affinity of a number of chemicals for the rainbow trout estr...

  16. Peptides in headlock – a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy

    PubMed Central

    Braun, Michael B.; Traenkle, Bjoern; Koch, Philipp A.; Emele, Felix; Weiss, Frederik; Poetz, Oliver; Stehle, Thilo; Rothbauer, Ulrich

    2016-01-01

    Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a β-sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once bound, the peptide is fastened by two nanobody side chains that clamp it in a headlock fashion. Exploiting this unusual binding mode, we generated a novel nanobody-derived capture and detection system. Matrix-coupled nanobody enables the fast and efficient isolation of epitope-tagged proteins from prokaryotic and eukaryotic expression systems. Additionally, the fluorescently labeled nanobody visualizes subcellular structures in different cellular compartments. The high-affinity-binding and modifiable peptide tag of this system renders it a versatile and robust tool to combine biochemical analysis with microscopic studies. PMID:26791954

  17. A soluble, high-affinity, interleukin-4-binding protein is present in the biological fluids of mice

    SciTech Connect

    Fernandez-Botran, R.; Vitetta, E.S. )

    1990-06-01

    Cytokines such as interleukin 4 (IL-4) play a key role in the regulation of immune responses, but little is known about how their multiple activities are regulated in vivo. In this report, we demonstrate that an IL-4-binding protein (IL-4BP) is constitutively present in the biological fluids of mice (serum, ascites fluid, and urine). Binding of {sup 125}I-labeled IL-4 to the IL-4BP is specific and saturable and can be inhibited by an excess of unlabeled IL-4 but not IL-2. The IL-4BP binds IL-4 with an affinity similar to that reported for the cellular IL-4 with an affinity similar to that reported for the cellular IL-4 receptor (K{sub d} {approx}7 {times} 10{sup {minus}11} M) and has a molecular mass of 30-40 kDa and pI values of 3.6-4.8. IL-4BP-containing biological fluids or purified IL-4BP competitively inhibit the binding of {sup 125}I-labeled IL-4 to mouse T or B cells and inhibit the biological activity of IL-4 but not IL-2. The serum levels of IL-4BP in severe combined immunodeficiency (SCID) mice are lower than those of normal mice. The above findings suggest that IL-4BP plays an important immunoregulatory role in vivo.

  18. Binding stoichiometry and affinity of the manganese-stabilizing protein affects redox reactions on the oxidizing side of photosystem II.

    PubMed

    Roose, Johnna L; Yocum, Charles F; Popelkova, Hana

    2011-07-12

    It has been reported previously that the two subunits of PsbO, the photosystem II (PSII) manganese stabilizing protein, have unique functions in relation to the Mn, Ca(2+), and Cl(-) cofactors in eukaryotic PSII [Popelkova; (2008) Biochemistry 47, 12593]. The experiments reported here utilize a set of N-terminal truncation mutants of PsbO, which exhibit altered subunit binding to PSII, to further characterize its role in establishing efficient O(2) evolution activity. The effects of PsbO binding stoichiometry, affinity, and specificity on Q(A)(-) reoxidation kinetics after a single turnover flash, S-state transitions, and O(2) release time have been examined. The data presented here show that weak rebinding of a single PsbO subunit to PsbO-depleted PSII repairs many of the defects in PSII resulting from the removal of the protein, but many of these are not sustainable, as indicated by low steady-state activities of the reconstituted samples [Popelkova; (2003) Biochemistry 42 , 6193]. High affinity binding of PsbO to PSII is required to produce more stable and efficient cycling of the water oxidation reaction. Reconstitution of the second PsbO subunit is needed to further optimize redox reactions on the PSII oxidizing side. Native PsbO and recombinant wild-type PsbO from spinach facilitate PSII redox reactions in a very similar manner, and nonspecific binding of PsbO to PSII has no significance in these reactions.

  19. 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. PMID:25810363

  20. Demonstration of high-affinity folate binding activity associated with the brush border membranes of rat kidney.

    PubMed Central

    Selhub, J; Rosenberg, I H

    1978-01-01

    Folate binding activity of high affinity was identified in the particulate fractions of rat kidney homogenates. This binding activity cofractionated with alkaline phosphatase and maltase, two brush border membranes markers. With an enriched preparation of brush border membranes, freed of endogenous folate by acid treatment, the binding of [3H]olate was found to be saturable (Kb = 4.2 X 10(-11)M) and rapid. Binding was optimal at pH 6.4-7.7. At neutral pH, competition for binding with [3H]folic acid required 1.45 equivalents of pteroylheptaglutamate, 6.25 equivalents of N5-methyltetrathydrofolate, 29 equivalents of methotrexate, and 125 equivalents of N5-formyltetrahydrofolate. At alkaline pH, N5-methyltetrahydrofolate was as effective a competitor as folic acid. In view of reports that renal tubular reabsorption of folate includes an initial tight binding step, the binding activity associated with the brush border membranes may participate in this process. PMID:28521

  1. Histidine-rich Glycoprotein Binds Fibrin(ogen) with High Affinity and Competes with Thrombin for Binding to the γ′-Chain*

    PubMed Central

    Vu, Trang T.; Stafford, Alan R.; Leslie, Beverly A.; Kim, Paul Y.; Fredenburgh, James C.; Weitz, Jeffrey I.

    2011-01-01

    Histidine-rich glycoprotein (HRG) is an abundant protein that binds fibrinogen and other plasma proteins in a Zn2+-dependent fashion but whose function is unclear. HRG has antimicrobial activity, and its incorporation into fibrin clots facilitates bacterial entrapment and killing and promotes inflammation. Although these findings suggest that HRG contributes to innate immunity and inflammation, little is known about the HRG-fibrin(ogen) interaction. By immunoassay, HRG-fibrinogen complexes were detected in Zn2+-supplemented human plasma, a finding consistent with a high affinity interaction. Surface plasmon resonance determinations support this concept and show that in the presence of Zn2+, HRG binds the predominant γA/γA-fibrinogen and the γ-chain elongated isoform, γA/γ′-fibrinogen, with Kd values of 9 nm. Likewise, 125I-labeled HRG binds γA/γA- or γA/γ′-fibrin clots with similar Kd values when Zn2+ is present. There are multiple HRG binding sites on fibrin(ogen) because HRG binds immobilized fibrinogen fragment D or E and γ′-peptide, an analog of the COOH terminus of the γ′-chain that mediates the high affinity interaction of thrombin with γA/γ′-fibrin. Thrombin competes with HRG for γ′-peptide binding and displaces 125I-HRG from γA/γ′-fibrin clots and vice versa. Taken together, these data suggest that (a) HRG circulates in complex with fibrinogen and that the complex persists upon fibrin formation, and (b) by competing with thrombin for γA/γ′-fibrin binding, HRG may modulate coagulation. Therefore, the HRG-fibrin interaction may provide a novel link between coagulation, innate immunity, and inflammation. PMID:21757718

  2. Histidine-rich glycoprotein binds fibrin(ogen) with high affinity and competes with thrombin for binding to the gamma'-chain.

    PubMed

    Vu, Trang T; Stafford, Alan R; Leslie, Beverly A; Kim, Paul Y; Fredenburgh, James C; Weitz, Jeffrey I

    2011-09-01

    Histidine-rich glycoprotein (HRG) is an abundant protein that binds fibrinogen and other plasma proteins in a Zn(2+)-dependent fashion but whose function is unclear. HRG has antimicrobial activity, and its incorporation into fibrin clots facilitates bacterial entrapment and killing and promotes inflammation. Although these findings suggest that HRG contributes to innate immunity and inflammation, little is known about the HRG-fibrin(ogen) interaction. By immunoassay, HRG-fibrinogen complexes were detected in Zn(2+)-supplemented human plasma, a finding consistent with a high affinity interaction. Surface plasmon resonance determinations support this concept and show that in the presence of Zn(2+), HRG binds the predominant γ(A)/γ(A)-fibrinogen and the γ-chain elongated isoform, γ(A)/γ'-fibrinogen, with K(d) values of 9 nm. Likewise, (125)I-labeled HRG binds γ(A)/γ(A)- or γ(A)/γ'-fibrin clots with similar K(d) values when Zn(2+) is present. There are multiple HRG binding sites on fibrin(ogen) because HRG binds immobilized fibrinogen fragment D or E and γ'-peptide, an analog of the COOH terminus of the γ'-chain that mediates the high affinity interaction of thrombin with γ(A)/γ'-fibrin. Thrombin competes with HRG for γ'-peptide binding and displaces (125)I-HRG from γ(A)/γ'-fibrin clots and vice versa. Taken together, these data suggest that (a) HRG circulates in complex with fibrinogen and that the complex persists upon fibrin formation, and (b) by competing with thrombin for γ(A)/γ'-fibrin binding, HRG may modulate coagulation. Therefore, the HRG-fibrin interaction may provide a novel link between coagulation, innate immunity, and inflammation.

  3. A photo-cleavable biotin affinity tag for the facile release of a photo-crosslinked carbohydrate-binding protein.

    PubMed

    Chang, Tsung-Che; Adak, Avijit K; Lin, Ting-Wei; Li, Pei-Jhen; Chen, Yi-Ju; Lai, Chain-Hui; Liang, Chien-Fu; Chen, Yu-Ju; Lin, Chun-Cheng

    2016-03-15

    The use of photo-crosslinking glycoprobes represents a powerful strategy for the covalent capture of labile protein complexes and allows detailed characterization of carbohydrate-mediated interactions. The selective release of target proteins from solid support is a key step in functional proteomics. We envisaged that light activation can be exploited for releasing labeled protein in a dual photo-affinity probe-based strategy. To investigate this possibility, we designed a trifunctional, galactose-based, multivalent glycoprobe for affinity labeling of carbohydrate-binding proteins. The resulting covalent protein-probe adduct is attached to a photo-cleavable biotin affinity tag; the biotin moiety enables specific presentation of the conjugate on streptavidin-coated beads, and the photolabile linker allows the release of the labeled proteins. This dual probe promotes both the labeling and the facile cleavage of the target protein complexes from the solid surfaces and the remainder of the cell lysate in a completely unaltered form, thus eliminating many of the common pitfalls associated with traditional affinity-based purification methods.

  4. Affinity selection of chemically modified proteins: role of lysyl residues in the binding of calmodulin to calcineurin

    SciTech Connect

    Manalan, A.S.; Klee, C.B.

    1987-03-10

    In affinity selection, calcineurin selects from a population of randomly modified calmodulins those species with which it prefers to interact. The method shows that acetylation of lysines affects calmodulin so as to interfere with its ability to interact with calcineurin. Monoacetylation of any lysine of calmodulin reduces its affinity for calcineurin by 5-10-fold. Multiple acetylations amplify the loss of affinity; none of the modifications are incompatible with activity. The lack of selective of calcineurin against any particular modified lysine indicates that the loss of affinity reflects changes induced by the removal of the charged groups and suggests an important role for electrostatic interactions in the cooperative structural transitions which calmodulin undergoes upon binding its target proteins or calcium. In the presence of calcineurin, a large and specific decrease in the rate of acetylation of Lys-75 and -148 of calmodulin is observed. The reactivity of the same residues is greatly increased in the presence of calcium alone. Their reactivity changes in opposite directions in response to calcium-induced or calcineurin-induced structural changes. The reactivity of other residues such as Lys-21, decreased in the presence of calcineurin but not calcium, is also affected by a conformational change which is induced specifically by calcineurin. Radiolabelled calmodulin was purified by HPLC.

  5. Focused pseudostatic hydrazone libraries screened by mass spectrometry binding assay: optimizing affinities toward γ-aminobutyric acid transporter 1.

    PubMed

    Sindelar, Miriam; Lutz, Toni A; Petrera, Marilena; Wanner, Klaus T

    2013-02-14

    Mass spectrometric (MS) binding assays, a powerful tool to determine affinities of single drug candidates toward chosen targets, were recently demonstrated to be suitable for the screening of compound libraries generated with reactions of dynamic combinatorial chemistry when rendering libraries pseudostatic. Screening of small hydrazone libraries targeting γ-aminobutyric acid transporter 1 (GAT1), the most abundant γ-aminobutyric acid (GABA) transporter in the central nervous system, revealed two nipecotic acid derived binders with submicromolar affinities. Starting from the biphenyl carrying hit as lead structure, the objective of the present study was to discover novel high affinity GAT1 binders by screening of biphenyl focused pseudostatic hydrazone libraries formed from hydrazine 10 and 36 biphenylcarbaldehydes 11c-al. Hydrazone 12z that carried a 2',4'-dichlorobiphenyl residue was found to be the most potent binder with low nanomolar affinity (pK(i) = 8.094 ± 0.098). When stable carba analogues of representative hydrazones were synthesized and evaluated, the best binder 13z was again displaying the 2',4'-dichlorobiphenyl moiety (pK(i) = 6.930 ± 0.021).

  6. Effects of Water Models on Binding Affinity: Evidence from All-Atom Simulation of Binding of Tamiflu to A/H5N1 Neuraminidase

    PubMed Central

    Nguyen, Trang Truc; Viet, Man Hoang

    2014-01-01

    The influence of water models SPC, SPC/E, TIP3P, and TIP4P on ligand binding affinity is examined by calculating the binding free energy ΔGbind of oseltamivir carboxylate (Tamiflu) to the wild type of glycoprotein neuraminidase from the pandemic A/H5N1 virus. ΔGbind is estimated by the Molecular Mechanic-Poisson Boltzmann Surface Area method and all-atom simulations with different combinations of these aqueous models and four force fields AMBER99SB, CHARMM27, GROMOS96 43a1, and OPLS-AA/L. It is shown that there is no correlation between the binding free energy and the water density in the binding pocket in CHARMM. However, for three remaining force fields ΔGbind decays with increase of water density. SPC/E provides the lowest binding free energy for any force field, while the water effect is the most pronounced in CHARMM. In agreement with the popular GROMACS recommendation, the binding score obtained by combinations of AMBER-TIP3P, OPLS-TIP4P, and GROMOS-SPC is the most relevant to the experiments. For wild-type neuraminidase we have found that SPC is more suitable for CHARMM than TIP3P recommended by GROMACS for studying ligand binding. However, our study for three of its mutants reveals that TIP3P is presumably the best choice for CHARMM. PMID:24672329

  7. Low density and high affinity of platelet [3H]paroxetine binding in women with bulimia nervosa.

    PubMed

    Ekman, Agneta; Sundblad-Elverfors, Charlotta; Landén, Mikael; Eriksson, Tomas; Eriksson, Elias

    2006-06-15

    Impaired serotonin transmission has been suggested to be implicated in the pathophysiology of bulimia nervosa. As an indirect measure of brain serotonergic activity, the binding of tritiated ligands to platelet serotonin transporters has been studied in bulimia nervosa as well as in other putatively serotonin-related psychiatric disorders. In this study, the density and affinity of platelet serotonin transporters were assessed in 20 women meeting the DSM-IV criteria for bulimia nervosa and in 14 controls without previous or ongoing eating disorder using [(3)H]paroxetine as a ligand. In comparison to controls, women with bulimia nervosa had a significantly reduced number of platelet binding sites (B(max) = 721 +/- 313 vs. 1145 +/- 293 fmol/mg protein) and an increase in the affinity for the ligand demonstrated by a lower dissociaton constant (K(d) = 33 +/- 10 vs. 44 +/- 10 pM). A significant correlation between B(max) and K(d) values was found in patients but not in controls. Our results support the notion that bulimia nervosa is associated with a reduction in platelet serotonin transporter density. In addition, our study is the first to report that this reduced transporter density in women with bulimia nervosa is accompanied by an increase in the affinity of the transporter for the ligand.

  8. DNA affinity labeling of adenovirus type 2 upstream promoter sequence-binding factors identifies two distinct proteins

    SciTech Connect

    Safer, B.; Cohen, R.B.; Garfinkel, S.; Thompson, J.A.

    1988-01-01

    A rapid affinity labeling procedure with enhanced specificity was developed to identify DNA-binding proteins. /sup 32/P was first introduced at unique phosphodiester bonds within the DNA recognition sequence. UV light-dependent cross-linking of pyrimidines to amino acid residues in direct contact at the binding site, followed by micrococcal nuclease digestion, resulted in the transfer of /sup 32/P to only those specific protein(s) which recognized the binding sequence. This method was applied to the detection and characterization of proteins that bound to the upstream promoter sequence (-50 to -66) of the human adenovirus type 2 major late promoter. We detected two distinct proteins with molecular weights of 45,000 and 116,000 that interacted with this promoter element. The two proteins differed significantly in their chromatographic and cross-linking behaviors.

  9. Affinity binding-guided fluorescent nanobiosensor for acetylcholinesterase inhibitors via distance modulation between the fluorophore and metallic nanoparticle.

    PubMed

    Zhang, Yaodong; Hei, Tingting; Cai, Yanan; Gao, Qunqun; Zhang, Qi

    2012-03-20

    The magnitude of fluorescence enhancement was found to depend strongly on the distance between fluorophores and metal nanostructures in metal-enhanced fluorescence (MEF). However, the precise placement of the particle in front of the molecule with nanometer accuracy and distance control is a great challenge. We describe a method using acetylcholinesterase (AChE) to modulate the distance between a gold nanoparticle (AuNP) and the fluorophore 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO). We found that DDAO is a reversible mixed type-I AChE inhibitor. DDAO binds to the peripheral anionic site and penetrates into the active gorge site of AChE via inhibition kinetics test and molecular docking study. The affinity ligand DDAO bound to AChE which was immobilized onto AuNPs, and its fluorescence was sharply enhanced due to MEF. The fluorescence was reduced by distance variations between the AuNP and DDAO, which resulted from other inhibitors competitively binding with AChE and partly or completely displacing DDAO. Experimental results show that changes in fluorescence intensity are related to the concentration of inhibitors present in the solution. In addition, the nanobiosensor has high sensitivity, with detection limits as low as 0.4 μM for paraoxon and 10 nM for tacrine, and also exhibits different reduction efficiencies for the two types of inhibitor. Thus, instead of an inhibition test, a new type of affinity binding-guided fluorescent nanobiosensor was fabricated to detect AChE inhibitors, determine AChE inhibitor binding mode, and screen more potent AChE inhibitors. The proposed strategy may be applied to other proteins or protein domains via changes in the affinity ligand.

  10. Elucidation of binding specificity of Jacalin toward O-glycosylated peptides: quantitative analysis by frontal affinity chromatography.

    PubMed

    Tachibana, Kouichi; Nakamura, Sachiko; Wang, Han; Iwasaki, Hiroko; Tachibana, Kahori; Maebara, Kanako; Cheng, Lamei; Hirabayashi, J; Narimatsu, H

    2006-01-01

    Jacalin, a lectin from the jackfruit Artocarpus integrifolia, has been known as a valuable tool for specific capturing of O-glycoproteins such as mucins and IgA1. Though its sugar-binding preference for T/Tn-antigens is well established, its detailed specificity has not been elucidated. In this study, we prepared a series of mucin-type glycopeptides using human glycosyltransferases, that is, ST6GalNAc1, Core1Gal-T1 and -T2, beta3Gn-T6, and Core2GnT1, and investigated their binding to immobilized Jacalin by frontal affinity chromatography (FAC). As a result, consistent with the previous observation, Jacalin showed high affinity for T-antigen (Core1) and Tn-antigen (alpha N-acetylgalactosamine)-attached peptides. Furthermore, we here show as novel findings that (1) Jacalin also showed significant affinity for Core3 and sialyl-T (ST)-attached peptides, but (2) Jacalin could not bind to Core2, Core6, and sialyl-Tn (STn)-attached peptides. The results were also confirmed by FAC using p-nitrophenyl (pNP)-derivatized saccharides. In conclusion, Jacalin binds to a GalNAcalpha1-peptide, in which C6-OH of alphaGalNAc is free (i.e., Core1, Tn, Core3, and ST), whereas it cannot recognize a GalNAcalpha1-peptide with a substitution at the C6 position (i.e., Core2, Core6, and STn). These findings provide useful information when applying jacalin for functional analysis of mucin-type glycoproteins and glycopeptides.

  11. Photoaffinity Labeling of High Affinity Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-Binding Proteins in Sea Urchin Egg*♦

    PubMed Central

    Walseth, Timothy F.; Lin-Moshier, Yaping; Jain, Pooja; Ruas, Margarida; Parrington, John; Galione, Antony; Marchant, Jonathan S.; Slama, James T.

    2012-01-01

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger that regulates calcium release from intracellular acidic stores. Recent studies have identified two-pore channels (TPCs) as endolysosomal channels that are regulated by NAADP; however, the nature of the NAADP receptor binding site is unknown. To further study NAADP binding sites, we have synthesized and characterized [32P-5-azido]nicotinic acid adenine dinucleotide phosphate ([32P-5N3]NAADP) as a photoaffinity probe. Photolysis of sea urchin egg homogenates preincubated with [32P-5N3]NAADP resulted in specific labeling of 45-, 40-, and 30-kDa proteins, which was prevented by inclusion of nanomolar concentrations of unlabeled NAADP or 5N3-NAADP, but not by micromolar concentrations of structurally related nucleotides such as NAD, nicotinic acid adenine dinucleotide, nicotinamide mononucleotide, nicotinic acid, or nicotinamide. [32P-5N3]NAADP binding was saturable and displayed high affinity (Kd ∼10 nm) in both binding and photolabeling experiments. [32P-5N3]NAADP photolabeling was irreversible in a high K+ buffer, a hallmark feature of NAADP binding in the egg system. The proteins photolabeled by [32P-5N3]NAADP have molecular masses smaller than the sea urchin TPCs, and antibodies to TPCs do not detect any immunoreactivity that comigrates with either the 45-kDa or the 40-kDa photolabeled proteins. Interestingly, antibodies to TPC1 and TPC3 were able to immunoprecipitate a small fraction of the 45- and 40-kDa photolabeled proteins, suggesting that these proteins associate with TPCs. These data suggest that high affinity NAADP binding sites are distinct from TPCs. PMID:22117077

  12. Novel thermo-responsive fucose binding ligands for glycoprotein purification by affinity precipitation.

    PubMed

    Arnold, Lindsay; Chen, Rachel

    2014-02-01

    Novel thermo-responsive affinity sugar binders were developed by fusing a bacterial fucose lectin with a thermo-responsive polypeptide. These designer affinity ligand fusions were produced using an Escherichia coli system capable of extracellular secretion of recombinant proteins and were isolated with a high recovery yield (95%) directly from growth medium by Inverse Temperature Cycling (ITC). With horse radish peroxidase (HRP) as a model protein, we demonstrate here that the designer thermo-responsive ligands are capable of interacting with glycans on a glycoprotein, a property that was used to develop a novel affinity precipitation method for glycoprotein purification. The method, requiring only simple process steps, affords full recovery of a target glycoprotein, and is effective at a target glycoprotein concentration as low as 1.4 pM in the presence of large amounts of contaminants. By developing other sugar binders in the similar fashion, the method should be highly useful for glycoprotein purification and detection.

  13. Novel thermo-responsive fucose binding ligands for glycoprotein purification by affinity precipitation.

    PubMed

    Arnold, Lindsay; Chen, Rachel

    2014-02-01

    Novel thermo-responsive affinity sugar binders were developed by fusing a bacterial fucose lectin with a thermo-responsive polypeptide. These designer affinity ligand fusions were produced using an Escherichia coli system capable of extracellular secretion of recombinant proteins and were isolated with a high recovery yield (95%) directly from growth medium by Inverse Temperature Cycling (ITC). With horse radish peroxidase (HRP) as a model protein, we demonstrate here that the designer thermo-responsive ligands are capable of interacting with glycans on a glycoprotein, a property that was used to develop a novel affinity precipitation method for glycoprotein purification. The method, requiring only simple process steps, affords full recovery of a target glycoprotein, and is effective at a target glycoprotein concentration as low as 1.4 pM in the presence of large amounts of contaminants. By developing other sugar binders in the similar fashion, the method should be highly useful for glycoprotein purification and detection. PMID:25271333

  14. High affinity neurexin binding to cell adhesion G-protein-coupled receptor CIRL1/latrophilin-1 produces an intercellular adhesion complex.

    PubMed

    Boucard, Antony A; Ko, Jaewon; Südhof, Thomas C

    2012-03-16

    The G-protein-coupled receptor CIRL1/latrophilin-1 (CL1) and the type-1 membrane proteins neurexins represent distinct neuronal cell adhesion molecules that exhibit no similarities except for one common function: both proteins are receptors for α-latrotoxin, a component of black widow spider venom that induces massive neurotransmitter release at synapses. Unexpectedly, we have now identified a direct binding interaction between the extracellular domains of CL1 and neurexins that is regulated by alternative splicing of neurexins at splice site 4 (SS4). Using saturation binding assays, we showed that neurexins lacking an insert at SS4 bind to CL1 with nanomolar affinity, whereas neurexins containing an insert at SS4 are unable to bind. CL1 competed for neurexin binding with neuroligin-1, a well characterized neurexin ligand. The extracellular sequences of CL1 contain five domains (lectin, olfactomedin-like, serine/threonine-rich, hormone-binding, and G-protein-coupled receptor autoproteolysis-inducing (GAIN) domains). Of these domains, the olfactomedin-like domain mediates neurexin binding as shown by deletion mapping. Cell adhesion assays using cells expressing neurexins and CL1 revealed that their interaction produces a stable intercellular adhesion complex, indicating that their interaction can be trans-cellular. Thus, our data suggest that CL1 constitutes a novel ligand for neurexins that may be localized postsynaptically based on its well characterized interaction with intracellular SH3 and multiple ankyrin repeats adaptor proteins (SHANK) and could form a trans-synaptic complex with presynaptic neurexins.

  15. The estimation of affinity constants for the binding of model peptides to DNA by equilibrium dialysis.

    PubMed Central

    Standke, K C; Brunnert, H

    1975-01-01

    The binding of lysine model peptides of the type Lys-X-Lys, Lys-X-X-Lys and Lys-X-X-X-Lys (X = different aliphatic and aromatic amino acids) has been studied by equilibrium dialysis. It was shown that the strong electrostatic binding forces generated by protonated amino groups of lysine can be distinguished from the weak forces stemming from neutral and aromatic spacer amino acids. The overall binding strength of the lysine model peptides is modified by these weak binding forces and the apparent binding constants are influenced more by the hydrophobic character of the spacer amino acid side chains than by the chainlength of the spacers. PMID:1187347

  16. The quantitative prediction of HLA-B*2705 peptide binding affinities using Support Vector Regression to gain insights into its role for the Spondyloarthropathies.

    PubMed

    Uslan, Volkan; Seker, Huseyin

    2015-01-01

    Computational methods are increasingly utilised in many immunoinformatics problems such as the prediction of binding affinity of peptides. The peptides could provide valuable insight into the drug design and development such as vaccines. Moreover, they can be used to diagnose diseases. The presence of human class I MHC allele HLA-B*2705 is one of the strong hypothesis that would lead spondyloarthropathies. In this paper, Support Vector Regression is used in order to predict binding affinity of peptides with the aid of experimentally determined peptide-MHC binding affinities of 222 peptides to HLA-B*2705 to get more insight into this problematic disease. The results yield a high correlation coefficient as much as 0.65 and the SVR-based predictive models can be considered as a useful tool in order to predict the binding affinities for newly discovered peptides.

  17. Computational Assay of H7N9 Influenza Neuraminidase Reveals R292K Mutation Reduces Drug Binding Affinity

    NASA Astrophysics Data System (ADS)

    Woods, Christopher J.; Malaisree, Maturos; Long, Ben; McIntosh-Smith, Simon; Mulholland, Adrian J.

    2013-12-01

    The emergence of a novel H7N9 avian influenza that infects humans is a serious cause for concern. Of the genome sequences of H7N9 neuraminidase available, one contains a substitution of arginine to lysine at position 292, suggesting a potential for reduced drug binding efficacy. We have performed molecular dynamics simulations of oseltamivir, zanamivir and peramivir bound to H7N9, H7N9-R292K, and a structurally related H11N9 neuraminidase. They show that H7N9 neuraminidase is structurally homologous to H11N9, binding the drugs in identical modes. The simulations reveal that the R292K mutation disrupts drug binding in H7N9 in a comparable manner to that observed experimentally for H11N9-R292K. Absolute binding free energy calculations with the WaterSwap method confirm a reduction in binding affinity. This indicates that the efficacy of antiviral drugs against H7N9-R292K will be reduced. Simulations can assist in predicting disruption of binding caused by mutations in neuraminidase, thereby providing a computational `assay.'

  18. Affinity of the enantiomers of. alpha. - and. beta. -cyclazocine for binding to the phencyclidine and. mu. opioid receptors

    SciTech Connect

    Todd, S.L.; Balster, R.L.; Martin, B.R. )

    1990-01-01

    The enantiomers in the {alpha} and {beta} series of cyclazocine were evaluated for their ability to bind to phencyclidine (PCP) and {mu}-opioid receptors in order to determine their receptor selectivity. The affinity of (-)-{beta}-cyclazocine for the PCP receptor was 1.5 greater than PCP itself. In contrast, (-)-{alpha}-cyclazocine, (+)-{alpha}-cyclazocine, and (+)-{beta}-cyclazocine were 3-, 5- and 138-fold less potent than PCP, respectively. Scatchard analysis of saturable binding of ({sup 3}H)Tyr-D-Ala-Gly-N-MePhe-Gly-ol (DAMGO) also exhibited a homogeneous population of binding sites with an apparent K{sub D} of 1.9 nM and an estimated Bmax of 117 pM. (3H)Tyr-D-Ala-Gly-N-MePhe-Gly-ol (DAMGO) binding studies revealed that (-)-{alpha}-cyclazocine (K{sub D} = 0.48 nM) was 31-, 1020- and 12,600-fold more potent than (-)-{beta}-cyclazocine, (+)-{alpha}-cyclazocine and (+)-{beta}-cyclazocine, respectively, for binding to the {mu}-opioid receptor. These data show that, although (-)-{beta}-cyclazocine is a potent PCP receptor ligand consistent with its potent PCP-like discriminative stimulus effects, it shows little selectivity for PCP receptor since it also potently displaces {mu}-opioid binding. However, these cyclazocine isomers, due to their extraordinary degree of stereoselectivity, may be useful in characterizing the structural requirements for benzomorphans having activity at the PCP receptor.

  19. Binding of the bioactive component Aloe dihydroisocoumarin with human serum albumin

    NASA Astrophysics Data System (ADS)

    Zhang, Xiu-Feng; Xie, Ling; Liu, Yang; Xiang, Jun-Feng; Tang, Ya-Lin

    2008-11-01

    Aloe dihydroisocoumarin, one of new components isolated from Aloe vera, can scavenge reactive oxygen species. In order to explore the mechanism of drug action at a molecular level, the binding of Aloe dihydroisocoumarin with human serum albumin (HSA) has been investigated by using fluorescence, ultraviolet (UV), circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy, fluorescence dynamics, and molecular dynamic docking for the first time. We observed a quenching of fluorescence of HSA in the presence of Aloe dihydroisocoumarin and also analyzed the quenching results using the Stern-Volmer equation and obtained high affinity binding to HSA. An isoemissive point at 414 nm is seen, indicating that the quenching of HSA fluorescence depends on the formation of Aloe dihydroisocoumarin-HSA complex, which is further confirmed by fluorescence dynamic result. From the CD and FT-IR results, it is apparent that the interaction of Aloe dihydroisocoumarin with HSA causes a conformational change of the protein, with the gain of α-helix, β-sheet and random coil stability and the loss of β-turn content. Data obtained by fluorescence spectroscopy, fluorescence dynamics, CD, and FTIR experiments along with the docking studies suggest that Aloe dihydroisocoumarin binds to residues located in subdomain IIA of HSA.

  20. [Cell-ELA-based determination of binding affinity of DNA aptamer against U87-EGFRvIII cell].

    PubMed

    Tan, Yan; Liang, Huiyu; Wu, Xidong; Gao, Yubo; Zhang, Xingmei

    2013-05-01

    A15, a DNA aptamer with binding specificity for U87 glioma cells stably overexpressing the epidermal growth factor receptor variant III (U87-EGFRvIII), was generated by cell systematic evolution of ligands by exponential enrichment (cell-SELEX) using a random nucleotide library. Subsequently, we established a cell enzyme-linked assay (cell-ELA) to detect the affinity of A15 compared to an EGFR antibody. We used A15 as a detection probe and cultured U87-EGFRvIII cells as targets. Our data indicate that the equilibrium dissociation constants (K(d)) for A15 were below 100 nmol/L and had similar affinity compared to an EGFR antibody for U87-EGFRvIII. We demonstrated that the cell-ELA was a useful method to determine the equilibrium dissociation constants (K(d)) of aptamers generated by cell-SELEX.

  1. Synthesis and dopamine D2-like receptor binding affinity of substituted 5-phenyl-pyrrole-3-carboxamides.

    PubMed

    Pinna, G A; Curzu, M M; Sechi, M; Chelucci, G; Maciocco, E

    1999-08-30

    A series of 5-p-substituted phenyl-pyrrole-3-carboxamide derivatives was designed as hybrid analogs of the dopamine D2-like 5-phenyl-pyrrole and heterocyclic carboxamide antipsychotics. The title compounds were synthesized and evaluated for dopamine D2-like receptor by means of [3H]YM-09151-2 receptor binding assay. The compound bearing a 1-ethyl-2-methyl-pyrrolidine moiety as the basic part of 5-phenyl-pyrrole-3-carboxamide derivative 1a together with its 2-chloro analog 1f were found to possess affinity in the low micromolar range. Substituted phenyl-pyrrolecarboxamides containing groups such as F, Cl, NO2, CH3, at the 4-position of the phenyl ring, gave ligands with lower D2-like affinity.

  2. Isolation of ubiquitinated substrates by tandem affinity purification of E3 ligase-polyubiquitin-binding domain fusions (ligase traps).

    PubMed

    Mark, Kevin G; Loveless, Theresa B; Toczyski, David P

    2016-02-01

    Ubiquitination is an essential protein modification that influences eukaryotic processes ranging from substrate degradation to nonproteolytic pathway alterations, including DNA repair and endocytosis. Previous attempts to analyze substrates via physical association with their respective ubiquitin ligases have had some success. However, because of the transient nature of enzyme-substrate interactions and rapid protein degradation, detection of substrates remains a challenge. Ligase trapping is an affinity purification approach in which ubiquitin ligases are fused to a polyubiquitin-binding domain, which allows the isolation of ubiquitinated substrates. Immunoprecipitation is first used to enrich for proteins that are bound to the ligase trap. Subsequently, affinity purification is used under denaturing conditions to capture proteins conjugated with hexahistidine-tagged ubiquitin. By using this protocol, ubiquitinated substrates that are specific for a given ligase can be isolated for mass spectrometry or western blot analysis. After cells have been collected, the described protocol can be completed in 2-3 d.

  3. Synthesis, characterization and binding affinities of rhenium(I) thiosemicarbazone complexes for the estrogen receptor (α/β).

    PubMed

    Núñez-Montenegro, Ara; Carballo, Rosa; Vázquez-López, Ezequiel M

    2014-11-01

    The binding affinities towards estrogen receptors (ERs) α and β of a set of thiosemicarbazone ligands (HL(n)) and their rhenium(I) carbonyl complexes [ReX(HL(n))(CO)3] (X=Cl, Br) were determined by a competitive standard radiometric assay with [(3)H]-estradiol. The ability of the coordinated thiosemicarbazone ligands to undergo deprotonation and the lability of the ReX bond were used as a synthetic strategy to obtain [Re(hpy)(L(n))(CO)3] (hpy=3- or 4-hydroxypyridine). The inclusion of the additional hpy ligand endows the new thiosemicarbazonate complexes with an improved affinity towards the estrogen receptors and, consequently, the values of the inhibition constant (Ki) could be determined for some of them. In general, the values of Ki for both ER subtypes suggest an appreciable selectivity towards ERα.

  4. A conserved docking site in MEKs mediates high-affinity binding to MAP kinases and cooperates with a scaffold protein to enhance signal transmission.

    PubMed

    Bardwell, A J; Flatauer, L J; Matsukuma, K; Thorner, J; Bardwell, L

    2001-03-30

    The recognition of mitogen-activated protein kinases (MAPKs) by their upstream activators, MAPK/ERK kinases (MEKs), is crucial for the effective and accurate transmission of many signals. We demonstrated previously that the yeast MAPKs Kss1 and Fus3 bind with high affinity to the N terminus of the MEK Ste7, and proposed that a conserved motif in Ste7, the MAPK-docking site, mediates this interaction. Here we show that the corresponding sequences in human MEK1 and MEK2 are necessary and sufficient for the direct binding of the MAPKs ERK1 and ERK2. Mutations in MEK1, MEK2, or Ste7 that altered conserved residues in the docking site diminished binding of the cognate MAPKs. Furthermore, short peptides corresponding to the docking sites in these MEKs inhibited MEK1-mediated phosphorylation of ERK2 in vitro. In yeast cells, docking-defective alleles of Ste7 were modestly compromised in their ability to transmit the mating pheromone signal. This deficiency was dramatically enhanced when the ability of the Ste5 scaffold protein to associate with components of the MAPK cascade was also compromised. Thus, both the MEK-MAPK docking interaction and binding to the Ste5 scaffold make mutually reinforcing contributions to the efficiency of signaling by this MAPK cascade in vivo. PMID:11134045

  5. Affinity chromatographic purification of tetrodotoxin by use of tetrodotoxin-binding high molecular weight substances in the body fluid of shore crab (Hemigrapsus sanguineus) as ligands.

    PubMed

    Shiomi, K; Yamaguchi, S; Shimakura, K; Nagashima, Y; Yamamori, K; Matsui, T

    1993-12-01

    A purification method for tetrodotoxin (TTX), based on affinity chromatography using the TTX-binding high mol. wt substances in the body fluid of shore crab (Hemigrapsus sanguineus) as ligands, was developed. This method was particularly useful for analysis of TTX in biological samples with low concentrations of TTX. The affinity gel prepared was highly specific for TTX, having no ability to bind 4-epi-TTX and anhydro-TTX as well as saxitoxin.

  6. Evidence for monomeric and oligomeric hormone-binding domains in affinity-purified gonadotropin receptor from rat ovary

    SciTech Connect

    Zhang, Q.Y.; Menon, K.M.J. )

    1989-11-01

    Rat ovarian lutropin/choriogonadotropin receptor was purified from a Triton X-100-solubilized membrane preparation by affinity chromatography with Affi-Gel 10 coupled to purified human choriogonadotropin. The affinity-purified receptor preparations contained a single class of high-affinity binding sites for {sup 125}I-labeled human choriogonadotropin, with an equilibrium dissociation constant (K{sub d}) of 2.5 {times} 10{sup {minus}9} M, which is comparable to the K{sub d} values for membrane-bound and solubilized receptors. The purified receptor appeared as two dominant bands with molecular weights of 135,000 and 92,000 after sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) under nonreducing conditions. When the individual affinity-purified receptor bands were electroeluted from the gel and analyzed again by SDS/PAGE under nonreducing conditions, both the M{sub r} 92,000 and the 135,000 proteins retained their original molecular form even when 8 M urea was included in the gel. However, when the electrophoretically purified M{sub r} 92,000 and 135,000 bands were subjected to SDS/PAGE under reducing conditions, the M{sub r} 135,000 species was almost completely converted to a M{sub r} 92,000 band, but the M{sub r} 92,000 species did not undergo any alteration in molecular weight. The results suggest that the lutropin/choriogonadotropin receptor from rat ovary exists in two molecular forms, and the higher molecular weight form appears to be composed of disulfide-linked M{sup r} 92,000 subunit, which comprises the hormone-binding domain.

  7. Affinity-purified tetanus neurotoxin interaction with synaptic membranes: properties of a protease-sensitive receptor component

    SciTech Connect

    Lazarovici, P.; Yavin, E.

    1986-11-04

    The pharmacokinetic interaction of an affinity-purified /sup 125/I-labeled tetanotoxin fraction with guinea pig brain synaptosomal preparations was investigated. Binding of tetanotoxin was time- and temperature-dependent, was proportional to protein concentration, and was saturable at about 8 x 10/sup -9/ M as estimated by a solid-surface binding assay. Binding was optimal at pH 6.5 under low ionic strength buffer and was almost entirely blocked by gangliosides or antitoxin. In analogy to intact nerve cells, binding of toxin to membranes resulted in a tight association operationally defined as sequestration. Binding and sequestration were abolished after membrane pretreatment with sialidase. The enzyme could not dissociate the membrane-bound toxin formed at 4 or 37/sup 0/C under low ionic strength conditions, which is in part compatible with internalization as defined in nerve cell cultures. In the latter system the toxin could be removed at 4/sup 0/C but not at 37/sup 0/C. Binding was significantly reduced upon pretreatment of guinea pig brain membranes by a variety of hydrolytic enzymes. It is proposed that, in addition to a ganglioside, interaction of tetanotoxin with synaptic membranes is facilitated by a protein and may also require an appropriate lipid environment. These latter membrane constituents may play a pivotal role in the sequestration of the toxin.

  8. The Structure of the Amyloid-[beta] Peptide High-Affinity Copper II Binding Site in Alzheimer Disease

    SciTech Connect

    Streltsov, Victor A.; Titmuss, Stephen J.; Epa, V. Chandana; Barnham, Kevin J.; Masters, Colin L.; Varghese, Joseph N.

    2008-11-03

    Neurodegeneration observed in Alzheimer disease (AD) is believed to be related to the toxicity from reactive oxygen species (ROS) produced in the brain by the amyloid-{beta} (A{beta}) protein bound primarily to copper ions. The evidence for an oxidative stress role of A{beta}-Cu redox chemistry is still incomplete. Details of the copper binding site in A{beta} may be critical to the etiology of AD. Here we present the structure determined by combining x-ray absorption spectroscopy (XAS) and density functional theory analysis of A{beta} peptides complexed with Cu{sup 2+} in solution under a range of buffer conditions. Phosphate-buffered saline buffer salt (NaCl) concentration does not affect the high-affinity copper binding mode but alters the second coordination sphere. The XAS spectra for truncated and full-length A{beta}-Cu{sup 2+} peptides are similar. The novel distorted six-coordinated (3N3O) geometry around copper in the A{beta}-Cu{sup 2+} complexes include three histidines: glutamic, or/and aspartic acid, and axial water. The structure of the high-affinity Cu{sup 2+} binding site is consistent with the hypothesis that the redox activity of the metal ion bound to A{beta} can lead to the formation of dityrosine-linked dimers found in AD.

  9. Using three-dimensional quantitative structure-activity relationships to examine estrogen receptor binding affinities of polychlorinated hydroxybiphenyls

    SciTech Connect

    Waller, C.L.; Minor, D.L.; McKinney, J.D.

    1995-07-01

    Certain phenyl-substituted hydrocarbons of environmental concern have the potential to disrupt the endocrine system of animals, apparently in association with their estrogenic properties. Competition with natural estrogens for the estrogen receptor is a possible mechanism by which such effects could occur. We used comparative molecular field analysis (CoMFA), a three-dimensional quantitative structure-activity relationship (QSAR) paradigm, to examine the underlying structural properties of ortho-chlorinated hydroxybiphenyl analogs known to bind to the estrogen receptor. The cross-validated and conventional statistical results indicate a high degree of internal predictability for the molecules included in the training data set. In addition to the phenolic (A) ring system, conformational restriction of the overall structure appears to play an important role in estrogen receptor binding affinity. Hydrophobic character as assessed using hydropathic interaction fields also contributes in a positive way to binding affinity. The CoMFA-derived QSARs may be useful in examining the estrogenic activity of a wider range of phenyl-substituted hydrocarbons of environmental concern. 37 refs., 2 figs., 2 tabs.

  10. Selection of ceramic fluorapatite-binding peptides from a phage display combinatorial peptide library: optimum affinity tags for fluorapatite chromatography.

    PubMed

    Islam, Tuhidul; Bibi, Noor Shad; Vennapusa, Rami Reddy; Fernandez-Lahore, Marcelo

    2013-08-01

    Peptide affinity tags have become efficient tools for the purification of recombinant proteins from biological mixtures. The most commonly used ligands in this type of affinity chromatography are immobilized metal ions, proteins, antibodies, and complementary peptides. However, the major bottlenecks of this technique are still related to the ligands, including their low stability, difficulties in immobilization, and leakage into the final products. A model approach is presented here to overcome these bottlenecks by utilizing macroporous ceramic fluorapatite (CFA) as the stationary phase in chromatography and the CFA-specific short peptides as tags. The CFA chromatographic materials act as both the support matrix and the ligand. Peptides that bind with affinity to CFA were identified from a randomized phage display heptapeptide library. A total of five rounds of phage selection were performed. A common N-terminal sequence was found in two selected peptides: F4-2 (KPRSMLH) and F5-4 (KPRSVSG). The peptide F5-4, displayed by more than 40% of the phages analyzed in the fifth round of selection, was subjected to further studies. Selectivity of the peptide for the chemical composition and morphology of CFA was assured by the adsorption studies. The dissociation constant, obtained from the F5-4/CFA adsorption isotherm, was in the micromolar range, and the maximum capacity was 39.4 nmol/mg. The chromatographic behavior of the peptides was characterized on a CFA stationary phase with different buffers. Preferential affinity and specific retention properties suggest the possible application of the phage-derived peptides as a tag in CFA affinity chromatography for enhancing the selective recovery of proteins.

  11. RELATIVE BINDING AFFINITY OF ENDOCRINE DISRUPTING CHEMICALS TO ESTROGEN RECEPTOR IN TWO SPECIES OF FRESHWATER FISH

    EPA Science Inventory

    The US EPA has been mandated to screen industrial chemicals and pesticides for potential endocrine activity. To evaluate the potential for chemicals to cause endocrine disruption in fish we have previously measured the affinity of a number of chemicals for the rainbow trout estr...

  12. The interaction of human serum albumin with selected lanthanide and actinide ions: Binding affinities, protein unfolding and conformational changes.

    PubMed

    Ali, Manjoor; Kumar, Amit; Kumar, Mukesh; Pandey, Badri N

    2016-04-01

    Human serum albumin (HSA), the most abundant soluble protein in blood plays critical roles in transportation of biomolecules and maintenance of osmotic pressure. In view of increasing applications of lanthanides- and actinides-based materials in nuclear energy, space, industries and medical applications, the risk of exposure with these metal ions is a growing concern for human health. In present study, binding interaction of actinides/lanthanides [thorium: Th(IV), uranium: U(VI), lanthanum: La(III), cerium: Ce(III) and (IV)] with HSA and its structural consequences have been investigated. Ultraviolet-visible, Fourier transform-infrared, Raman, Fluorescence and Circular dichroism spectroscopic techniques were applied to study the site of metal ions interaction, binding affinity determination and the effect of metal ions on protein unfolding and HSA conformation. Results showed that these metal ions interacted with carbonyl (CO..:)/amide(N..-H) groups and induced exposure of aromatic residues of HSA. The fluorescence analysis indicated that the actinide binding altered the microenvironment around Trp214 in the subdomain IIA. Binding affinity of U(VI) to HSA was slightly higher than that of Th(IV). Actinides and Ce(IV) altered the secondary conformation of HSA with a significant decrease of α-helix and an increase of β-sheet, turn and random coil structures, indicating a partial unfolding of HSA. A correlation was observed between metal ion's ability to alter HSA conformation and protein unfolding. Both cationic effects and coordination ability of metal ions seemed to determine the consequences of their interaction with HSA. Present study improves our understanding about the protein interaction of these heavy ions and their impact on its secondary structure. In addition, binding characteristics may have important implications for the development of rational antidote for the medical management of health effects of actinides and lanthanides.

  13. CB2 cannabinoid receptor agonist enantiomers HU-433 and HU-308: An inverse relationship between binding affinity and biological potency

    PubMed Central

    Smoum, Reem; Baraghithy, Saja; Chourasia, Mukesh; Breuer, Aviva; Mussai, Naama; Attar-Namdar, Malka; Kogan, Natalya M.; Raphael, Bitya; Bolognini, Daniele; Cascio, Maria G.; Marini, Pietro; Pertwee, Roger G.; Shurki, Avital; Mechoulam, Raphael; Bab, Itai

    2015-01-01

    Activation of the CB2 receptor is apparently an endogenous protective mechanism. Thus, it restrains inflammation and protects the skeleton against age-related bone loss. However, the endogenous cannabinoids, as well as Δ9-tetrahydrocannabinol, the main plant psychoactive constituent, activate both cannabinoid receptors, CB1 and CB2. HU-308 was among the first synthetic, selective CB2 agonists. HU-308 is antiosteoporotic and antiinflammatory. Here we show that the HU-308 enantiomer, designated HU-433, is 3–4 orders of magnitude more potent in osteoblast proliferation and osteoclast differentiation culture systems, as well as in mouse models, for the rescue of ovariectomy-induced bone loss and ear inflammation. HU-433 retains the HU-308 specificity for CB2, as shown by its failure to bind to the CB1 cannabinoid receptor, and has no activity in CB2-deficient cells and animals. Surprisingly, the CB2 binding affinity of HU-433 in terms of [3H]CP55,940 displacement and its effect on [35S]GTPγS accumulation is substantially lower compared with HU-308. A molecular-modeling analysis suggests that HU-433 and -308 have two different binding conformations within CB2, with one of them possibly responsible for the affinity difference, involving [35S]GTPγS and cAMP synthesis. Hence, different ligands may have different orientations relative to the same binding site. This situation questions the usefulness of universal radioligands for comparative binding studies. Moreover, orientation-targeted ligands have promising potential for the pharmacological activation of distinct processes. PMID:26124120

  14. CB2 cannabinoid receptor agonist enantiomers HU-433 and HU-308: An inverse relationship between binding affinity and biological potency.

    PubMed

    Smoum, Reem; Baraghithy, Saja; Chourasia, Mukesh; Breuer, Aviva; Mussai, Naama; Attar-Namdar, Malka; Kogan, Natalya M; Raphael, Bitya; Bolognini, Daniele; Cascio, Maria G; Marini, Pietro; Pertwee, Roger G; Shurki, Avital; Mechoulam, Raphael; Bab, Itai

    2015-07-14

    Activation of the CB2 receptor is apparently an endogenous protective mechanism. Thus, it restrains inflammation and protects the skeleton against age-related bone loss. However, the endogenous cannabinoids, as well as Δ(9)-tetrahydrocannabinol, the main plant psychoactive constituent, activate both cannabinoid receptors, CB1 and CB2. HU-308 was among the first synthetic, selective CB2 agonists. HU-308 is antiosteoporotic and antiinflammatory. Here we show that the HU-308 enantiomer, designated HU-433, is 3-4 orders of magnitude more potent in osteoblast proliferation and osteoclast differentiation culture systems, as well as in mouse models, for the rescue of ovariectomy-induced bone loss and ear inflammation. HU-433 retains the HU-308 specificity for CB2, as shown by its failure to bind to the CB1 cannabinoid receptor, and has no activity in CB2-deficient cells and animals. Surprisingly, the CB2 binding affinity of HU-433 in terms of [(3)H]CP55,940 displacement and its effect on [(35)S]GTPγS accumulation is substantially lower compared with HU-308. A molecular-modeling analysis suggests that HU-433 and -308 have two different binding conformations within CB2, with one of them possibly responsible for the affinity difference, involving [(35)S]GTPγS and cAMP synthesis. Hence, different ligands may have different orientations relative to the same binding site. This situation questions the usefulness of universal radioligands for comparative binding studies. Moreover, orientation-targeted ligands have promising potential for the pharmacological activation of distinct processes. PMID:26124120

  15. The interaction of human serum albumin with selected lanthanide and actinide ions: Binding affinities, protein unfolding and conformational changes.

    PubMed

    Ali, Manjoor; Kumar, Amit; Kumar, Mukesh; Pandey, Badri N

    2016-04-01

    Human serum albumin (HSA), the most abundant soluble protein in blood plays critical roles in transportation of biomolecules and maintenance of osmotic pressure. In view of increasing applications of lanthanides- and actinides-based materials in nuclear energy, space, industries and medical applications, the risk of exposure with these metal ions is a growing concern for human health. In present study, binding interaction of actinides/lanthanides [thorium: Th(IV), uranium: U(VI), lanthanum: La(III), cerium: Ce(III) and (IV)] with HSA and its structural consequences have been investigated. Ultraviolet-visible, Fourier transform-infrared, Raman, Fluorescence and Circular dichroism spectroscopic techniques were applied to study the site of metal ions interaction, binding affinity determination and the effect of metal ions on protein unfolding and HSA conformation. Results showed that these metal ions interacted with carbonyl (CO..:)/amide(N..-H) groups and induced exposure of aromatic residues of HSA. The fluorescence analysis indicated that the actinide binding altered the microenvironment around Trp214 in the subdomain IIA. Binding affinity of U(VI) to HSA was slightly higher than that of Th(IV). Actinides and Ce(IV) altered the secondary conformation of HSA with a significant decrease of α-helix and an increase of β-sheet, turn and random coil structures, indicating a partial unfolding of HSA. A correlation was observed between metal ion's ability to alter HSA conformation and protein unfolding. Both cationic effects and coordination ability of metal ions seemed to determine the consequences of their interaction with HSA. Present study improves our understanding about the protein interaction of these heavy ions and their impact on its secondary structure. In addition, binding characteristics may have important implications for the development of rational antidote for the medical management of health effects of actinides and lanthanides. PMID:26821345

  16. CB2 cannabinoid receptor agonist enantiomers HU-433 and HU-308: An inverse relationship between binding affinity and biological potency.

    PubMed

    Smoum, Reem; Baraghithy, Saja; Chourasia, Mukesh; Breuer, Aviva; Mussai, Naama; Attar-Namdar, Malka; Kogan, Natalya M; Raphael, Bitya; Bolognini, Daniele; Cascio, Maria G; Marini, Pietro; Pertwee, Roger G; Shurki, Avital; Mechoulam, Raphael; Bab, Itai

    2015-07-14

    Activation of the CB2 receptor is apparently an endogenous protective mechanism. Thus, it restrains inflammation and protects the skeleton against age-related bone loss. However, the endogenous cannabinoids, as well as Δ(9)-tetrahydrocannabinol, the main plant psychoactive constituent, activate both cannabinoid receptors, CB1 and CB2. HU-308 was among the first synthetic, selective CB2 agonists. HU-308 is antiosteoporotic and antiinflammatory. Here we show that the HU-308 enantiomer, designated HU-433, is 3-4 orders of magnitude more potent in osteoblast proliferation and osteoclast differentiation culture systems, as well as in mouse models, for the rescue of ovariectomy-induced bone loss and ear inflammation. HU-433 retains the HU-308 specificity for CB2, as shown by its failure to bind to the CB1 cannabinoid receptor, and has no activity in CB2-deficient cells and animals. Surprisingly, the CB2 binding affinity of HU-433 in terms of [(3)H]CP55,940 displacement and its effect on [(35)S]GTPγS accumulation is substantially lower compared with HU-308. A molecular-modeling analysis suggests that HU-433 and -308 have two different binding conformations within CB2, with one of them possibly responsible for the affinity difference, involving [(35)S]GTPγS and cAMP synthesis. Hence, different ligands may have different orientations relative to the same binding site. This situation questions the usefulness of universal radioligands for comparative binding studies. Moreover, orientation-targeted ligands have promising potential for the pharmacological activation of distinct processes.

  17. A Mechanism for Actin Filament Severing by Malaria Parasite Actin Depolymerizing Factor 1 via a Low Affinity Binding Interface*

    PubMed Central

    Wong, Wilson; Webb, Andrew I.; Olshina, Maya A.; Infusini, Giuseppe; Tan, Yan Hong; Hanssen, Eric; Catimel, Bruno; Suarez, Cristian; Condron, Melanie; Angrisano, Fiona; NebI, Thomas; Kovar, David R.; Baum, Jake

    2014-01-01

    Actin depolymerizing factor (ADF)/cofilins are essential regulators of actin turnover in eukaryotic cells. These multifunctional proteins facilitate both stabilization and severing of filamentous (F)-actin in a concentration-dependent manner. At high concentrations ADF/cofilins bind stably to F-actin longitudinally between two adjacent actin protomers forming what is called a decorative interaction. Low densities of ADF/cofilins, in contrast, result in the optimal severing of the filament. To date, how these two contrasting modalities are achieved by the same protein remains uncertain. Here, we define the proximate amino acids between the actin filament and the malaria parasite ADF/cofilin, PfADF1 from Plasmodium falciparum. PfADF1 is unique among ADF/cofilins in being able to sever F-actin but do so without stable filament binding. Using chemical cross-linking and mass spectrometry (XL-MS) combined with structure reconstruction we describe a previously overlooked binding interface on the actin filament targeted by PfADF1. This site is distinct from the known binding site that defines decoration. Furthermore, total internal reflection fluorescence (TIRF) microscopy imaging of single actin filaments confirms that this novel low affinity site is required for F-actin severing. Exploring beyond malaria parasites, selective blocking of the decoration site with human cofilin (HsCOF1) using cytochalasin D increases its severing rate. HsCOF1 may therefore also use a decoration-independent site for filament severing. Thus our data suggest that a second, low affinity actin-binding site may be universally used by ADF/cofilins for actin filament severing. PMID:24371134

  18. Structural heterogeneity of the alpha subunits of the nicotinic acetylcholine receptor in relation to agonist affinity alkylation and antagonist binding.

    PubMed

    Ratnam, M; Gullick, W; Spiess, J; Wan, K; Criado, M; Lindstrom, J

    1986-07-29

    The structural basis for the heterogeneity of the two agonist binding sites of the Torpedo californica acetylcholine receptor with respect to antagonist binding and reactivity toward affinity alkylating reagents was investigated. There is one agonist binding site on each of the two alpha subunits in a receptor monomer. One of these sites is easily affinity labeled with bromoacetylcholine, while more extreme conditions are required to label the other. Evidence is presented that the site which is easily labeled with bromoacetylcholine is the site with higher affinity for the antagonist d-tubocurarine. Digestion of purified alpha subunits with staphylococcal V8 protease gave two limit fragments with apparent molecular weights of 17K and 19K. Both of these fragments began at residue 46 of the alpha sequence, and both reacted with monoclonal antibodies specific for the sequence alpha 152-159 but not with antibodies specific for alpha 235-242. Their tryptic peptide maps and reactivity with a number of monoclonal antibodies were virtually identical. Only the 17-kilodalton (17-kDa) fragments stained heavily for sugars with Schiff's reagent. However, both fragments bound 125I-labeled concanavalin A. Complete removal of carbohydrate detectable with concanavalin A from V8 protease digests of alpha subunits resulted in two fragments of lower apparent molecular weights, indicating that these fragments differed not only in carbohydrate content but also in their C-termini or by another covalent modification. Covalent labeling of one of the two agonist sites of the intact receptor with bromo[3H]acetylcholine followed by digestion with V8 protease resulted in labeling of only the 19-kDa fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Enhanced DR5 binding capacity of nanovectorized TRAIL compared to its cytotoxic version by affinity chromatography and molecular docking studies.

    PubMed

    Zakaria, Albatoul; Picaud, Fabien; Guillaume, Yves Claude; Gharbi, Tijani; Micheau, Olivier; Herlem, Guillaume

    2016-09-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis of cancer cells when bound to its cognate receptors, TRAIL-R1 and TRAIL-R2 (DR4 and DR5), without being toxic to healthy cells. Nanovectorized TRAIL (abbreviated as NPT) is 10 to 20 times more efficient than one of the most potent soluble TRAIL used in preclinical studies (His-TRAIL). To determine whether differences in affinity may account for NPT superiority, a thermodynamic study was undertaken to evaluate NPT versus TRAIL binding affinity to DR5. Docking calculations showed that TRAIL in homotrimer configuration was more stable than in heterotrimer, because of the presence of one Zn ion in its structure. Indeed, TRAIL trimers can have head-to-tail orientations when Zn is missing. Altogether these data suggest that TRAIL homotrimer structures are predominant in solution and then are grafted on NPT. When docked to DR5, NPT carrying TRAIL homotrimer leads to a more stable complex than TRAIL monomer-based NPT. To comfort these observations, the extracellular domain of DR5 was immobilized on a chromatographic support using an "in situ" immobilization technique. The determination of the thermodynamic data (enthalpy ∆H° and entropy ∆S°*) of TRAIL and NPT binding to DR5 showed that the binding mechanism was pH dependent. The affinity of NPT to DR5 increased with pH, and the ionized energy was more important for NPT than for soluble TRAIL. Moreover, because of negative values of ∆H° and ∆S°* quantities, we demonstrated that van der Waals and hydrogen bonds governed the strong NPT-DR5 association for pH > 7.4 (as for TRAIL alone). Copyright © 2016 John Wiley & Sons, Ltd. PMID:26952193

  20. Distinct sites on the G-actin molecule bind group-specific component and deoxyribonuclease I.

    PubMed Central

    Goldschmidt-Clermont, P J; Galbraith, R M; Emerson, D L; Marsot, F; Nel, A E; Arnaud, P

    1985-01-01

    Addition of group-specific component (Gc) to G-actin with or without deoxyribonuclease I (DNAase) led to formation of binary complexes (Gc-G-actin) and ternary complexes (Gc-G-actin-DNAase) respectively. The electrophoretic mobility of ternary complexes, as shown by crossed and rocket immunoelectrophoresis, was slower than that of binary complexes, although both were faster than native Gc. In gradient polyacrylamide-gel electrophoresis, such complexes could again be resolved, apparently on the basis of relative molecular size: Gc-G-actin-DNAase (Mr approx. 131000), Gc-G-actin (Mr approx. 98000) and Gc (Mr approx. 56000). In contrast, the pI of ternary complex was indistinguishable by isoelectric focusing from that of binary complex, even though both were clearly more acidic than native Gc. The affinity of Gc for G-actin (affinity constant, Ka, 1.9 X 10(8) M-1) was not significantly altered by additional interaction with DNAase (Ka, 1.5 X 10(8)M-1), and both binary and ternary complexes still bound 25-hydroxycholecalciferol. In addition, the inhibitory effect of G-actin on DNAase activity was not discernibly affected by interaction with Gc. These results demonstrate that the various molecular forms of Gc can be distinguished by physicochemical parameters, and that Gc and DNAase bind to distinct sites on G-actin and can interact both independently and contemporaneously with this molecule. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:4040363

  1. The binding of pentapeptides to biological and synthetic high affinity heparin.

    PubMed

    Flengsrud, Ragnar; Antonsen, Simen Gjelseth

    2015-11-01

    Pentapeptides have been shown to bind the synthetic heparin fondaparinux (Arixtra) as well the biological heparins dalteparin (Fragmin) and salmon heparin. In contrast to heparin binding consensus sequences, the pentapeptides are acidic or neutral, with no arginine or histidine residue. The peptides showed an effect on in vitro heparin anti-factor X activity with a reduction of fondaparinux activity by 65-95%. Heparin binding was further studied by using peptide solid phase chromatography and NMR analysis.

  2. Affinity, stoichiometry and cooperativity of heterochromatin protein 1 (HP1) binding to nucleosomal arrays

    NASA Astrophysics Data System (ADS)

    Teif, Vladimir B.; Kepper, Nick; Yserentant, Klaus; Wedemann, Gero; Rippe, Karsten

    2015-02-01

    Heterochromatin protein 1 (HP1) participates in establishing and maintaining heterochromatin via its histone-modification-dependent chromatin interactions. In recent papers HP1 binding to nucleosomal arrays was measured in vitro and interpreted in terms of nearest-neighbour cooperative binding. This mode of chromatin interaction could lead to the spreading of HP1 along the nucleosome chain. Here, we reanalysed previous data by representing the nucleosome chain as a 1D binding lattice and showed how the experimental HP1 binding isotherms can be explained by a simpler model without cooperative interactions between neighboring HP1 dimers. Based on these calculations and spatial models of dinucleosomes and nucleosome chains, we propose that binding stoichiometry depends on the nucleosome repeat length (NRL) rather than protein interactions between HP1 dimers. According to our calculations, more open nucleosome arrays with long DNA linkers are characterized by a larger number of binding sites in comparison to chains with a short NRL. Furthermore, we demonstrate by Monte Carlo simulations that the NRL dependent folding of the nucleosome chain can induce allosteric changes of HP1 binding sites. Thus, HP1 chromatin interactions can be modulated by the change of binding stoichiometry and the type of binding to condensed (methylated) and non-condensed (unmethylated) nucleosome arrays in the absence of direct interactions between HP1 dimers.

  3. Zn2+ Mediates High Affinity Binding of Heparin to the αC Domain of Fibrinogen*

    PubMed Central

    Fredenburgh, James C.; Leslie, Beverly A.; Stafford, Alan R.; Lim, Teresa; Chan, Howard H.; Weitz, Jeffrey I.

    2013-01-01

    The nonspecific binding of heparin to plasma proteins compromises its anticoagulant activity by reducing the amount of heparin available to bind antithrombin. In addition, interaction of heparin with fibrin promotes formation of a ternary heparin-thrombin-fibrin complex that protects fibrin-bound thrombin from inhibition by the heparin-antithrombin complex. Previous studies have shown that heparin binds the E domain of fibrinogen. The current investigation examines the role of Zn2+ in this interaction because Zn2+ is released locally by platelets and both heparin and fibrinogen bind the cation, resulting in greater protection from inhibition by antithrombin. Zn2+ promotes heparin binding to fibrinogen, as determined by chromatography, fluorescence, and surface plasmon resonance. Compared with intact fibrinogen, there is reduced heparin binding to fragment X, a clottable plasmin degradation product of fibrinogen. A monoclonal antibody directed against a portion of the fibrinogen αC domain removed by plasmin attenuates binding of heparin to fibrinogen and a peptide analog of this region binds heparin in a Zn2+-dependent fashion. These results indicate that the αC domain of fibrinogen harbors a Zn2+-dependent heparin binding site. As a consequence, heparin-catalyzed inhibition of factor Xa by antithrombin is compromised by fibrinogen to a greater extent when Zn2+ is present. These results reveal the mechanism by which Zn2+ augments the capacity of fibrinogen to impair the anticoagulant activity of heparin. PMID:23990470

  4. Evidence that the low-affinity folate-binding protein in erythrocyte hemolysate is identical to hemoglobin

    SciTech Connect

    Hansen, S.I.; Holm, J.; Lyngbye, J.

    1981-07-01

    Gel filtration studies on erythrocyte hemolysate demonstrated the presence of a folate binding protein, apparently of the low-affinity type, that co-elutes with hemoglobin. Further, the folate binder eluted with a low salt concentration after DEAE-Sepharose CL-6B anion-exchange chromatography of erythrocyte hemolysate at pH 6.3. The chromatographic behavior of hemoglobin labeled with (3H)folate was so similar to that of the present binder as to suggest that the folate binder in erythrocytes is in fact hemoglobin.

  5. Electron paramagnetic resonance spectroscopic measurement of Mn2+ binding affinities to the hammerhead ribozyme and correlation with cleavage activity.

    PubMed

    Horton, T E; Clardy, D R; DeRose, V J

    1998-12-22

    Efficient phosphodiester bond cleavage activity by the hammerhead ribozyme requires divalent cations. Toward understanding this metal ion requirement, the Mn2+-binding properties of hammerhead model ribozymes have been investigated under dilute solution conditions, using electron paramagnetic resonance spectroscopy (EPR) to detect free Mn2+ in the presence of added ribozyme. Numbers and affinities of bound Mn2+ were obtained at pH 7.8 (5 mM triethanolamine) in the presence of 0, 0.1, and 1.0 M NaCl for an RNA-DNA model consisting of a 13-nucleotide DNA "substrate" hybridized to a 34-nucleotide RNA "enzyme" [Pley, H. W., Flaherty, K. M., and McKay, D. B. (1994) Nature 372, 68-74]. In 0.1 M NaCl, two classes of Mn2+ sites are found with n1 = 3.7 +/- 0.4, Kd(1) = 4 +/- 1 microM (type 1) and n2 = 5.2 +/- 0.4, Kd(2) = 460 +/- 130 microM (type 2). The high-affinity type 1 sites are confirmed for an active RNA-RNA hybrid (34-nucleotide RNA enzyme:13-nucleotide RNA substrate) by EPR measurements at low Mn2+ concentrations. Decreasing NaCl concentration results in an increased number of bound Mn2+ per hammerhead. By contrast, a binding titration in 1 M NaCl indicates that a single Mn2+ site with apparent Kd approximately 10 microM is populated in low concentrations of Mn2+, and apparent cooperative effects at higher Mn2+ concentrations result in population of a similar total number of Mn2+ sites (n1 = 8-10) as found in 0.1 M NaCl. Mn2+-dependent activity profiles are similar for the active RNA-RNA hybrid in 0.1 and 1 M NaCl. Correlation with binding affinities determined by EPR indicates that hammerhead activity in 0.1 M NaCl is only observed after all four of the high-affinity Mn2+ sites are occupied, rises with population of the type 2 sites, and is independent of Mn2+ concentrations corresponding to > 8-9 Mn2+ bound per hammerhead. Equivalent measurements in 1 M NaCl demonstrate a rise in activity with the cooperative transition observed in the Mn2+ binding curve. These

  6. Determination of binding constants by affinity capillary electrophoresis, electrospray ionization mass spectrometry and phase-distribution methods

    PubMed Central

    Chen, Zhi; Weber, Stephen G.

    2008-01-01

    Many methods for determining intermolecular interactions have been described in the literature in the past several decades. Chief among them are methods based on spectroscopic changes, particularly those based on absorption or nuclear magnetic resonance (NMR) [especially proton NMR (1H NMR)]. Recently, there have been put forward several new methods that are particularly adaptable, use very small quantities of material, and do not place severe requirements on the spectroscopic properties of the binding partners. This review covers new developments in affinity capillary electrophoresis, electrospray ionization mass spectrometry (ESI-MS) and phasetransfer methods. PMID:19802330

  7. Target binding to S100B reduces dynamic properties and increases Ca2+-binding affinity for wild type and EF-hand mutant proteins

    PubMed Central

    Liriano, Melissa A.; Varney, Kristen M.; Wright, Nathan T.; Hoffman, Cassandra L.; Toth, Eric A.; Ishima, Rieko; Weber, David J.

    2012-01-01

    Mutations in the second EF-hand (D61N, D63N, D65N, E72A) of S100B were used to study its Ca2+-binding and dynamic properties in the absence and presence of abound target, TRTK-12. With D63NS100B as an exception (D63NKD = 50 ± 9 µM), Ca2+-binding to EF2-hand mutants were reduced by more than 8-fold in the absence of TRTK-12 (D61NKD = 412 ± 67 µM; D65NKD = 968 ± 171 µM; E72AKD = 471 ± 133 µM), when compared to wild-type protein (WTKD = 56 ± 9 µM). For the TRTK-12 complexes, the Ca2+-binding affinity to wild type (WT+TRTKKD = 12 ± 10 µM) and the EF2 mutants were increased by 5- to 19-fold versus in the absence of target (D61N+TRTKKD = 29 ± 1.2 µM; D63N+TRTKKD = 10 ± 2.2 µM; D65N+TRTKKD = 73 ± 4.4 µM; E72A+TRTKKD = 18 ± 3.7 µM). In addition, Rex, as measured using relaxation dispersion for side chain 15N resonances of Asn63 (D63NS100B) was reduced upon TRTK-12 binding when measured by nuclear magnetic resonance (NMR). Likewise, backbone motions on multiple time scales (ps-ms) throughout wild type, D61NS100B D63NS100B, and D65NS100B were lowered upon binding TRTK-12. However, the X-ray structures of Ca2+-bound (2.0 Å) and TRTK-bound (1.2 Å) D63NS100B showed no change in Ca2+ coordination, so these and analogous structural data for the wild-type protein could not be used to explain how target binding increased Ca2+-binding affinity in solution. Thus, a model for how S100B-TRTK12 complex formation increases Ca2+ binding is discussed, which considers changes in protein dynamics upon binding the target TRTK-12. PMID:22824086

  8. Genetically encoded photocrosslinkers locate the high-affinity binding site of antidepressant drugs in the human serotonin transporter.

    PubMed

    Rannversson, Hafsteinn; Andersen, Jacob; Sørensen, Lena; Bang-Andersen, Benny; Park, Minyoung; Huber, Thomas; Sakmar, Thomas P; Strømgaard, Kristian

    2016-01-01

    Despite the well-established role of the human serotonin transporter (hSERT) in the treatment of depression, the molecular details of antidepressant drug binding are still not fully understood. Here we utilize amber codon suppression in a membrane-bound transporter protein to encode photocrosslinking unnatural amino acids (UAAs) into 75 different positions in hSERT. UAAs are incorporated with high specificity, and functionally active transporters have similar transport properties and pharmacological profiles compared with wild-type transporters. We employ ultraviolet-induced crosslinking with p-azido-L-phenylalanine (azF) at selected positions in hSERT to map the binding site of imipramine, a prototypical tricyclic antidepressant, and vortioxetine, a novel multimodal antidepressant. We find that the two antidepressants crosslink with azF incorporated at different positions within the central substrate-binding site of hSERT, while no crosslinking is observed at the vestibular-binding site. Taken together, our data provide direct evidence for defining the high-affinity antidepressant binding site in hSERT. PMID:27089947

  9. Genetically encoded photocrosslinkers locate the high-affinity binding site of antidepressant drugs in the human serotonin transporter

    PubMed Central

    Rannversson, Hafsteinn; Andersen, Jacob; Sørensen, Lena; Bang-Andersen, Benny; Park, Minyoung; Huber, Thomas; Sakmar, Thomas P.; Strømgaard, Kristian

    2016-01-01

    Despite the well-established role of the human serotonin transporter (hSERT) in the treatment of depression, the molecular details of antidepressant drug binding are still not fully understood. Here we utilize amber codon suppression in a membrane-bound transporter protein to encode photocrosslinking unnatural amino acids (UAAs) into 75 different positions in hSERT. UAAs are incorporated with high specificity, and functionally active transporters have similar transport properties and pharmacological profiles compared with wild-type transporters. We employ ultraviolet-induced crosslinking with p-azido-L-phenylalanine (azF) at selected positions in hSERT to map the binding site of imipramine, a prototypical tricyclic antidepressant, and vortioxetine, a novel multimodal antidepressant. We find that the two antidepressants crosslink with azF incorporated at different positions within the central substrate-binding site of hSERT, while no crosslinking is observed at the vestibular-binding site. Taken together, our data provide direct evidence for defining the high-affinity antidepressant binding site in hSERT. PMID:27089947

  10. Understanding Ion Binding Affinity and Selectivity in β-Parvalbumin Using Molecular Dynamics and Mean Spherical Approximation Theory.

    PubMed

    Kucharski, Amir N; Scott, Caitlin E; Davis, Jonathan P; Kekenes-Huskey, Peter M

    2016-08-25

    Parvalbumin (PV) is a globular calcium (Ca(2+))-selective protein expressed in a variety of biological tissues. Our computational studies of the rat β-parvalbumin (β-PV) isoform seek to elucidate the molecular thermodynamics of Ca(2+) versus magnesium (Mg(2+)) binding at the protein's two EF-hand motifs. Specifically, we have utilized molecular dynamics (MD) simulations and a mean-field electrolyte model (mean spherical approximation (MSA) theory) to delineate how the EF-hand scaffold controls the "local" thermodynamics of Ca(2+) binding selectivity over Mg(2+). Our MD simulations provide the probability density of metal-chelating oxygens within the EF-hand scaffolds for both Ca(2+) and Mg(2+), as well the conformational strain induced by Mg(2+) relative to Ca(2+) binding. MSA theory utilizes the binding domain oxygen and charge distributions to predict the chemical potential of ion binding, as well as their corresponding concentrations within the binding domain. We find that the electrostatic and steric contributions toward ion binding were similar for Mg(2+) and Ca(2+), yet the latter was 5.5 kcal/mol lower in enthalpy when internal strain within the EF hand was considered. We therefore speculate that beyond differences in dehydration energies for the Ca(2+) versus Mg(2+), strain induced in the β-PV EF hand by cation binding significantly contributes to the nearly 10,000-fold difference in binding affinity reported in the literature. We further complemented our analyses of local factors governing cation binding selectivity with whole-protein (global) contributions, such as interhelical residue-residue contacts and solvent exposure of hydrophobic surface. These contributions were found to be comparable for both Ca(2+)- and Mg(2+)-bound β-PV, which may implicate local factors, EF-hand strain, and dehydration, in providing the primary means of selectivity. We anticipate these methods could be used to estimate metal binding thermodynamics across a broad range of

  11. Characterization of the high affinity binding of epsilon toxin from Clostridium perfringens to the renal system.

    PubMed

    Dorca-Arévalo, Jonatan; Martín-Satué, Mireia; Blasi, Juan

    2012-05-25

    Epsilon toxin (ε-toxin), produced by Clostridium perfringens types B and D, causes fatal enterotoxaemia in livestock. In the renal system, the toxin binds to target cells before oligomerization, pore formation and cell death. Still, there is little information about the cellular and molecular mechanism involved in the initial steps of the cytotoxic action of ε-toxin, including the specific binding to the target sensitive cells. In the present report, the binding step of ε-toxin to the MDCK cell line is characterized by means of an ELISA-based binding assay with recombinant ε-toxin-green fluorescence protein (ε-toxin-GFP) and ε-prototoxin-GFP. In addition, different treatments with Pronase E, detergents, N-glycosidase F and beta-elimination on MDCK cells and renal cryosections have been performed to further characterize the ε-toxin binding. The ELISA assays revealed a single binding site with a similar dissociation constant (K(d)) for ε-toxin-GFP